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Sample records for avidin

  1. Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4.

    Science.gov (United States)

    Repo, Susanna; Paldanius, Tiina A; Hytönen, Vesa P; Nyholm, Thomas K M; Halling, Katrin K; Huuskonen, Juhani; Pentikäinen, Olli T; Rissanen, Kari; Slotte, J Peter; Airenne, Tomi T; Salminen, Tiina A; Kulomaa, Markku S; Johnson, Mark S

    2006-10-01

    The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.

  2. Improved avidin-biotin-peroxidase complex (ABC) staining.

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    Cattoretti, G; Berti, E; Schiró, R; D'Amato, L; Valeggio, C; Rilke, F

    1988-02-01

    A considerable intensification of the avidin-biotin-peroxidase complex staining system (ABC) was obtained by sequentially overlaying the sections to be immunostained with an avidin-rich and a biotin-rich complex. Each sequential addition contributed to the deposition of horseradish peroxidase on the immunostained site and allowed the subsequent binding of a complementary complex. With this technique a higher dilution of the antisera could be used and minute amounts of antigen masked by the fixative could be demonstrated on paraffin sections.

  3. Bifunctional avidin with covalently modifiable ligand binding site.

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    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  4. Chemical linkage to injected tissues is a distinctive property of oxidized avidin.

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    Rita De Santis

    Full Text Available We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation.

  5. A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

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    Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

    2016-03-01

    Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells.

  6. Endogenous avidin biotin activity (EABA in thyroid pathology: immunohistochemical study

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    Nikiel Barbara

    2009-04-01

    Full Text Available Abstract Background Immunohistochemical methods based on the high affinity of avidin and biotin (e.g. ABC, LSAB are characterized by high sensitivity and are widely used for detection of immunologic reaction. However, a non-specific reaction, observed in frozen tissues and in paraffin-embedded material, increasing after heat induced epitope retrieval (HIER, and caused either by endogenous biotin or any another chemical compound with high affinity for avidin, may lead to diagnostic mistakes. The aim of our investigation is to study presence of endogenous avidin biotin activity (EABA in thyrocytes originating from various thyroid pathological lesions (neoplastic and non-neoplastic. Materials and methods The immunohistochemical study was performed on paraffin-embedded specimens of surgically resected thyroid tissue from 97 patients with thyroid diseases: 65 patients with papillary carcinoma (PTC, 11 patients with nodular goiter in whom features of benign papillary hyperplasia were found, 9 with lymphocytic thyroiditis (LT, 8 with follicular adenoma, and 4 patients with follicular carcinoma. In PTC immunohistochemical study was performed both in primary tumors and in lymph node metastases. After HIER, incubation with streptavidin from LSAB+ (DakoCytomation kit was done. Results Strong cytoplasmic EABA was observed in 56 of 65 (87.5% PTC and in oxyphilic cells in 8 of 9 cases of LT. Significant correlation between EABA in primary PTC tumor and EABA in lymph node metastases was stated. Normal surrounding thyroid tissues showed absence or weak EABA. Aberrant intranuclear localization of biotin was noted in morules of cribriform-morular variant of PTC. No statistically significant correlation between patient's age, sex, metastases presence and EABA was observed. Conclusion Among thyroid lesions, false positive reactions are highly probable in papillary thyroid carcinoma and in lymphocytic thyroiditis if immunohistochemical detection is used on systems

  7. Bone Tissue Engineering by Using Calcium Phosphate Glass Scaffolds and the Avidin-Biotin Binding System.

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    Kim, Min-Chul; Hong, Min-Ho; Lee, Byung-Hyun; Choi, Heon-Jin; Ko, Yeong-Mu; Lee, Yong-Keun

    2015-12-01

    Highly porous and interconnected scaffolds were fabricated using calcium phosphate glass (CPG) for bone tissue engineering. An avidin-biotin binding system was used to improve osteoblast-like cell adhesion to the scaffold. The scaffolds had open macro- and micro-scale pores, and continuous struts without cracks or defects. Scaffolds prepared using a mixture (amorphous and crystalline CPG) were stronger than amorphous group and crystalline group. Cell adhesion assays showed that more cells adhered, with increasing cell seeding efficiency to the avidin-adsorbed scaffolds, and that cell attachment to the highly porous scaffolds significantly differed between avidin-adsorbed scaffolds and other scaffolds. Proliferation was also significantly higher for avidin-adsorbed scaffolds. Osteoblastic differentiation of MG-63 cells was observed at 3 days, and MG-63 cells in direct contact with avidin-adsorbed scaffolds were positive for type I collagen, osteopontin, and alkaline phosphatase gene expression. Osteocalcin expression was observed in the avidin-adsorbed scaffolds at 7 days, indicating that cell differentiation in avidin-adsorbed scaffolds occurred faster than the other scaffolds. Thus, these CPG scaffolds have excellent biological properties suitable for use in bone tissue engineering.

  8. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

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    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  9. Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

    Institute of Scientific and Technical Information of China (English)

    Gui-Ping Li; Yong-Xian Wang; Kai Huang; Hui Zhang; Chun-Fu Zhang

    2005-01-01

    AIM: To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome.METHODS: Purified anti-CEA monoclonal antibody (McAb)was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeledbiotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg) followed by intravenous injection of cold avidin (80 μg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg), another control group (non-treated group) only received 0.1 mL normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed.RESULTS: Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA Mc Ab-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the nontreated group, treated groups with and without avidin chase showed significant anti

  10. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

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    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  11. Reproductive performance and oviductal expression of avidin and avidin-related protein-2 in young and old broiler breeder hens orally exposed to supplementary biotin.

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    Daryabari, H; Akhlaghi, A; Zamiri, M J; Mianji, G Rahimi; Pirsaraei, Z Ansari; Deldar, H; Eghbalian, A N

    2014-09-01

    Published data on the probable involvement of avidin and avidin-related protein-2 (AVR2) in sustaining sperm viability in sperm storage tubules in 38-wk-old turkeys, and the high affinity of avidin or its analogs to biotin suggest that supplementary biotin may increase oviductal avidin and AVR2 expression, thereby attenuating the adverse effect of aging on hen reproductive performance. Broiler breeder hens (n = 120) were randomly assigned to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg of biotin/L of drinking water from 30 to 33 (young) and 53 to 56 (old) wk of age, and artificially inseminated to determine their reproductive performance. At the end of each period of biotin administration, 8 hens from each treatment group were killed for RNA extraction from the uterovaginal junction. Egg production was lower in the old hens (44%) compared with the young ones (82%), and biotin supplementation increased egg production only in the latter. Administering supplementary biotin to young hens increased their oviductal expression of AVR2, which was much higher in the old hens (1.0 and 4.6 for young and old groups, respectively). Fertility rate was not different between young and old hens, and was increased (4.4%) at the higher level of biotin supplementation. Hatchability and hatchling quality were not affected by biotin supplementation. Embryonic mortality between 17 to 21 d of incubation was higher in young (5.2%) compared with old (1.4%) birds. Egg fertility rate showed a moderate correlation (P biotin supplementation on AVR2 expression, and the relationship between biotin administration and oviductal expression of avidin and AVR2 was dependent on the hen's age, being higher in the young hens.

  12. Biotin-Avidin Based Universal Cell-Matrix Interaction for Promoting Three-Dimensional Cell Adhesion.

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    Dou, Xiao-Qiu; Zhang, Jia; Feng, Chuanliang

    2015-09-23

    To promote cell adhesion in three-dimensional (3D) extracellular matrix (ECM) is crucial for avoiding cell anoikis, which is one of the most important issues for fundamental cell biology. Herein, a biotin-avidin based universal cell-matrix interaction for different types of cells is developed in order to achieve the promoted adhesion in 3D ECM. For the purpose, biotinylated nanofibrous hydrogels are constructed by coassembling 1,4-benzyldicarboxamide (C2) based non-biotinylated and biotinylated supramolecular gelators. The used cells are modified by avidin (AV-cells) through biotinylating cells and then interacting with avidin. After in situ encapsulating AV-cells in the hydrogels, the adhered amount can be increased by tens of percent even with adding several percentages of the biotinylated C2 gelators in the coassembly due to the specific biotin-avidin interaction. Reverse transcription polymerase chain reaction (RT-PCR) confirms that AV-cells can proliferate without varying gene expression and denaturation. Compared with the interaction between RGD and cells, this avidin-biotin interaction should be much more universal and it is feasible to be employed to promote cell adhesion for most types of cells in 3D matrix.

  13. Quantitative label-free characterization of avidin-biotin assemblies on silanized glass.

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    Chen, Li-Jung; Seo, Jeong Hyun; Eller, Michael J; Verkhoturov, Stanislav V; Shah, Sunny S; Revzin, Alexander; Schweikert, Emile A

    2011-09-15

    In this study, a time-of-flight secondary ion mass spectrometer TOF-SIMS, operating in the event-by-event bombardment/detection mode was used to characterize avidin-biotin assemblies on silane-modified glass substrates. SIMS was used to analyze several variants of the biointerface, including avidin physically adsorbed on a monofunctional acryl silane surface and covalently attached on monofunctional (amine terminated) and bifunctional (amine and acryl terminated) silanes. The goal of these studies was to determine density of avidin and biotin layers chemically or physically adsorbed on silanized glass substrate. An individual impact of a C(60) projectile used in this study creates a hemispherical crater (∼10 nm in diameter) and emits large numbers of secondary ions from the same nanovolume. Thus, a single impact enables one to unfold distinct secondary ions that span the thickness of the assembled film. This method was used to monitor the presence of glass, silane, and protein ions and to estimate the thickness and density of the avidin layer. In addition, we employed the double coincidence mass spectrometry approach to identify ions coemitted from a specific stratum of the biointerface. This approach was used to determine density of biotin and avidin immobilization while eliminating interferences from isobaric ions that originated from other constituents on the surface. Overall, novel TOF-SIMS quantitative approaches employed here were useful for examining complex biointerfaces and determining both lateral and in depth composition of the film.

  14. Versatile bio-ink for covalent immobilization of chimeric avidin on sol-gel substrates.

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    Heikkinen, Jarkko J; Kivimäki, Liisa; Määttä, Juha A E; Mäkelä, Inka; Hakalahti, Leena; Takkinen, Kristiina; Kulomaa, Markku S; Hytönen, Vesa P; Hormi, Osmo E O

    2011-10-15

    A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films.

  15. Specific detection of avidin-biotin binding using liquid crystal droplets.

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    Khan, Mashooq; Park, Soo-Young

    2015-03-01

    Poly(acrylicacid-b-4-cynobiphenyl-4'-undecylacrylate) (PAA-b-LCP)-functionalized 4-cyano-4'-pentylbiphenyl (5CB) droplets were made by using microfluidic technique. The PAA chains on the 5CB droplets, were biotinylated, and used to specifically detect avidin-biotin binding at the 5CB/aqueous interface. The avidin-biotin binding was characterized by the configurational change (from radial to bipolar) of the 5CB droplets, as observed through a polarized optical microscope. The maximum biotinylation was obtained by injecting a >100 μg/mL biotin aqueous solution, which enabled a limit of detection of 0.5 μg/mL avidin. This droplet biosensor could specifically detect avidin against other proteins such as bovine serum albumin, lysozyme, hemoglobin, and chymotrypsinogen solutions. Avidin detection with 5CBPAA-biotin droplets having high sensitivity, specificity, and stability demonstrates new applications of the functionalized liquid crystal droplets that can detect specific proteins or other analytes through a ligand/receptor model.

  16. Surface-charge differentiation of streptavidin and avidin by atomic force microscopy-force spectroscopy.

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    Almonte, Lisa; Lopez-Elvira, Elena; Baró, Arturo M

    2014-09-15

    Chemical information can be obtained by using atomic force microscopy (AFM) and force spectroscopy (FS) with atomic or molecular resolution, even in liquid media. The aim of this paper is to demonstrate that single molecules of avidin and streptavidin anchored to a biotinylated bilayer can be differentiated by using AFM, even though AFM topographical images of the two proteins are remarkably alike. At physiological pH, the basic glycoprotein avidin is positively charged, whereas streptavidin is a neutral protein. This charge difference can be determined with AFM, which can probe electrostatic double-layer forces by using FS. The force curves, owing to the electrostatic interaction, show major differences when measured on top of each protein as well as on the lipid substrate. FS data show that the two proteins are negatively charged. Nevertheless, avidin and streptavidin can be clearly distinguished, thus demonstrating the sensitivity of AFM to detect small changes in the charge state of macromolecules.

  17. pH-dependent deformations of the energy landscape of avidin-like proteins investigated by single molecule force spectroscopy.

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    Köhler, Melanie; Karner, Andreas; Leitner, Michael; Hytönen, Vesa P; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

    2014-08-18

    Avidin and avidin-like proteins are widely used in numerous techniques since the avidin-biotin interaction is known to be very robust and reliable. Within this study, we investigated this bond at the molecular level under harsh conditions ranging from very low to very high pH values. We compared avidin with streptavidin and a recently developed avidin-based mutant, chimeric avidin. To gain insights of the energy landscape of these interactions we used a single molecule approach and performed the Single Molecule Force Spectroscopy atomic force microscopy technique. There, the ligand (biotin) is covalently coupled to a sharp AFM tip via a distensible hetero-bi-functional crosslinker, whereas the receptor of interest is immobilized on the probe surface. Receptor-ligand complexes are formed and ruptured by repeatedly approaching and withdrawing the tip from the surface. Varying both pulling velocity and pH value, we could determine changes of the energy landscape of the complexes. Our results clearly demonstrate that avidin, streptavidin and chimeric avidin are stable over a wide pH range although we could identify differences at the outer pH range. Taking this into account, they can be used in a broad range of applications, like surface sensors at extreme pH values.

  18. Recording intracellular molecular events from the outside: glycosylphosphatidylinositol-anchored avidin as a reporter protein for in vivo imaging.

    NARCIS (Netherlands)

    Lehmann, S.A.; Garayoa, E.G.; Blanc, A.; Keist, R.; Schibli, R.; Rudin, M.

    2011-01-01

    With the emergence of multimodal imaging strategies, genetically encoded reporters that can be flexibly combined with any imaging modality become highly attractive. Here we describe the use of glycosylphosphatidylinositol (GPI)-anchored avidin, an avidin moiety targeted to the extracellular side of

  19. Virus immobilization on biomaterial scaffolds through biotin-avidin interaction for improving bone regeneration.

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    Hu, Wei-Wen; Wang, Zhuo; Krebsbach, Paul H

    2016-02-01

    To spatially control therapeutic gene delivery for potential tissue engineering applications, a biotin-avidin interaction strategy was applied to immobilize viral vectors on biomaterial scaffolds. Both adenoviral vectors and gelatin sponges were biotinylated and avidin was applied to link them in a virus-biotin-avidin-biotin-material (VBABM) arrangement. The tethered viral particles were stably maintained within scaffolds and SEM images illustrated that viral particles were evenly distributed in three-dimensional (3D) gelatin sponges. An in vivo study demonstrated that transgene expression was restricted to the implant sites only and transduction efficiency was improved using this conjugation method. For an orthotopic bone regeneration model, adenovirus encoding BMP-2 (AdBMP2) was immobilized to gelatin sponges before implanting into critical-sized bone defects in rat calvaria. Compared to gelatin sponges with AdBMP2 loaded in a freely suspended form, the VBABM method enhanced gene transfer and bone regeneration was significantly improved. These results suggest that biotin-avidin immobilization of viral vectors to biomaterial scaffolds may be an effective strategy to facilitate tissue regeneration.

  20. Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

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    Strzelczyk, Paweł; Bujacz, Grzegorz

    2016-04-01

    Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

  1. Biotin avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

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    Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

    2007-04-01

    Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5 mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11).

  2. Highly sensitive fluorescence detection of avidin/streptavidin with an optical interference mirror slide.

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    Yasuda, Mitsuru; Akimoto, Takuo

    2012-01-01

    This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

  3. Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures.

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    Gown, A M; Garcia, R; Ferguson, M; Yamanaka, E; Tippens, D

    1986-03-01

    We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.

  4. The biodistribution and Kinetics of the Samarium-153 labeled avidin, streptavidin and biotin

    Institute of Scientific and Technical Information of China (English)

    LI Gui-ping; ZHU Cheng-mo; JIANG Xu-feng; FENG Guo-wei; ZHANG Sheng-guo

    2001-01-01

    Objective: To label avidin (Av) or streptavidin (SA) with 153Sm by taking advantage of the high binding affinity of biotin to Av or SA. Methods: A biotin derivative (DTPA-biotin) was radiolabelled with 153Sm and then bound to Av or SA. The in vivo kinetics and biodistribution of 153Sm-labeled Ay, SA and DTPA-biotin were studied in rats and mice.Results: 153Sm-Av was characterized by rapid clearance from the blood with high liver and renal uptake; 153Sm-SA was cleared from the blood slowly with high retention in the liver, spleen and kidney, whereas 153Sm-DTPA-biotin metabolism was accelerated, and its excretion was mainly through the kidney. Conclusion: The biodistribution difference of SA and Av may provide an experimental basis for the selection of different components of avidin-biotin system in pretageting radioimmunoimaging and radioimmunotherapy.

  5. Oral administration of supplementary biotin differentially influences the fertility rate and oviductal expression of avidin and avidin-related protein-2 in low- and high-fertility broiler line hens.

    Science.gov (United States)

    Daryabari, H; Akhlaghi, A; Zamiri, M J; Pirsaraei, Z Ansari; Mianji, G Rahimi; Deldar, H; Eghbalian, A N

    2015-02-01

    Probable involvement of avidin and avidin-related protein-2 (AVR2) in sperm viability in the sperm storage tubules of turkeys has been suggested. The high affinity of biotin to avidin and its analogs is also well documented. The present study aimed to determine the effect of oral biotin on reproductive performance and oviductal mRNA expression of avidin and AVR2 in 2 broiler hen lines with different fertility rates. Low-fertility (line B) and high-fertility (line D) hens (n=144) were randomly allotted to receive 0 (T0), 0.30 (T1), or 0.45 (T2) mg/L biotin in drinking water from 30 through 33 wk of age. The reproductive performance of the hens was evaluated using artificial insemination. At the end of the treatment period, 24 hens per line were killed to assay the expression of avidin and AVR2 in the uterovaginal junction. Supplementary biotin increased egg production from 73.5% for T0 to 87.8% for T2. Hens administered with biotin in line B, but not in line D, showed an increase (8.4%) in fertility rate. Hatchability, chick quality, and overall embryonic mortality were not different among the experimental groups. Real-time PCR data showed that both avidin (P=0.0013) and AVR2 (Phens receiving the high biotin level recorded respectively a 3.9 and 15.3% increase in avidin and AVR2 mRNA expression, although biotin did not affect these traits in line D hens. Control hens in line D had a dramatically higher AVR2 expression record (7.4-fold) compared with the control hens in line B. The correlation coefficients of fertility rate and avidin expression were 0.73 and 0.66 in lines B and D, respectively. However, the correlation of fertility and AVR2 (r=0.65) was significant for line D hens only. Overall, fertility rate and oviductal expression of avidin and AVR2 were dichotomously affected by oral biotin in low- and high-fertility line hens, where only low-fertility birds showed improvements in these attributes.

  6. Screening Effect of PEG on Avidin Binding to Liposome Surface Receptors

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, Ole G.; Jørgensen, Kent

    2000-01-01

    This study investigates the screening effect of poly(ethylene glycol)-phospholipids (PE-PEG) on the interaction of avidin with PEGylated liposomes containing surface-bound biotin ligands. The influence of grafting density and lipopolymer chain length is examined. A simple fluorescence assay....... and it is found that the screening effect of PE-PEG(5000) is stronger than that for PE-PEG(2000). Thus, the results reveal that both the grafting density and the polymer length of the PE-PEC lipopolymers are of importance for the ability of water-soluble macromolecules to reach the surface of PEG liposomes...

  7. Label free optical sensor for Avidin based on single gold nanoparticles functionalized with aptamers.

    Science.gov (United States)

    Hernandez, Frank Jeyson; Dondapati, Srujan Kumar; Ozalp, V Cengiz; Pinto, Alessandro; O'Sullivan, Ciara K; Klar, Thomas A; Katakis, Ioannis

    2009-04-01

    Optical spectroscopy of a single gold nanoparticle, functionalized with an aptamer, is used to sense the specific binding of avidin. Herewith, the field of single noble metal nanoparticle biosensors is extended to the important field of aptamer based assays. The sensitivity of this initial, but not yet optimized apta-nano-sensor is in the range of 20 nM. Due to its nanoscopic size, this single nanoparticle based apta-sensor may be used in nanoscopic volumes such as in array type assays or even inside cells.

  8. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

    Science.gov (United States)

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  9. Utilization of chip-based CE for avidin determination in transgenic tobacco and its comparison with square-wave voltammetry and standard gel electrophoresis

    Science.gov (United States)

    Avidin transgenic plants are a potential tool for providing resistance against various species of insect pests due to the sequestration of vitamin H (biotin) in the plant from the insect pests. In this project we compared three techniques for avidin determination in transgenic tobacco plants, a nove...

  10. Combining a sensor and a pH-gated nanopore based on an avidin-biotin system.

    Science.gov (United States)

    Lepoitevin, Mathilde; Nguyen, Gael; Bechelany, Mikhael; Balanzat, Emmanuel; Janot, Jean-Marc; Balme, Sebastien

    2015-04-01

    Here we propose a new approach to tailor nanopores, which combines both pH gating and sensing properties. This strategy is based on PEG like-avidin grafting in nanopores designed by atomic layer deposition (ALD). Below pH 5 the nanopore is blocked. We show that the PEG chains are at the origin of these properties.

  11. Biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of chondrocyte adhesion to scaffolds.

    Science.gov (United States)

    Lin, Hong; Zhou, Jian; Shen, Longxiang; Ruan, Yuhui; Dong, Jian; Guo, Changan; Chen, Zhengrong

    2014-04-01

    The clinical need for improved treatment options for patients with cartilage injuries has motivated tissue-engineering studies aimed at the in vitro generation of cell-based implants with functional properties. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to the scaffold. In the present study, chondrocyte-scaffold constructs were engineered by planting porcine chondrocytes into nonporous chitosan membranes and 3D porous chitosan scaffolds that were treated with or without biotin-conjugated anti-CD44 antibody-avidin binding system and avidin-biotin binding system. The spreading area, cell exfoliation rates, cell proliferation rates, histological analysis, DNA and glycosaminoglycan (GAG) content, and mRNA expression were investigated to evaluate the efficiency of biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of cell adhesion to scaffolds in the cartilage tissue. The results showed that the biotin-conjugated anti-CD44 antibody-avidin binding system improved cell adhesion to scaffolds effectively. These studies suggest that this binding system has the potential to provide improved tissue-engineered cartilage for clinical applications.

  12. Applicability of avidin protein coated mesoporous silica nanoparticles as drug carriers in the lung

    Science.gov (United States)

    van Rijt, S. H.; Bölükbas, D. A.; Argyo, C.; Wipplinger, K.; Naureen, M.; Datz, S.; Eickelberg, O.; Meiners, S.; Bein, T.; Schmid, O.; Stoeger, T.

    2016-04-01

    Mesoporous silica nanoparticles (MSNs) exhibit unique drug delivery properties and are thus considered as promising candidates for next generation nano-medicines. In particular, inhalation into the lungs represents a direct, non-invasive delivery route for treating lung disease. To assess MSN biocompatibility in the lung, we investigated the bioresponse of avidin-coated MSNs (MSN-AVI), as well as aminated (uncoated) MSNs, after direct application into the lungs of mice. We quantified MSN distribution, clearance rate, cell-specific uptake, and inflammatory responses to MSNs within one week after instillation. We show that amine-functionalized (MSN-NH2) particles are not taken up by lung epithelial cells, but induced a prolonged inflammatory response in the lung and macrophage cell death. In contrast, MSN-AVI co-localized with alveolar epithelial type 1 and type 2 cells in the lung in the absence of sustained inflammatory responses or cell death, and showed preferential epithelial cell uptake in in vitro co-cultures. Further, MSN-AVI particles demonstrated uniform particle distribution in mouse lungs and slow clearance rates. Thus, we provide evidence that avidin functionalized MSNs (MSN-AVI) have the potential to serve as versatile biocompatible drug carriers for lung-specific drug delivery.Mesoporous silica nanoparticles (MSNs) exhibit unique drug delivery properties and are thus considered as promising candidates for next generation nano-medicines. In particular, inhalation into the lungs represents a direct, non-invasive delivery route for treating lung disease. To assess MSN biocompatibility in the lung, we investigated the bioresponse of avidin-coated MSNs (MSN-AVI), as well as aminated (uncoated) MSNs, after direct application into the lungs of mice. We quantified MSN distribution, clearance rate, cell-specific uptake, and inflammatory responses to MSNs within one week after instillation. We show that amine-functionalized (MSN-NH2) particles are not taken up

  13. Diagnosis of internal acariasis with avidin-biotin system enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    Rong-Bo Zhang; Yong Huang; Chao-Pin Li; Yu-Bao Cui

    2004-01-01

    AIM: To explore the value of avidin-biotin system enzymelinked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis.METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA.The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA).RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABCELISA was statistically higher than that with SPA-ELISA (X2=13.50, P<0.01).CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis.

  14. A hypersensitive biotin-avidin-TRFIA for quantitative detection of ANA-Ig(GAM) and its clinical application.

    Science.gov (United States)

    Liu, Jie; Ye, Yan; Hu, Zhigang; Zou, Yaohong; Chen, Guoqian; Yu, Lei

    2013-01-01

    We demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) with high sensitivity and wide range for quantitative detection of ANA-Ig(GAM) antibodies using a biotin-avidin amplification system. The immunoassay was conducted by following procedures for a typical sandwich immunoreactions with cell nucleus form Hela and the Eu(3+)-labeled biotin combined with biotinylated mouse anti-human Ig(GAM) served as the solid nuclear antigen for ANA and the tracer, respectively. The sensitivity, specificity, and stability of the kit were evaluated and comparison with the classical enzyme-linked immunosorbent assay (ELISA) kit was also made. The average intra-assay and interassay CVs detected by the established ANA-Ig(GAM) biotin-avidin-TRFIA were 4.21% and 6.34%, respectively. The lower detection limit was 2.24 U/mL, and the mean recovery rate was 100.74%. The good measurable range of the established biotin-avidin-TRFIA was within 1.95-64,000 U/mL, while it was only within 32.5-4000 U/mL using an ELISA kit. The values determined by the biotin-avidin-TRFIA and ELISA correlated well (R2 = 0.989). The positive rate of healthy volunteers and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC), Sjögren's syndrome (SS), scleroderma, and mixed connective tissue disease (MCTD) was 0, 100%, 18.5%, 100%, 37.9%, 90.9%, and 92%, respectively. We conclude that the biotin-avidin-TRFIA we developed gives promise for greater sensitivity and accurate detection for ANA-Ig(GAM) in diagnosing and monitoring autoimmune disorders.

  15. Towards the development of a direct electrochemical biodetector of avidin based on the poly(chloro amino β-styryl terthiophene)-coated glassy carbon electrode

    KAUST Repository

    Mehenni, Hakim

    2012-03-30

    In this study, a simple and direct biodetector was proposed, which was based on biotin immobilized onto a conducting polymer-coated electrode, for the detection of avidin, a highly stable glycoprotein found in egg-whites. Biotin was immobilized onto the electrode by covalent coupling to the primary amine group on the poly 3′-(3-chloro-4-amino-β-styryl)-(2,2′: 5′,2″-terthiophene) (PCAST), and the biotinavidin interaction was monitored by cyclic voltammetry. Incubation of the PCAST/biotin-modified-coated electrode with avidin in a phosphate buffered saline solution caused a significant change to its cyclic voltammogram, which was explained by the binding of avidin by biotin, and resulted in restricted ion transfer to and from the conducting polymer. This change was then utilized to detect avidin at 4 × 10 -6molL -1. © 2012 CSIRO.

  16. Improved Magnetic Resonance Molecular Imaging of Tumor Angiogenesis by Avidin-Induced Clearance of Nonbound Bimodal Liposomes

    Directory of Open Access Journals (Sweden)

    Geralda A.F. van Tilborg

    2008-12-01

    Full Text Available Angiogenic, that is, newly formed, blood vessels play an important role in tumor growth and metastasis and are a potential target for tumor treatment. In previous studies, the αvβ3 integrin, which is strongly expressed in angiogenic vessels, has been used as a target for Arg-Gly-Asp (RGD-functionalized nanoparticulate contrast agents for magnetic resonance imaging-based visualization of angiogenesis. In the present study, the target-to-background ratio was increased by diminishing the nonspecific contrast enhancement originating from contrast material present in the blood pool. This was accomplished by the use of a so-called avidin chase, which allowed rapid clearance of non-bound paramagnetic RGD-biotin-liposomes from the blood circulation. C57BL/6 mice, bearing a B16F10 mouse melanoma, received RGD-functionalized or untargeted biotin-liposomes, which was followed by avidin infusion or no infusion. Precontrast, postcontrast, and postavidin T1-weighted magnetic resonance images were acquired at 6.3 T. Postcontrast images showed similar percentages of contrast-enhanced pixels in the tumors of mice that received RGD-biotin-liposomes and biotin-liposomes. Post avidin infusion this percentage rapidly decreased to precontrast levels for biotin-liposomes, whereas a significant amount of contrast-enhanced pixels remained present for RGD-biotin-liposomes. These results showed that besides target-associated contrast agent, the circulating contrast agent contributed significantly to the contrast enhancement as well. Ex vivo fluorescence microscopy confirmed association of the RGD-biotin-liposomes to tumor endothelial cells both with and without avidin infusion, whereas biotin-liposomes were predominantly found within the vessel lumen. The clearance methodology presented in this study successfully enhanced the specificity of molecular magnetic resonance imaging and opens exciting possibilities for studying detection limits and targeting kinetics of site

  17. Development of an avidin sensor based on the poly(methoxy amino-β-styryl terthiophene)-coated glassy carbon electrode

    KAUST Repository

    Mehenni, Hakim

    2012-03-01

    In this study, a simple and direct biosensor was proposed, which was based on biotin immobilized onto a conducting polymer-coated electrode, for the determination of avidin, a highly stable glycoprotein found in egg whites. Biotin was immobilized onto the electrode by covalent coupling to the primary amine group on poly-3′-(2-methoxy-5-amino-β-styryl)-(2,2′: 5′,2″-terthiophene) (PMAST), and the biotin-avidin interaction was monitored by square-wave voltammetry. Incubation of the PMAST/biotin-modified coated electrode with avidin in a phosphate-buffered saline solution caused a significant change to its square-wave voltammogram, which was explained by the binding of avidin by biotin, and resulted in restricted ion transfer to and from the conducting polymer. This change was then utilized to determine avidin. Importantly, we found a linear relationship for the avidin sensor in the range of 4 × 10 -14 to 3 × 10 -4 mol/L, and the detection limit was determined to be approximately 10 -14 mol/L. © 2012 Published by NRC Research Press.

  18. Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index.

    Science.gov (United States)

    Esmenjaud, D; Walter, B; Minot, J C; Voisin, R; Cornuet, P

    1993-09-01

    The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.

  19. NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching.

    Science.gov (United States)

    Lippert, Lisa G; Hallock, Jeffrey T; Dadosh, Tali; Diroll, Benjamin T; Murray, Christopher B; Goldman, Yale E

    2016-03-16

    We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.

  20. Influences of dietary biotin and avidin on growth, survival, deficiency syndrome and hepatic gene expression of juvenile Nile tilapia Oreochromis niloticus.

    Science.gov (United States)

    Sarker, Pallab Kumer; Yossa, Rodrigue; Karanth, Santhosh; Ekker, Marc; Vandenberg, Grant W

    2012-08-01

    This study was undertaken to assess the interactive effects of dietary biotin and avidin on growth, feed conversion, survival and deficiency syndrome of tilapia and to determine the influence of dietary biotin deficiency on the expression of key genes related to biotin metabolism in tilapia. Six iso-nitrogenous and iso-energetic diets based on a common purified basal diet (vitamin-free casein as the protein source) were prepared for this study. The six dietary groups were 0 g avidin with 0 mg biotin (A0B0), 0 g avidin with 0.06 mg biotin/kg diet (A0B1), four avidin-supplemented diets incorporating at a incremental concentrations 0.25, 0.5, 1.0 and 2.0 g/kg diet with 0.06 mg biotin/kg diet (A15B1, A30B1, A60B1 and A120B1). Fish were hand-fed three times a day to apparent satiation for 12 weeks. Each diet was fed to three replicate groups of fish. Fish were kept in glass aquaria in a recirculating aquaculture system under standardized environmental conditions. Growth was significantly higher in fish that received the biotin-supplemented diet (A0B1), compared to diets lacking biotin or supplemented with avidin. Tilapia fed higher concentration of avidin-supplemented diets (A60B1 and A120B1) showed significant growth depression and displayed severe deficiency syndromes such as lethargy, anorexia, circular swimming and convulsions, which ultimately lead to death. There was a strong proportional linear relationship between the avidin content of the diet and feed conversion ratio, FCR (y = 0.43x + 0.135; r = 0.960; P protein efficiency ratio, PER (y = -0.309x + 2.195; r = 0.961; P levels of biotinidase, pyruvate carboxylase, propionyl-CoA carboxylase-A and propionyl-CoA carboxylase-B transcripts were noted in fish fed all graded level of avidin-supplemented diets. A broken-line analysis indicated that feeding tilapia a diet with 44.5 times more avidin than the dietary biotin requirement can induce deficiency syndromes including retarded growth, when

  1. SPECT/NIRF Dual Modality Imaging for Detection of Intraperitoneal Colon Tumor with an Avidin/Biotin Pretargeting System.

    Science.gov (United States)

    Dong, Chengyan; Yang, Sujuan; Shi, Jiyun; Zhao, Huiyun; Zhong, Lijun; Liu, Zhaofei; Jia, Bing; Wang, Fan

    2016-01-06

    We describe herein dual-modality imaging of intraperitoneal colon tumor using an avidin/biotin pretargeting system. A novel dual-modality probe, (99m)Tc-HYNIC-lys(Cy5.5)-PEG4-biotin, was designed, synthesized and characterized. Single-photon emission computed tomography/ computed tomography (SPECT/CT) imaging and near infrared fluorescence (NIRF) imaging were developed using intraperitoneal LS180 human colon adenocarcinoma xenografts. Following avidin preinjection for 4 hours, (99m)Tc-HYNIC-lys(Cy5.5)-PEG4-biotin could successfully detect colon tumors of different sizes inside the abdominal region using both modalities, and the imaging results showed no differences. Biodistribution studies demonstrated that the tumors had a very high uptake of the probe (99m)Tc-HYNIC-lys(Cy5.5)-PEG4-biotin (12.74 ± 1.89% ID/g at 2 h p.i.), and the clearance from blood and other normal tissues occured very fast. The low tumor uptake in the non-pretargeted mice (1.63 ± 0.50% ID/g at 2 h p.i.) and tumor cell staining results showed excellent tumor binding specificity of the pretargeting system. The ability of the novel probe to show excellent imaging quality with high tumor-to-background contrast, a high degree of binding specificity with tumors and excellent in vivo biodistribution pharmacokinetics should prove that the avidin/biotin based dual-modality pretargeting probe is a promising imaging tool during the entire period of tumor diagnosis and treatment.

  2. A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris.

    Science.gov (United States)

    Jungo, Carmen; Schenk, Jonas; Pasquier, Muriel; Marison, Ian William; von Stockar, Urs

    2007-08-01

    The advantages of mixed feeds of sorbitol and methanol for the production of recombinant proteins with Pichia pastoris were analyzed quantitatively. The influence of the methanol-sorbitol ratio in the feed medium was investigated on growth stoichiometry and recombinant protein productivity with a P. pastoris Mut(+) strain secreting avidin by performing a transient nutrient gradient in continuous cultivation at a dilution rate of 0.03h(-1). Results showed that mixed feeds of sorbitol and methanol instead of methanol as sole carbon source can improve the productivity in recombinant avidin due to increased biomass yields during mixed substrate growth. The highest volumetric avidin productivity was achieved at a methanol fraction of 43%C-molC-mol(-1) in the feed medium: the volumetric avidin productivity was 1.3-fold higher than during chemostat culture on methanol. The heat production and the oxygen consumption rates were reduced by about 38% for a given dry cell weight concentration at this methanol fraction, features which are very useful for high cell density cultures. Results were in good agreement with a high cell density fed-batch culture performed with a mixed feed of 43% methanol and 57% sorbitol C-molC-mol(-1) at a specific growth rate of 0.03h(-1) during the induction phase. Moreover, it was confirmed that sorbitol accumulation in the culture medium does not affect recombinant avidin productivity, which can especially be advantageous during large-scale cultures where transient substrate accumulation can result from imperfect mixing.

  3. Intraoperative avidination for radionuclide treatment as a radiotherapy boost in breast cancer: results of a phase II study with {sup 90}Y-labeled biotin

    Energy Technology Data Exchange (ETDEWEB)

    Paganelli, Giovanni; De Cicco, Concetta; Carbone, Giuseppe; Pacifici, Monica [European Institute of Oncology, Division of Nuclear Medicine, Milan (Italy); Ferrari, Mahila E.; Cremonesi, Marta; Di Dia, Amalia [European Institute of Oncology, Division of Medical Physics, Milan (Italy); Pagani, Gianmatteo; Galimberti, Viviana; Luini, Alberto [European Institute of Oncology, Division of Senology, Milan (Italy); Leonardi, Maria Cristina; Ferrari, Annamaria; Orecchia, Roberto [European Institute of Oncology, Division of Radiotherapy, Milan (Italy); De Santis, Rita [Sigma-Tau SpA R and D, Rome (Italy); Zurrida, Stefano [European Institute of Oncology, Division of Senology, Milan (Italy); University of Milan School of Medicine, Milan (Italy); Veronesi, Umberto [European Institute of Oncology, Scientific Director, Milan (Italy)

    2010-02-15

    External beam radiotherapy (EBRT) after conservative surgery for early breast cancer requires 5-7 weeks. For elderly patients and those distant from an RT center, attending for EBRT may be difficult or impossible. We investigated local toxicity, cosmetic outcomes, and quality of life in a new breast irradiation technique - intraoperative avidination for radionuclide therapy (IART) - in which avidin is administered to the tumor bed and {sup 90}Y-labelled biotin later administered intravenously to bind the avidin and provide irradiation. Reduced duration EBRT (40 Gy) is given subsequently. After surgery, 50 (ten patients), 100 (15 patients) or 150 mg (ten patients) of avidin was injected into the tumor bed. After 12-24 h, 3.7 GBq {sup 90}Y-biotin (beta source for therapeutic effect) plus 185 MBq {sup 111}In-biotin (gamma source for imaging and dosimetry) was infused slowly. Whole-body scintigraphy and SPECT/CT images were taken for up to 30 h. Shortened EBRT started 4 weeks later. Local toxicity was assessed by RTOG scale; quality of life was assessed by EORTC QOL-30. Of 35 patients recruited (mean age 63 years; range 42-74) 32 received IART plus EBRT. 100 mg avidin provided 19.5 {+-} 4.0 Gy to the tumor bed and was considered the optimum dose. No side-effects of avidin or {sup 90}Y-biotin occurred, with no hematological or local toxicity. Local G3 toxicity occurred in 3/32 patients during EBRT. IART plus EBRT was well accepted, with good cosmetic outcomes and maintained quality of life. IART plus reduced EBRT can accelerate irradiation after conservative breast surgery. (orig.)

  4. Avidin-biotin-based approach to forming heterotypic cell clusters and cell sheets on a gas-permeable membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hamon, M; Ozawa, T; Montagne, K; Kojima, N; Ishii, R; Sakai, Y [Institute of Industrial Science (IIS), University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 (Japan); Yamaguchi, S; Nagamune, T [Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ushida, T, E-mail: mzh0026@auburn.edu [Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2011-09-15

    Implantation of sheet-like liver tissues is a promising method in hepatocyte-based therapies, because angiogenesis is expected to occur upon implantation from the surrounding tissues. In this context, we introduce here a new methodology for the formation of a functional thick hepatic tissue usable for cell sheet technology. First, we report the formation of composite tissue elements in suspension culture. Composite elements were composed of human hepatoma Hep G2 cells and mouse NIH/3T3 fibroblasts which are important modulators for thick-tissue formation. To overcome the very low attachment and organization capability between different cells in suspension, we synthesized a new cell-to-cell binding molecule based on the avidin-biotin binding system that we previously applied to attach hepatocytes on artificial substrata. This newly synthesized biotin-conjugated biocompatible anchoring molecule was inserted in the plasma membrane of both cell types. NIH/3T3 cells were further conjugated with avidin and incubated with biotin-presenting Hep G2 cells to form highly composite tissue elements. Then, we seeded those elements on highly gas-permeable membranes at their closest packing density to induce the formation of a thick, composite, functional hepatic tissue without any perfusion. This methodology could open a new way to engineer implantable thick liver tissue sheets where different cell types are spatially organized and well supplied with oxygen.

  5. In vitro evaluation of avidin antibody pretargeting using 211At-labeled and biotinylated poly-L-lysine as effector molecule

    DEFF Research Database (Denmark)

    Frost, Sofia H L; Jensen, Holger Lau; Lindegren, Sture

    2010-01-01

    Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled po...

  6. Maskless localized patterning of biomolecules on carbon nanotube microarray functionalized by ultrafine atmospheric pressure plasma jet using biotin-avidin system

    Science.gov (United States)

    Abuzairi, Tomy; Okada, Mitsuru; Purnamaningsih, Retno Wigajatri; Poespawati, Nji Raden; Iwata, Futoshi; Nagatsu, Masaaki

    2016-07-01

    Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.

  7. Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

    Directory of Open Access Journals (Sweden)

    Buda A

    2015-01-01

    Full Text Available Andrea Buda,1,* Sonia Facchin,1,* Elisa Dassie,2 Elisabetta Casarin,3 Mark A Jepson,4 Helmut Neumann,5 Giorgia Hatem,1 Stefano Realdon,6 Renata D’Incà,1 Giacomo Carlo Sturniolo,1 Margherita Morpurgo3 1Department of Surgical, Oncological, and Gastroenterological Sciences, University of Padova, 2Department of Molecular Medicine, University of Padova, Padova, Italy; 3Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; 4School of Biochemistry and Wolfson Bioimaging Facility, University of Bristol, Bristol, UK; 5Ludwig Demlig Endoscopic Center of Excellence, ESGE Endoscopy Training Center, University of Erlangen-Nuremberg, Erlangen, Germany; 6Veneto Institute of Oncology IOV-IRCCS, Padova, Italy *These authors contributed equally to this work Abstract: Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent-labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of

  8. Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

    Directory of Open Access Journals (Sweden)

    Ting-Yu Angela Liao

    Full Text Available Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

  9. Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

    Science.gov (United States)

    Dou, Shuping; Virostko, John; Greiner, Dale L; Powers, Alvin C; Liu, Guozheng

    2015-08-03

    Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry. In an ongoing pretargeting project, we designed a trifunctional antibody (Ab) that concomitantly carried a biotin and a DNA analogue (hereafter termed MORF). The biotin and the MORF were fused into one structure prior to conjugation to the Ab for the concomitant attachment. Because it was known that avidin-bound Ab molecules leave the circulation rapidly, this design would theoretically allow complete clearance by avidin. The clearability of the trifunctional Ab was determined by calculating the blood MORF concentration ratio of avidin-treated Ab to non-avidin-treated Ab using mice injected with these compounds. In theory, any compromised clearability should be due to the presence of impurities. In vitro, we measured the biotinylated percentage of the Ab-reacting (MORF-biotin)⊃-NH2 modifier, by addition of streptavidin to the radiolabeled (MORF-biotin)⊃-NH2 samples and subsequent high-performance liquid chromatography (HPLC) analysis. On the basis of our previous quantitative understanding, we predicted that the clearability of the Ab would be equal to the biotinylation percentage measured via HPLC. We validated this prediction within a 3% difference. In addition to the high avidin-induced clearability of the trifunctional Ab (up to ∼95%) achieved by the design, we were able to predict the required quality of the (MORF-biotin)⊃-NH2 modifier for any given in vivo clearability. This approach may greatly reduce the steps and time currently required in pharmaceutical development in the process of synthesis, chemical analysis, in

  10. 3D dosimetry in patients with early breast cancer undergoing Intraoperative Avidination for Radionuclide Therapy (IART {sup registered}) combined with external beam radiation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Ferrari, Mahila E.; Cremonesi, Marta; Di Dia, Amalia; Botta, Francesca; Pedroli, Guido [European Institute of Oncology, Division of Medical Physics, Milan (Italy); De Cicco, Concetta; Calabrese, Michele; Paganelli, Giovanni [European Institute of Oncology, Division of Nuclear Medicine, Milan (Italy); Sarnelli, Anna [IRCCS Istituto Romagnolo per lo Studio e la Cura dei Tumori, Medical Physics Unit, Meldola, FC (Italy); Pedicini, Piernicola [Centro Regionale Oncologico Basilicata (IRCCS-CROB), Department of Radiation Oncology, Rionero in Vulture, PZ (Italy); Orecchia, Roberto [European Institute of Oncology, Division of Radiotherapy, Milan (Italy)

    2012-11-15

    Intraoperative Avidination for Radionuclide Therapy (IART {sup registered}) is a novel targeted radionuclide therapy recently used in patients with early breast cancer. It is a radionuclide approach with {sup 90}Y-biotin combined with external beam radiotherapy (EBRT) to release a boost of radiation in the tumour bed. Two previous clinical trials using dosimetry based on the calculation of mean absorbed dose values with the hypothesis of uniform activity distribution (MIRD 16 method) assessed the feasibility and safety of IART {sup registered}. In the present retrospective study, a voxel dosimetry analysis was performed to investigate heterogeneity in distribution of the absorbed dose. The aim of this work was to compare dosimetric and radiobiological evaluations derived from average absorbed dose vs. voxel absorbed dose approaches. We evaluated 14 patients who were injected with avidin into the tumour bed after conservative surgery and 1 day later received an intravenous injection of 3.7 GBq of {sup 90}Y-biotin (together with 185 MBq {sup 111}In-biotin for imaging). Sequential images were used to estimate the absorbed dose in the target region according to the standard dosimetry method (SDM) and the voxel dosimetry method (VDM). The biologically effective dose (BED) distribution was also evaluated. Dose/volume and BED volume histograms were generated to derive equivalent uniform BED (EUBED) and equivalent uniform dose (EUD) values. No ''cold spots'' were highlighted by voxel dosimetry. The median absorbed-dose in the target region was 20 Gy (range 15-27 Gy) by SDM, and the median EUD was 20.4 Gy (range 16.5-29.4 Gy) by the VDM; SDM and VDM estimates differed by about 6 %. The EUD/mean voxel absorbed dose ratio was >0.9 in all patients, indicative of acceptable uniformity in the target. The median BED and EUBED values were 21.8 Gy (range 15.9-29.3 Gy) and 22.8 Gy (range 17.3-31.8 Gy), respectively. VDM highlighted the absence of significant

  11. Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

    Science.gov (United States)

    Hynninen, Ville; Vuori, Leena; Hannula, Markku; Tapio, Kosti; Lahtonen, Kimmo; Isoniemi, Tommi; Lehtonen, Elina; Hirsimäki, Mika; Toppari, J. Jussi; Valden, Mika; Hytönen, Vesa P.

    2016-01-01

    A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications. PMID:27381834

  12. Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

    Science.gov (United States)

    Hynninen, Ville; Vuori, Leena; Hannula, Markku; Tapio, Kosti; Lahtonen, Kimmo; Isoniemi, Tommi; Lehtonen, Elina; Hirsimäki, Mika; Toppari, J. Jussi; Valden, Mika; Hytönen, Vesa P.

    2016-07-01

    A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications.

  13. A comparative evaluation of avidin-biotin ELISA and micro SNT for detection of antibodies to infectious bovine rhinotracheitis in cattle population of Odisha, India

    Directory of Open Access Journals (Sweden)

    Priyaranjan Das

    2014-08-01

    Full Text Available Aim: The present study was undertaken to serologically detect Infectious Bovine Rhinotracheitis (IBR in the cattle population of Odisha, India using micro-Serum neutralization test (micro SNT and Avidin-Biotin Enzyme linked immuno sorbent assay (AB ELISA and finding out their comparative efficacy to serve as a suitable diagnostic tool in field condition. Materials and Methods: The study was carried out using serum samples (n=180 collected randomly from cattle populations of nine districts of Odisha. Similarly vaginal swabs (n=26 from cattle having history of repeat breeding, abortion, vulvo-vaginitis and nasal swabs (n=8 from calves with respiratory symptoms and nasal discharge were collected aseptically, to ascertain the circulation of virus among the cattle population. Results: Virus isolation by cell culture and subsequent confirmation by polymerase chain reaction confirmed four isolates. Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively. The sensitivity and specificity of AB ELISA test was found to be 88.23% and 95.70% respectively taking micro SNT as gold standard and the kappa value between the two tests was 0.75. Conclusion: Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT and AB ELISA respectively, thus highlighting the circulation of virus among the livestock population of Odisha and that AB ELISA could be more efficiently applied for the sero-diagnosis of IBR virus infections at field conditions, with demand for more study on faster, efficient and large scale screening of the infected animals.

  14. Effect of biotin and pantothenic acid on performance and concentrations of avidin-binding substances in blood and milk of lactating dairy cows.

    Science.gov (United States)

    Ferreira, Gonzalo; Brown, Alston N; Teets, Christy L

    2015-09-01

    We hypothesized that pantothenic acid reduces the absorption of biotin in lactating dairy cows. Therefore, the objective of this study was to evaluate the plausible interaction between biotin and pantothenic acid on production performance and concentration of avidin-binding substances (ABS), an indicator of biotin concentration, in blood and milk of lactating dairy cows. Eight primiparous and 16 multiparous Holstein cows were assigned to 1 of 4 diet sequences in a replicated 4×4 Latin square design with 18-d periods. Cows were housed in a freestall barn and fed once daily (0730 h) by means of a Calan gate system (American Calan Inc., Northwood, NH). Treatments consisted of a control diet that contained no B-vitamins, a biotin diet that contained 0.87 mg of biotin per kilogram of dry matter (DM), a pantothenic acid diet that contained 21 mg of pantothenic acid per kilogram of DM, and a biotin plus pantothenic acid diet that contained 0.87 mg of biotin and 21 mg of calcium pantothenic acid per kilogram of DM. Four different concentrates were prepared in a commercial feed mill. These concentrates were mixed with corn silage and grass hay and delivered ad libitum as a total mixed ration. Biotin supplementation did not affect DM intake, milk yield, or milk fat, protein, lactose, and milk-urea-nitrogen concentrations. Fat, protein, and lactose yields were not affected by treatments. The fat-to-protein ratio was Biotin supplementation did not increase the concentration of ABS in plasma. The supplementation of pantothenic acid did not affect the concentration of ABS in plasma when either supplemented alone or in combination with biotin. Biotin supplementation increased the concentration of ABS in milk relative to control. Contrary to our hypothesis, the supplementation of pantothenic acid did not decrease the concentration of ABS in milk relative to the control. When cows were supplemented with both biotin and pantothenic acid, the concentration of ABS in milk was similar

  15. Ultrastructural localization of intracellular immunoglobulins in Epon-embedded human lymph nodes. An immunoelectron microscopic investigation using the immunogold staining (IGS) and the avidin-biotin-peroxidase complex (ABC) methods.

    Science.gov (United States)

    Viale, G; Dell'Orto, P; Braidotti, P; Coggi, G

    1985-05-01

    The ultrastructural localization of intracellular immunoglobulins on ultrathin sections of glutaraldehyde-fixed, postosmicated, and Epon-embedded human lymph nodes has been achieved using such highly sensitive immunocytochemical techniques as immunogold staining and avidin-biotin-peroxidase complex. These immunoelectron microscopic techniques allow the identification of intracellular immunoglobulins without affecting the ultrastructural morphology of the tissue, since they do not require any pretreatment of the sections with proteolytic enzymes or deresinating agents. Therefore, immunoglobulins can be precisely localized in the cell organelles; structures whose morphology is well preserved. The availability of a reliable postembedding staining procedure for the ultrastructural localization of immunoglobulins is of definite value for investigations on human lymphoid tissue, both normal and pathological.

  16. 生物素-亲和素技术锚定肝素于胰岛表面*☆%Heparin anchored to the surface of islet by avidin-biotin technique

    Institute of Scientific and Technical Information of China (English)

    田晓辉; 李杨; 丁小明; 宋焕瑾; 冯新顺; 薛武军

    2013-01-01

    BACKGROUND:Islet capil aries are damaged in the process of islet isolation, thereby affecting the nutrient supply of islets after transplantation. Heparin has a very important significance for the regeneration of blood vessels;meanwhile, heparin is commonly used in the clinical islet transplantation to inhibit thrombosis. But systemic heparin can increase the risk of bleeding. The avidin has two strong binding sites of biotin and heparin respectively. OBJECTIVE:To improve islet revascularization and decrease risk of bleeding resulting from heparin systemic application through anchoring the heparin on the surface of islet with avidin-biotin technique based on the characteristics of avidin. METHODS:Adult human pancreas were isolated and purified with Ricordi automation method, then the islets were incubated and cultured with 0, 0.5, 1, 1.5, 2 g/L biotin (including biotin-N-hydroxysuccinimide ester, N-hydroxy-succinimido-6-biotinyl amido hexanoate, biocytin hydrazine, biotin hydrazide and TFP-biotin), 1 g/L avidin, and 0.5, 1.0, 1.5 and 2.0 g/L heparin, the change of heparin was observed. RESULTS AND CONCLUSION:TFP-biotin had the best effect to mediate the islet surface heparinization, and there was no significant difference in the activity of islet before and after heparinization (P>0.05);the heparinized and unheparinized heparin islets had the similar insulin release reaction (P>0.05). Biotin-avidin technique is a safe and effective islet surface heparinization treatment method.%  背景:胰岛微血管在胰岛分离过程中被破坏,进而影响移植后胰岛的营养供应。肝素对于血管的再生具有非常重要的意义;同时,临床胰岛移植多应用肝素来抑制血栓形成,但全身肝素化增加了出血的风险。而亲和素同时具备生物素和肝素2个较强的结合位点。  目的:利用亲和素这一特性,应用生物素-亲和素技术,将肝素锚定于胰岛的表面,促进胰

  17. 单抗CD44-生物素-亲和素系统提高软骨细胞与支架黏附能力的研究%CD44 monoclonal antibody-biotin-avidin binding system for the improvement of cell adhesion to scaffolds

    Institute of Scientific and Technical Information of China (English)

    林红; 周健; 沈龙祥; 阮玉辉; 戴文达; 郭常安; 陈峥嵘

    2012-01-01

    目的 观察单抗CD44-生物素(Biotin) -亲和素(Avidin)绑定系统在构建组织工程软骨中能否提高软骨细胞与支架的黏附能力.方法 分别制备软骨细胞二维和三维培养体系,分3组:A:壳聚糖+软骨细胞;B:生物素+亲和素化壳聚糖+软骨细胞;C:生物素化单抗CD44+亲和素化壳聚糖+软骨细胞.分别测定细胞展平面积、细胞接种脱落率、细胞增殖率,逆转录-聚合酶链反应(RT-PCR)法检测Ⅱ型胶原(ColⅡ)、聚集蛋白聚糖(aggrecan )、sox9的mRNA表达,组织学切片观察壳聚糖支架内细胞黏附生长情况.结果 细胞展平面积、细胞增殖率及ColⅡ、aggrecan、sox9的mRNA表达量皆为:C组>B组>A组(P<0.05).细胞接种脱落率:A组为C组的3.71倍、为B组的2.17倍,而B组为C组的1.71倍,A组>B组>C组(P<0.05).组织切片显示细胞在支架内C组增殖旺盛、胞外基质表达较丰富,A组增殖较弱、胞外基质表达较弱,B组介于C与A组间.结论 单抗CD44-Biotin-Avidin绑定系统能比Biotin-Avidin绑定系统更显著的提高软骨细胞与支架的黏附能力,促进组织工程软骨种子细胞的增殖和软骨细胞表型的表达.%Objective To oberserve the efficiency of CD44 monoclonal antibody-biotin-avidin binding system for the improvement of cells adhesion to scaffolds in the cartilage tissue engineering.Methods The chondrocytes were cultured in two-dimensional and three-dimensional culture system respectively.And in each system the cells were divided into 3 groups:Group A,the chondrocytes were seeded routinely on the chitosan membrane or scaffolds ; Group B,the chondrocytes were seeded on the chitosan membrane or scaffolds with biotin-avidin binding system; Group C,the chondrocytes were seeded on the chitosan membrane or scaffolds with CD44 monoclonal antibody-biotin-avidin binding system.The spreading area in the two-dimensional culture system,cell exfoliation rate and cell proliferation rate in the

  18. 奥曲肽-葡聚糖-亲和素的偶联及其与177Lu-DTPA-BIS-BIOTIN的体外结合%177Lu-DTPA-BIS-BIOTIN Binding of Octreotide-dextran-avidinated PANC-1 Cell Lines in Vitro

    Institute of Scientific and Technical Information of China (English)

    邓新荣; 杜进; 翟士桢; 沈亦佳; 罗志福

    2011-01-01

    以葡聚糖为载体,奥曲肽为导向分子合成了生长抑素配体化合物奥曲肽-葡聚糖-亲和素(Tyr3-oct-reotide-dxtran 40-avidin,TOC-Dx40-Av);在体外模拟肿瘤预定位二步法以177Lu-DTPA-BIS-BIOTIN对TOC-Dx40-Av培养的PANC-1细胞的结合特性进行了研究.将长满人源胰腺癌细胞PANC-1的24孔板置于含有TOC-Dx40-Av的缓冲液中培养,2h后洗去上清液,再用含不同浓度177Lu- DTPA-BIS-BIOTIN(177Lu-DT-PA-BIS-BIOTIN的摩尔质量范围为48.8~391 pmol)的缓冲液继续培养细胞,使177Lu-DTPA-BIS-BIOTIN 与细胞上的亲和素结合,测定细胞上结合的放射性计数,考察TOC-Dx40-Av与177 Lu-DTPA-BIS-BIOTIN的结合特性以评价合成的大分子亲和素连接物的活性.实验结果显示,奥曲肽-葡聚糖-亲和素的化学纯度>99%,其中亲和素的含量为6.46 g/L.体外细胞结合实验结果表明,177Lu-DTPA-BIS-BIOTIN能快速与细胞上连接的亲和素结合,生物素与亲和素的摩尔比约为1∶1达到平衡.%Tyr3-octreotide, dextran-40 and avidin were used to prepare octreotide-dextran-avidin (TOC-Dx40-Av). DTPA-BIS-BIOTIN was labelled with 177Lu. The in vitro soma-tostatin receptor binding study was carried out by pretargeted method using TOC-Dx40-Av and 177Lu-DTPA-BIS-BIOTIN. The 24 well cell culture plates were prepared with PANC-1 cell monolayer and then incubated with TOC-Dx40-Av. After two washed with PBS, the cells were incubated with different concentration of 177 Lu-DTPA-BIS-BIOTIN (48. 8~ 391 pmol). Cells uptake was evaluated with y counter. The results showed that the chemi- cal purity of TOC-Dx40-Av was over 99%. The results also showed that TOC-Dx40-Av remained high receptor binding affinity to somatostatin receptor which indicated that TOC-Dx40-Av could bind to 177 Lu-DTPA-BIS-BIOTIN with the molar ratio of 1 ? 1 on the cell surface.

  19. A biotin-avidin enzyme-linked immunosorbent assay for studying platelet membrane glycoprotein Ⅱb/Ⅲa, CD62p expression and its clinical application%BA-ELISA定量检测人血小板膜糖蛋白Ⅱb/Ⅲa、CD62p的表达及其临床应用

    Institute of Scientific and Technical Information of China (English)

    张有涛; 赵益明; 朱明清; 蒋敏; 程寅峰; 史进方; 顾国浩; 阮长耿

    2012-01-01

    Objective To develop a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) method for detecting platelet membrane glycoprotein function, and evaluate its clinical application. Methods With the monoclonal antibodies (mAb) recognizing GPⅡb/Ⅲa (7E3) , CD62p (SZ51) and biotin-avidin system, to develop a biotin-avidin-ELISA ( BA-ELISA). The levels of GP Ⅱ b/ Ⅲ a and CD62p were measured in patients with acute myocardial infarction ( AMI) , acute cerebral infarction (ACI) , diabetes mellitus ( DM) or in the healthy people. This BA-ELISA was compared with flow cytometry ( FCM). Inhibition of membrane glycoprotein expression was evaluated with inhibitory mAb, SZ21 and aspirin (acetylsalicylic acid,ASA) respectively. Results The BA-ELISA detected platelet count were as low as 3. 13 × 109/L or 6. 25 × 109/L in the platelet-rich plasma (PRP) of the specimens. Both of the interassay and intraassay coefficient variation ( CV) were less than 10%. Adenosine diphosphate ( ADP) -induced, or non-ADP-induced GP Ⅱ b/ Ⅲ a and CD62p expression in AMI, ACI, DM were significantly higher than those in the controls ( all P < 0.01). Either SZ21 or aspirin inhibited the expression of GP H b/ M a and CD62p induced by ADP. A high correlation was showed between BA-ELISA and FCM methods. Conclusion These observations indicate that BA-ELISA is a sensitive and high-throughput assay for evaluating platelet membrane glycoprotein expression and activation extent. This method is suitable to screen inhibitors/activators of platelet activation and has a potential in use for diagnostic purposes.%目的 建立测定血小板膜糖蛋白功能的生物素-亲和素-酶联免疫吸附(BA-ELISA)方法,并探讨其临床应用价值.方法 采用抗血小板膜糖蛋白Ⅱb/Ⅲa( GPⅡb/Ⅲa)单抗7E3和抗血小板CD62p单抗SZ51及生物素-亲和素系统建立BA-ELISA方法,并应用该方法检测50名健康志愿者、30例急性心肌梗死(AMI)、30例急性脑梗死(ACI)和30例

  20. Immunoradiometric assay for carcinoembryonic antigenusing avidin-biotin separation technique

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    A sensitive, specific, noncompetitive, sandwich-typeradioimmunoassay for carcinoembryonic antigen (CEA) has been developedin our laboratory, which can be performedconveniently. The assay involves two monoclonal antibodies, selected for highaffinity and specificity and also for reaction against antigenic sites on CEA that aredistal from each other. One of these antibodies was labeled with125I and the other wasconjugated covalently to biotin. Polystyrene tubes were conjugated covalently toavidin. These tubes represent a rapid, simple method for separating the CEA-boundantibody from the free antibody. The biotin-antibody-CEA-125I-labeled antibodycomplexes bind to the tubes and CEA concentration is directly related to counts perminute. This assay can detect the CEA at a concentration of 0.22 μg/L in serum.

  1. 生物素-亲和素介导以KDR为靶点的脂质体超声造影剂体外靶向实验研究%Preparation of biotin-avidin mediated KDR-targeted liposome ultrasound contrast agent and targeted experiment in vitro

    Institute of Scientific and Technical Information of China (English)

    李颖嘉; 何洁; 孙学刚; 杨莉; 宾建平; 文戈

    2010-01-01

    Objective To prepare a new kind of targeted liposome ultrasound contrast agent with small peptide K237 as the ligand which can combine specifically with KDR which is the main receptor of VEGF.and to test its capability in vitro. Methods Targeted bubbles(P-Bio-Av-Bio-Mbs) were formed through "biotin-avidin" bridge grafting, then they were incubated respectively with LOVO, HUVECs and LS174T which were KDR positive or negative expressed in various cells,meanwhile incubated LOVO cells with FITC- P-Bio-Av-Bio-Mbs,FITC-P-Mbs and FITC-Mbs respectively. After that, the rosette formation rate and fluorescence intensity of the combination between microbubbles and cells were observed with microscope and fluorescence microscope. After being incubated with small peptide K237 of 10 μg and 50 μg, LOVO cells were incubated with P-Bio-Av-Bio-Mbs for observing the distribution of microbubbles. Results In KDR sharply positive expressed LOVO cells, the surrounding rosette formation rate was as high as 90. 52% with the fluorescence intensity of grade 3, and it was 53. 46% with grade 2 fluorescence intensity rate in KDR positive expressed HUVECs cells, while in KDR negative expressed LS174T cells, there were few microbubbles surrounded with rosette formation rate of 5. 57% and fluorescence intensity rate of grade 0-1, therefore there were significant statistic differences in rosette formation rate among groups ( P < 0.05). After LOVO cells combined with FITC-P-Bio-Av-Bio-Mbs, FITC-P-Mbs and FITC-Mbs respectively,there were significant differences in their rosette formation rate, namely 89.62%, 7. 56% , 0 with the fluorescence rate of 3,0 - 1 and 0 respectively. Targeted cells pretreated with 10 pg K237 showed significant decreased rosette formation,and there was no formation in 50 ?g pretreated group. Conclusions KDR-Targeted liposome contrast agent with small peptide K237 liganded has been successfully prepared through biotin-avidin mediation and could combine specifically and high

  2. 基于亲和素-生物素双夹心体系测定外源性蛋白血清动态浓度的非抗体依赖新方法%A Novel Method for Assessment of Dynamic Concentration of Exogenous Protein in Serum: an Antibody-independent Sandwich System Based on Avidin-biotin Interaction

    Institute of Scientific and Technical Information of China (English)

    余颖欣; 刘艳君; 焦德龙; 文李艳; 徐江平; 富宁

    2013-01-01

    For the assessment of the dynamic concentration of exogenous protein or peptide in serum, an antibody- independent sandwich system based on the avidin-biotin interaction was established and validated. The exogenous protein or polypeptide labeled with biotin was added to the microplate coated with streptavidin, and followed by adding HRP-streptavidin to complete the sandwich system. This sandwich system was validated with respect to specificity, sensitivity, accuracy (recovery) and reproducibility. While the sensitivity of detection could reach to 0.3125 μg/L, the sensitivity and the range of detection can also be adjusted by changing coating concentration of streptavidin. Recoveries ranged from 97.82% to 107.29%, and the intra- and inter-assay variation was < 5.76% and < 8.42%, respectively. Thus, the well-validated streptavidin sandwich system was successfully applied to determine dynamic concentrations of Biotin-HAS (human serum albumin) and Biotin-OVM (Ovomucoid) in mouse serum, respectively. Most importantly, this novel method, where generation of antibody or radionuclide is not needed, may provide a new choice for assessment or monitoring of exogenous protein or peptide in pharmacokinetics study.%建立一种非抗体依赖的检测外源性蛋白多肽分子代谢动力学血清浓度及动态变化的新方法.链亲和素为捕获分子,加入待测生物素标记蛋白或合成肽,辣根过氧化物酶标记链亲和素为检测分子,构成链亲和素-生物素标记大分子-酶链亲和素的双夹心体系,并进行特异性、敏感性、准确性及稳定性评价.本检测体系灵敏度高,可达0.3125 μg/L,且可通过改变亲和素包被浓度调整检出敏感度与检测范围.准确性回收率为97.82%~107.92%,批内、批间变异系数分别<5.76%和<8.42%,并成功应用在生物素标记人血清白蛋白(biotin-HSA)与生物素标记鸡卵黏蛋白(biotin-OVM)的小鼠血清浓度动态检测.本方法不依赖抗体

  3. Establishment and application of biotin-avidin-system-time-resolved fluoroimmunoassay for the quantitive detection of antinuclear antibody-Ig%生物素-亲合素系统-时间分辨荧光免疫分析法检测抗核抗体方法学的建立和临床应用

    Institute of Scientific and Technical Information of China (English)

    胡志刚; 刘洁; 陈国千; 邹耀红; 俞蕾

    2012-01-01

    Objective To establish an analytical method with high sensitivity and wide range in biotin-avidin and time-resolved fluoroimmunoassay system for the quantitative detection of antinuclear antibody (ANA)-Ig (GAM).Methods ANA in standard preparation or sample was combined with solid nuclear antigen,biotinylation antibody and the europium(Eu3+)-labelled avidin to form the compounds of solid nuclear antigen-antibody-biotinylation antibody-SA-Eu3+.The fluorescence enhancement fluid was added to dis-sociate the Eu3+,the content of ANA was directly proportional to fluorescence intensity,the BAS-TRFLA was established for the quantitative detection of ANA-Ig (GAM).The sensitivity,specificity,reliability and range of detection were evaluated.Semm of 50 blood donor,105 patients with systemic lupus erythematosus (SLE),109 patients with rheumatoid arthritis (RA),25 patients with PBC,29 patients with Sj(o)gren's syndrome (SS),23 patients with scleroderma,25 patients with MCTD were included in this study.The positive rate was calculated.Results The inner-group difference between high,medium and low densities mixture serum was 3.13%,3.74% and 5.76% and the inter-group precision rate was 5.31%,6.25% and 7.46% in BAS-TRFLA.The sensitivity of TRFIA was better than that of the ELISA method.The low detection limit was 2.24 U/ml.The mean recovery rate was 100.74%.The results measured by the TRFIA and ELISA methods were closely correlated (R2=0.978).The positive rate of blood donor,and patient with SLE,RA,PBC,SS,scleroderma and MCTD were 0,100%,23%,96%,38%,91%,92% respectively.When compared with ELISA,the detection range of TRFIA was wider,and stability was better.Conclusion BAS-TRFIA is a stable method for detection of ANA with high sensitive and wide range of detection.It is important for the early diagnosis of autoimmune disease and monitoring the treatment efficacy of autoimmune disease.And this method may be widely used in clinical laboratories in the future

  4. AGGREGATION AND FUSION OF PLANT-PROTOPLASTS AFTER SURFACE-LABELING WITH BIOTIN AND AVIDIN

    NARCIS (Netherlands)

    VANKESTEREN, WJP; MOLEMA, E; TEMPELAAR, MJ

    1993-01-01

    In mass electrofusion systems with aggregation of protoplasts by alignment, the yield and composition of fusion products can be predicted by a simple model. Through computer simulation, upper limits were found for the yield of binary and multi fusions. To overcome constraints on binary products, sur

  5. Design and solid phase synthesis of new DOTA conjugated (+)-biotin dimers planned to develop molecular weight-tuned avidin oligomers.

    Science.gov (United States)

    Pratesi, Alessandro; Ginanneschi, Mauro; Melani, Fabrizio; Chinol, Marco; Carollo, Angela; Paganelli, Giovanni; Lumini, Marco; Bartoli, Mattia; Frediani, Marco; Rosi, Luca; Petrucci, Giorgio; Messori, Luigi; Papini, Anna Maria

    2015-04-07

    Chemical modifications of the biotin carrier in pretargeted avidin–biotin radionuclide therapy may be of paramount importance for tuning the amount of the radioactivity delivered to cancer cells by labelled biotins. We report here the synthesis of a collection of new synthetic DOTA-constructs bearing two (+)-biotin molecules (bis-biotins), designed for the creation of multimeric Av units (tetramers) bonded to the antibody. All the syntheses were carried out following the solid phase strategy and growing the molecules on a Rink Amide resin. The biotin heads are connected through spacers containing PEG or non-PEG residues. Molecular modelling calculations suggested that the Av cross-linking ability of the bis-biotins depends mainly on the spacers length, with the best results being expected for arms affording distances in the range of 10–25 Å between the biotin carboxylate atoms, in the fully extended conformation. SEC-HPLC MALLS analysis of the products of our Av/bis-biotin reaction mixtures have confirmed this hypothesis. The bis-biotin 16, where the non-PEG linker ensured a distance of 26.7 Å between the biotin moieties, gave about 50% of Av oligomers while the shorter analogue 18 (19.5 Å) afforded 100% of an Av polymer containing about 21 protein units. Remarkably, the solubility of both the bis-biotins, i.e.16 and 18, in aqueous solutions was good and they showed excellent stability against the action of peptidases.

  6. Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin-biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochemical staining of human testicular tissue.

    Science.gov (United States)

    Davidoff, M S; Schulze, W; Holstein, A F

    1991-01-01

    An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP-combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker.

  7. Potential Use of Proteinase Inhibitors, Avidin, and Other Bio-reagents for Synergizing Bt Performance and Delaying Resistance Development to Bt

    Science.gov (United States)

    After being ingested by target insects, the insecticidal proteins from Bacillus thuringiensis (Bt) need to go through a proteolytic process by insect midgut proteinases to become activated. At the same time, Bt can be hydrolyzed and degraded by midgut proteinases to become non-toxic to target insect...

  8. Laser-guided direct writing: a novel method to deposit biomolecules for biosensors arrays.

    Science.gov (United States)

    Xu, Juntao; Grant, Sheila A; Pastel, Robert L

    2003-01-01

    In this paper, we present a potential biomolecular patterning method, laser-guided direct writing guidance (LGDW), which may be utilized to deposit organic and bioactive particles for biosensor arrays. The instrumentation and operation of the LGDW system is introduced and the system settings used to achieve deposition are reported. The biomolecule, avidin, was deposited onto a substrate using LGDW to evaluate the possible damage from the laser on the biomolecules. The functionality of avidin after laser-based guidance was examined by exposing the deposited avidin molecules to its ligand, biotin. The results show some avidin retained its affinity to biotin after LGDW demonstrating little damage to the biomolecules.

  9. Requirements of Cultured Mammalian Cells for Vitamin B12 and Biotin

    Science.gov (United States)

    power M vitamin B12 for optimal growth in a chemically defined medium. Requirement of HeLa cells for biotin was demonstrated initially with avidin, a...biotin inactivator. The inhibitory activity of avidin on growth of HeLa cells was reversible by addition of biotin. Serial passage of both HeLa and L

  10. Targeted and ultrasound-triggered cancer cell injury using perfluorocarbon emulsion-loaded liposomes endowed with cancer cell-targeting and fusogenic capabilities.

    Science.gov (United States)

    Ninomiya, Kazuaki; Yamashita, Takahiro; Tanabe, Yamato; Imai, Miki; Takahashi, Kenji; Shimizu, Nobuaki

    2016-01-01

    This study investigated the targeting and ultrasound-triggered injury of cancer cells using anticancer drug-free liposomes that contained an emulsion of perfluoropentane (ePFC5) and were co-modified with avidin as a targeting ligand for cancer cells and the hemagglutinating virus of Japan (HVJ) envelope to promote liposome fusion with the cells. These liposomes are designated as ePFC5-loaded avidin/HVJ liposomes. ePFC5-loaded liposomes were sensitized to ultrasound irradiation. Liposomes modified with avidin alone (avidin liposomes) showed binding to MCF-7 human breast cancer cells, and liposomes modified with HVJ envelope alone (HVJ liposomes) were found to fuse with MCF-7 cells. The irradiation of MCF-7 cells with 1 MHz ultrasound (30s, 1.2 W/cm(2), duty ratio 30%) combined with ePFC5-loaded avidin/HVJ liposomes resulted in a decrease in cell viability at 1h after irradiation to 43% of that of controls without ultrasound irradiation or liposomes. The cell viability was lower than that of cells treated with ultrasound irradiation with ePFC5-loaded avidin liposomes or ePFC5-loaded HVJ liposomes. This indicates that co-modification of liposome with avidin and HVJ envelope could enhance ultrasound-induced cell injury in the presence of ePFC5-loaded liposomes.

  11. A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

    Science.gov (United States)

    Martin, Harry; Murray, Colleen; Christeller, John; McGhie, Tony

    2008-10-01

    A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

  12. Experiment list: SRX149198 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available || cell cycle profile=asynchronous http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/eachData/bw/SRX149198.bw ...avidin-coated beads used to pulldown biotinlyated PH) || facs sorting antibody=H3

  13. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

    NARCIS (Netherlands)

    E. de Boer (Ernie); P. Rodriguez (Patrick); E. Bonte (Edgar); J. Krijgsveld (Jeroen); E. Katsantoni (Eleni); A.J.R. Heck (Albert); F.G. Grosveld (Frank); J. Strouboulis (John)

    2003-01-01

    textabstractProteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated temp

  14. Design of a reversible biotin analog and applications in protein labeling, detection, and isolation.

    Science.gov (United States)

    Ying, Lai-Qiang; Branchaud, Bruce P

    2011-08-14

    To expand the applicability of the biotin-(strept)avidin system, a biotin analog with reversible binding under non-denaturing conditions has been designed, and its applications in protein labeling, detection, and isolation have been evaluated.

  15. Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

    Science.gov (United States)

    Meir, Amit; Bayer, Edward A.; Livnah, Oded

    2012-01-01

    Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application. PMID:22493427

  16. Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

    Science.gov (United States)

    Takakura, Yoshimitsu; Oka, Naomi; Tsunashima, Masako

    2014-01-01

    Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity.

  17. Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane

    Directory of Open Access Journals (Sweden)

    Magnone Joshua

    2011-10-01

    Full Text Available Abstract Background The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed. Results A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655. Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R2 = .9973 while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R2 = .999. Conclusion A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.

  18. Acoustically-active microbubbles conjugated to liposomes: characterization of a proposed drug delivery vehicle.

    Science.gov (United States)

    Kheirolomoom, Azadeh; Dayton, Paul A; Lum, Aaron F H; Little, Erika; Paoli, Eric E; Zheng, Hairong; Ferrara, Katherine W

    2007-04-23

    A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes, each containing 5% DSPE-PEG2kBiotin, was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. High-speed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse in a manner similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37 degrees C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.

  19. Observation of adsorption behavior of biomolecules on ferroelectric crystal surfaces with polarization domain patterns

    Science.gov (United States)

    Nakayama, Tomoaki; Isobe, Akiko; Ogino, Toshio

    2016-08-01

    Lithium tantalate (LiTaO3) is one of the ferroelectric crystals that exhibit spontaneous polarization domain patterns on its surface. We observed the polarization-dependent adsorption of avidin molecules, which are positively charged in a buffer solution at pH 7.0, on LiTaO3 surfaces caused by electrostatic interaction at an electrostatic double layer using atomic force microscopy (AFM). Avidin adsorption in the buffer solution was confirmed by scratching the substrate surfaces using the AFM cantilever, and the adsorption patterns were found to depend on the avidin concentration. When KCl was added to the buffer solution to weaken the electrostatic double layer interaction between avidin molecules and LiTaO3 surfaces, adsorption domain patterns disappeared. From the comparison between the adsorption and chemically etched domain patterns, it was found that avidin molecule adsorption is enhanced on negatively polarized domains, indicating that surface polarization should be taken into account in observing biomolecule behaviors on ferroelectric crystals.

  20. Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

    Institute of Scientific and Technical Information of China (English)

    ZHAO,Jin-Fu(赵金富); WANG,Yong-Cheng(王永成); LI,Yuan-Zong(李元宗); CHANG,Wen-Bao(常文保)

    2004-01-01

    Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.

  1. Characterization of the chemical architecture of carbon-fiber microelectrodes. 1. Carboxylates.

    Science.gov (United States)

    Pantano, P; Kuhr, W G

    1991-07-15

    A new method to characterize the chemical architecture of a carbon-fiber microelectrode surface is described. Derivatization of carboxyl groups on the carbon surface with a poly(oxyalkalene)diamine (Jeffamine ED-600), followed by biotinylation of the free amine, allowed the attachment of a fluorescein isothiocyanate (FITC) conjugate of ExtrAvidin. The fluorescence observed after excitation at 488 nm was imaged with a fluorescence microscope equipped with a CCD camera, yielding a spatial map of the distribution of modified carboxyl groups on the surface of the carbon fiber with 0.5-micron resolution. Colloidal gold particles (15 nm diameter) coated with ExtrAvidin were used in place of the FITC-ExtrAvidin, and the carbon-fiber surface was imaged with scanning electron microscopy on a submicron scale. This selective information regarding surface-bound functional groups (i.e. carboxylates) has proven invaluable toward the rational design of novel sensors based on surface-modified ultramicroelectrodes.

  2. Directed capture of enzymes and bacteria on bioplastic films.

    Science.gov (United States)

    Koepsel, Richard R; Russell, Alan J

    2003-01-01

    The development of smart coatings for a variety of uses depends on the ability of the coating material to perform specific functions. We have used water dispersible polyurethane preparations for the immobilization of binding proteins under mild conditions. In these experiments, antibodies against the enzyme beta-galactosidase or the bacterium Escherichia coli were immobilized in polyurethane coatings and then used to effectively capture their cognate antigen. Further, a second, more general, capture protocol was developed which involves the incorporation of the protein avidin in the plastics. This system efficiently captures biotinylated beta-galactosidase. Biotinylated anti-E. coli antibody captured by avidin bioplastics resulted in a nearly 5-fold increase in the number of bound bacteria when compared to blank polyurethane. The use of avidin in a bioplastic allows any biotinylated antibody to be applied to all or part of the surface resulting in a patterning of capture agents on a preformed surface.

  3. Intermolecular forces and energies between ligands and receptors.

    Science.gov (United States)

    Moy, V T; Florin, E L; Gaub, H E

    1994-10-14

    The recognition mechanisms and dissociation pathways of the avidin-biotin complex and of actin monomers in actin filaments were investigated. The unbinding forces of discrete complexes of avidin or streptavidin with biotin analogs are proportional to the enthalpy change of the complex formation but independent of changes in the free energy. This result indicates that the unbinding process is adiabatic and that entropic changes occur after unbinding. On the basis of the measured forces and binding energies, an effective rupture length of 9.5 +/- 1 angstroms was calculated for all biotin-avidin pairs and approximately 1 to 3 angstroms for the actin monomer-monomer interaction. A model for the correlation among binding forces, intermolecular potential, and molecular function is proposed.

  4. Biofunctionalization of polyelectrolyte microcapsules with biotinylated polyethylene glycol-grafted liposomes.

    Science.gov (United States)

    Gao, Jie; Reibetanz, Uta; Venkatraman, Subbu; Neu, Björn

    2011-08-11

    Hollow polyelectrolyte microcapsules (PEMC) are prepared using layer-by-layer self-assembly of polyelectrolytes on melamine formaldehyde templates, followed by template dissolution, and subsequent coating with biotinylated polyethylene glycol-grafted liposomes. These potential site-specific carrier systems show a high specificity for NeutrAvidin binding and a strong resistance against unspecific protein binding. It is concluded that this design with NeutrAvidin as the outermost layer of such capsules provides an ideal platform for the biofunctionalization of PEMC as drug delivery systems or as artificial cell-like structures for biomimetic studies.

  5. Recent development of biotin conjugation in biological imaging, sensing, and target delivery.

    Science.gov (United States)

    Ren, Wen Xiu; Han, Jiyou; Uhm, Soojin; Jang, Yu Jin; Kang, Chulhun; Kim, Jong-Hoon; Kim, Jong Seung

    2015-07-04

    Despite encouraging results from preliminary studies of anticancer therapies, the lack of tumor specificity remains an important issue in the modern pharmaceutical industry. New findings indicate that biotin or biotin-conjugates could be favorably assimilated by tumor cells that over-express biotin-selective transporters. Furthermore, biotin can form stable complexes with avidin and its bacterial counterpart streptavidin. The strong bridging between avidin and biotin moieties on other molecules is a proven adaptable tool with broad biological applications. Under these circumstances, a biotin moiety is certainly an attractive choice for live-cell imaging, biosensing, and target delivery.

  6. Lipid domain formation and ligand-receptor distribution in lipid bilayer membranes investigated by atomic force microscopy

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, O.G.; Jørgensen, K.

    2002-01-01

    A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid-supported l......A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid...

  7. A Simple Bioconjugate Attachment Protocol for Use in Single Molecule Force Spectroscopy Experiments Based on Mixed Self-Assembled Monolayers

    Directory of Open Access Journals (Sweden)

    Myriam Ouberai

    2012-10-01

    Full Text Available Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin.

  8. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, JianQing

    1997-01-01

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin `chase` in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs.

  9. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Reimert, C M; Venge, P; Kharazmi, A;

    1991-01-01

    were subsequently raised in rabbits. The ELISA utilizes the biotin/avidin method and measures ECP within the range 15-1000 ng/l. The intra- and interassay coefficients of variation were 6% and 10%, respectively, and the recoveries of 12 and 25 pg of purified ECP added to diluted serum samples were 108...

  10. Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine

    DEFF Research Database (Denmark)

    Reimert, C M; Minuva, U; Kharazmi, A;

    1991-01-01

    subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation...

  11. SELECTIVE ELECTROFUSION OF CONJUGATED CELLS IN FLOW

    NARCIS (Netherlands)

    SCHUT, TCB; KRAAN, YM; BARLAG, W; DELEIJ, L; DEGROOTH, BG; GREVE, J

    1993-01-01

    Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and Dil, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the h

  12. Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe

    DEFF Research Database (Denmark)

    Harsløf, Mads; Müller, Christoph Felix; Rohrberg, Julie;

    2015-01-01

    BACKGROUND: Biocytin has found numerous uses as a neuronal tracer, since it shows both antero- and retrograde transport in neuronal tracts. The main advantage of biocytin lies in the comprehensive intracellular distribution of the molecule, and in effective detection using avidin-based reactions....

  13. Synthesis of a Functionalized Polypyrrole Coated Electrotextile for Use in Biosensors

    Directory of Open Access Journals (Sweden)

    Andre Senecal

    2012-11-01

    Full Text Available An electrotextile with a biosensing focus composed of conductive polymer coated microfibers that contain functional attachment sites for biorecognition elements was developed. Experiments were conducted to select a compound with a pendant functional group for inclusion in the polymer, a fiber platform, and polymerization solvent. The effects of dopant inclusion and post-polymerization wash steps were also analyzed. Finally, the successful attachment of avidin, which was then used to capture biotin, to the electrotextile was achieved. The initial results show a nonwoven fiber matrix can be successfully coated in a conductive, functionalized polymer while still maintaining surface area and fiber durability. A polypropylene fiber platform with a conductive polypyrrole coating using iron (III chloride as an oxidant, water as a solvent, and 5-sulfosalicylic acid as a dopant exhibited the best coating consistency, material durability, and lowest resistance. Biological attachment of avidin was achieved on the fibers through the inclusion of a carboxyl functional group via 3-thiopheneacetic acid in the monomer. The immobilized avidin was then successfully used to capture biotin. This was confirmed through the use of fluorescent quantum dots and confocal microscopy. A preliminary electrochemical experiment using avidin for biotin detection was conducted. This technology will be extremely useful in the formation of electrotextiles for use in biosensor systems.

  14. The typing of Staphylococcus epidermidis by a lectin-binding assay

    DEFF Research Database (Denmark)

    Jarløv, J O; Hansen, J E; Rosdahl, V T

    1992-01-01

    plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71...

  15. Characterization of Hematopoietic Transcription Factor Complexes in Erythroid Cells

    NARCIS (Netherlands)

    P.J.F. Rodriguez

    2006-01-01

    textabstractEfficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Due to biotin’s very high affinity for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexe

  16. DETECTION OF DNA DAMAGE USING A FIBEROPTIC BIOSENSOR

    Science.gov (United States)

    A rapid and sensitive fiber optic biosensor assay for radiation-induced DNA damage is reported. For this assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed...

  17. Rapid Mastitis Detection assay on porous nitrocellulose membrane slides

    NARCIS (Netherlands)

    Mujawar, L.H.; Moers, A.P.H.A.; Norde, W.; Amerongen, van A.

    2013-01-01

    We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase a

  18. Rapid mastitis detection assay on porous nitrocellulose membrane slides

    NARCIS (Netherlands)

    Mujawar, Liyakat Hamid; Moers, Antoine; Norde, Willem; van Amerongen, Aart

    2013-01-01

    We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase a

  19. Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hofer, Manuel; Adamsmaier, Stefan [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Zanten, Thomas S. van [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Chtcheglova, Lilia A. [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Manzo, Carlo [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Duman, Memed [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Mayer, Barbara [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Ebner, Andreas [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Moertelmaier, Manuel; Kada, Gerald [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria); Garcia-Parajo, Maria F. [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); ICREA-Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona (Spain); Hinterdorfer, Peter, E-mail: peter.hinterdorfer@jku.at [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kienberger, Ferry [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria)

    2010-05-15

    We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2{sup o}. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

  20. Development of a functionalized Xenon biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Spence, Megan M.; Ruiz, E. Janette; Rubin, Seth M.; Lowery, Thomas J.; Winssinger, Nicolas; Schultz, Peter G.; Wemmer, David E.; Pines, Alexander

    2004-03-25

    NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of remote amplified detection. Here we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of functionalized xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

  1. A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity

    DEFF Research Database (Denmark)

    Mikkelsen, J D; Larsen, P J; Sørensen, G G;

    1994-01-01

    -immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer...

  2. Surface potential variations on a silicon nanowire transistor in biomolecular modification and detection

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Chia-Chang; Chiang, Pei-Ling; Lin, Tsung-Wu; Chen, Yit-Tsong [Institute of Atomic and Molecular Sciences, Academia Sinica, PO Box 23-166, Taipei 106, Taiwan (China); Sun, Chih-Jung; Tsai, Ming-Hsueh [Department of Chemistry, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan (China); Chang, Yun-Chorng, E-mail: ychang6@mail.ncku.edu.tw, E-mail: ytcchem@ntu.edu.tw [Institute of Electro-Optical Science and Engineering, National Cheng Kung University, No. 1, Ta-Hsueh Road, Tainan 701, Taiwan (China)

    2011-04-01

    Using a silicon nanowire field-effect transistor (SiNW-FET) for biomolecule detections, we selected 3-(mercaptopropyl)trimethoxysilane (MPTMS), N-[6-(biotinamido)hexyl]-3{sup '}-(2{sup '}-pyridyldithio) propionamide (biotin-HPDP), and avidin, respectively, as the designated linker, receptor, and target molecules as a study model, where the biotin molecules were modified on the SiNW-FET to act as a receptor for avidin. We applied high-resolution scanning Kelvin probe force microscopy (KPFM) to detect the modified/bound biomolecules by measuring the induced change of the surface potential ({Delta}{Phi}{sup s}) on the SiNW-FET under ambient conditions. After biotin-immobilization and avidin-binding, the {Delta}{Phi}{sup s} on the SiNW-FET characterized by KPFM was demonstrated to correlate to the conductance change inside the SiNW-FET acquired in aqueous solution. The {Delta}{Phi}{sup s} values on the SiNW-FET caused by the same biotin-immobilization and avidin-binding were also measured from drain current versus gate voltage curves (I{sub d}-V{sub g}) in both aqueous condition and dried state. For comparison, we also study the {Delta}{Phi}{sup s} values on a Si wafer caused by the same biotin-immobilization and avidin-binding through KPFM and {zeta} potential measurements. This study has demonstrated that the surface potential measurement on a SiNW-FET by KPFM can be applied as a diagnostic tool that complements the electrical detection with a SiNW-FET sensor. Although the KPFM experiments were carried out under ambient conditions, the measured surface properties of a SiNW-FET are qualitatively valid compared with those obtained by other biosensory techniques performed in liquid environment.

  3. An innovative pre-targeting strategy for tumor cell specific imaging and therapy

    Science.gov (United States)

    Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

    2015-08-01

    A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging

  4. Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system.

    Science.gov (United States)

    Ahmed, Raju; Spikings, Emma; Zhou, Shaobo; Thompsett, Andrew; Zhang, Tiantian

    2014-04-01

    Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin-avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin-streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin-streptavidin detection system.

  5. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    Science.gov (United States)

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  6. Biotin-conjugated fusogenic liposomes for high-quality cell purification.

    Science.gov (United States)

    Hersch, Nils; Wolters, Benjamin; Ungvari, Zoltan; Gautam, Tripti; Deshpande, Dhruva; Merkel, Rudolf; Csiszar, Anna; Hoffmann, Bernd; Csiszár, Agnes

    2016-01-01

    Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.

  7. Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu; Sugiura, Ryuta; Sasaki, Nobuaki; Misawa, Nobuo; Tero, Ryugo; Urisu, Tsuneo; Gardiner, Alastair T; Cogdell, Richard J; Hashimoto, Hideki; Nango, Mamoru

    2011-07-11

    The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

  8. Preliminary measurement results of biotinylated BSA detection of a low cost optical cavity based biosensor using differential detection

    Science.gov (United States)

    Cowles, Peter; Joy, Cody; Bujana, Antonio; Rho, DongGee; Kim, Seunghyun

    2016-03-01

    We report an optical cavity based biosensor using a novel differential detection method for point-of-care applications. Two laser diodes allow for multiplexing capability along with the ability to enhance the responsivity using differential detection. The laser wavelengths are chosen so that the optical intensities of two lasers change monotonically with opposite slopes upon the adsorption of desired biomarkers. The cavity width, PMMA thickness, and silver thickness have been optimized to achieve a large change in scaled differential value. We chose biotinylated BSA detection with Avidin as a receptor molecule to demonstrate the proposed design. Avidin is attached directly to the PMMA layer by physisorption. Then, biotinylated BSA is introduced to the sample and the intensities of the laser diodes are measured by a sCMOS camera. A change in the scaled differential value will correlate to the binding of biotinylated BSA. In this presentation, we will discuss simulation results, fabrication procedures, and preliminary measurement results.

  9. Highly sensitive optical biosensor based on silicon-microring-resonator-loaded Mach–Zehnder interferometer

    Science.gov (United States)

    Yoshida, Soichiro; Ishihara, Shintaro; Arakawa, Taro; Kokubun, Yasuo

    2017-04-01

    We propose and demonstrate a novel biosensor based on a silicon-single-microring-resonator-loaded Mach–Zehnder interferometer (MRR-MZI), and discuss the design of the sensor theoretically. Owing to the combination of an MZI and the enhanced phase change in a microring resonator (MRR), high sensitivity is expected to be realized. The designed MRR-MZI sensor is fabricated using a CMOS-compatible process, and its sensing characteristics are measured using ethanol solutions with a concentration of less than 3 wt % and avidin solutions. The sensitivity of the MRR-MZI to changes in the environmental refractive index is increased by approximately 50 times compared with that of a simple MRR. In addition, avidin solution with a concentration as low as 20 pM was successfully detected.

  10. Methodological Study of Cell Separation with Domestic Immunomagnetic Beads

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To establish the method of cell separation with domestic immuomagnetic beads, three methods were investigated. Direct method, SPA method and Biotin-Avidin method were applied to separate cell strain Hut-78 and CD4 positive cells. Separation rate of strain Hut-78 was more than 90 % in direct method. Detachment rate with papain was over 95 %. Cell activity was well retained. SPA method and Biotin-Avidin methods were also effective, but the direct method was superior to the other two techniques. Before separated by the direct method, CD4 positive cells constituted 46.4 %±6.4 % of mononuclear cells (MNC), but in eliminated suspension there was only 6.2 %±2.3 % CD4 positive cells left. In the separated part, 80.6 %±7.2 % of the cells combined with the beads. It is concluded that the direct method in separating cells had high sensitivity and specificity.

  11. Radio frequency plasma polymer coatings for affinity capture MALDI mass spectrometry.

    Science.gov (United States)

    Li, Meiling; Timmons, Richard B; Kinsel, Gary R

    2005-01-01

    Surface modification of MALDI probes is an attractive approach for combining bioaffinity isolation of targeted biomolecules with mass spectrometric analysis of the captured species. In this work, we demonstrate that a polymer thin film, produced by pulsed rf plasma polymerization of allylamine and deposited directly on a MALDI probe, can be subsequently biotinylated to develop a bioaffinity capture MALDI probe. The synthesis and characterization of the probe by XPS, FT-IR, and AFM is described, and the selective isolation of avidin from a three-component mixture of avidin, lysozyme, and cytochrome c is presented. These initial results offer encouragement for the further exploration of rf plasma polymer deposition as a novel approach for the development of on-probe affinity capture MALDI probes.

  12. Development of an electrochemical DNA biosensor for detection of specific Mycobacterium tuberculosis sequence based on poly(L-glutamic acid) modified electrode

    Indian Academy of Sciences (India)

    MERVE YESIL; SONER DONMEZ; FATMA ARSLAN

    2016-11-01

    An electrochemical DNA biosensor was developed by avidin-biotin interaction of a biotinylated probe and avidin-attached, poly(L-glutamic) acid coated pencil graphite electrode (PGA/PGE) for detection of specific Mycobacterium tuberculosis DNA sequence. The discrimination of fully complementary hybridization and mismatch hybridization was carried out by electrochemical reduction current of Meldola’s Blue (MDB) in square wave voltammetry (SWV). The calibration graph of the DNA biosensor was linear between 1.5–12.5 nM and the detection limit was calculated as 1.3 nM. The proposed biosensor successfully discriminated short andlong oligonucleotides related to DNA sequence of Mycobacterium tuberculosis in optimal condition.

  13. Protein adsorption to graphene surfaces controlled by chemical modification of the substrate surfaces.

    Science.gov (United States)

    Kamiya, Yasutaka; Yamazaki, Kenji; Ogino, Toshio

    2014-10-01

    We have investigated effects of the support substrate surfaces on properties of the attached graphene flakes by observing protein adsorption to the graphene surfaces on SiO2/Si substrates that are modified with self-assembled monolayers to control their hydrophilicity. Using atomic force microscopy operated in aqueous environment, we found that high-density clusters of agglomerated avidin molecules form on the graphene flakes in the areas supported by a hydrophobic substrate surface, whereas very low density of large avidin clusters form at the edge of graphene flakes in the area supported by a hydrophilic surface. These results demonstrate that hydrophilicity of the support surface affects hydrophilicity of the graphene surface also in aqueous environment and that surface modification of the support substrate is a useful technique to control protein adsorption phenomena on graphene surfaces for realization of high sensitive graphene biosensors.

  14. Versatility of immunohistochemical reactions: comprehensive survey of detection systems.

    Science.gov (United States)

    Mokrý, J

    1996-01-01

    The field of immunohistochemistry comprises histological methods enabling detection of tissue antigens via specific antibodies. Although all these techniques take an advantage of a large specificity of antibody to a particular tissue antigen there are many different approaches for enhancement and visualization of the signal. The aim of the present review article was to briefly outline the historical milestones that made the rapid progress of this discipline possible and give a comprehensive survey of immunohistochemical methods applicable to biomedical research. The survey starts with a description of the direct immunohistochemical method and then pays attention to a huge number of indirect methods. For better explanation of principles of individual techniques, the text is accompanied with graphical schemes. The highest attention is given to immunohistochemical methods that are most generally used, i.e. enzyme anti-enzyme complex methods (e.g. Peroxidase Anti-Peroxidase/PAP/ or Alkaline Phosphatase Anti-Alkaline Phosphatase /APAAP/) and methods based on avidin-biotin interactions (Bridged Avidin-Biotin/BRAB/, Avidin-Biotin Complex /ABC/, Labelled Avidin-Biotin/LAB/). Nevertheless, the principles of other immunohistochemical methods like two- or three-step indirect immunohistochemical methods, methods based on protein A-antibody interaction and Hapten Antibody Anti-Hapten method (HAAH), are also thoroughly characterized. Usefulness of each method for a specific utilization, its advantages and disadvantages are mentioned and compared with the latest immunohistochemical techniques, like Multi-Layered Peroxidase-Labelled Antibody (MLP) and water soluble polymer conjugates, e.g. Enhanced Polymer One-Step staining (EPOS) or EnVision. The last paragraphs are devoted to immunohistochemical amplification systems (Catalyzed Signal Amplification/CSA/ and label anti-label) that dramatically increase sensitivity of detection systems and enable to compare sensitivity of

  15. Oligomeric Neuronal Protein Aggregates as Biomarkers for Traumatic Brain Injury (TBI) and Alzheimer Disease (AD)

    Science.gov (United States)

    2013-10-01

    avidin-hors utilizing pos /ml) and 1/ le dilution concentratio ration and 1 signal was or the target e due to lim One of th ic a-syn lev ht at 4oC...or 13  ditions for ylation me A system. r target dete ing monom n, we are ab etect up to f e radish pe t- mortem P 140 (0.1mg gave better n (Figure

  16. 13.5.Toxic and drug-induced liver disease

    Institute of Scientific and Technical Information of China (English)

    1992-01-01

    920133 Measurement of human plasma abnormal prothrombin by biotin-avidin (BA)ELISA in the diagnosis of hepatocellularcarcinoma.HU Dachun (胡大春),et al.DeptChem,Basic Med Sci,Shanhai Med Univ,200032.Chin J Cancer 1991; 10 (4): 283-285.After the removal of fibrinogen and prothrom-bin by bentoite and barium citrate,the abnormal

  17. Pre-targeted immunodetection in glioma patients: tumour localization and single-photon emission tomography imaging of [[sup 99m]Tc ]PnAO-biotin

    Energy Technology Data Exchange (ETDEWEB)

    Paganelli, G. (INB-CNR, Milan Univ. (Italy). Dept. of Nuclear Medicine Scientific Inst. H San Raffaele, Milan (Italy)); Magnani, P. (INB-CNR, Milan Univ. (Italy). Dept. of Nuclear Medicine Scientific Inst. H San Raffaele, Milan (Italy)); Zito, F. (INB-CNR, Milan Univ. (Italy). Dept. of Nuclear Medicine Scientific Inst. H San Raffaele, Milan (Italy)); Lucignani, G. (INB-CNR, Milan Univ. (Italy). Dept. of Nuclear Medicine Scientific Inst. H San Raffaele, Milan (Italy)); Sudati, F. (INB-CNR, Milan Univ. (Italy). Dept. of Nuclear Medicine Scientific Inst. H San Raffaele, Milan (Italy)); Truci, G. (Div. of Neurology, Milan Univ. (Italy) Scientific Inst. H San Raffaele, Milan (Italy)); Motti, E. (Div. of Neurosurgery, Milan Univ. (Italy) Scientific Inst. H San Raffaele, Milan (Italy)); Terreni, M. (Dept. of Pathology, Scientific Inst. H San Raffaele, Milan (Italy)); Pollo, B. (Dept. of Pathology, Scientific Inst. G. Besta, Milan (Italy)); Giovanelli, M. (Div. of Neurosurgery, Milan

    1994-04-01

    We have developed a three-step pre-targeting method using the avidin-biotin system. The rationale of this technique consists in vivo labelling of biotinylated MoAbs targeted onto tumour deposits, when most of the unbound antibodies have been cleared from the bloodstream as avidin-bound complexes. The anti-tenascin MoAb BC2, specific for the majority of gliomas, was biotinylated and 1 mg was administered i.v. in 20 patients with histologically documented cerebral lesions. After 24-36 h, 5 mg avidin was injected i.v. followed 24 h later by a third i.v. injection of 0.2 mg PnAO-biotin labelled with 15-20 mCi technetium-99m. No evidence of toxicity was observed. Whole-body biodistribution was measured at 20 min, 3 h and 5 h post-injection. [[sup 99m]Tc]PnAO-biotin had a fast blood clearance and was primarily excreted through the biliary system. A dedicated single-photon emission tomography system was used to acquire brain tomographic images 1-2 h after the administration of [[sup 99m]Tc]PnAO-biotin. Tumours were detected in 15/18 glioma patients with a tumour to non-tumour ratio of up 14:1. This three-step method, based on the sequential adminsitration of anti-tenascin MoAb BC2, avidin and [[sup 99m]Tc]PnAO-biotin, can support computed tomography or magnetic resonance imaging for the diagnosis and follow-up of patients with glioma. (orig./MG)

  18. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

    OpenAIRE

    2003-01-01

    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demon...

  19. Histological assessment of the early enthesitis lesion in spondyloarthropathy

    OpenAIRE

    McGonagle, D.; Marzo-Ortega, H; O'Connor, P; Gibbon, W; HAWKEY, P; Henshaw, K; Emery, P

    2002-01-01

    Methods: Clinically evident acute enthesopathy was confirmed by magnetic resonance imaging and ultrasonography in four cases of plantar fasciitis and one case of patellar tendon enthesitis. Ultrasound guided biopsy of insertional points was carried out with a Jamshedi needle. Control tissue was obtained from two subjects undergoing spinal grafting surgery. Standard histochemistry and immunohistochemistry analysis using the avidin-biotin immunoperoxidase complex method employing markers agains...

  20. Diagnóstico del parvovirus canino-2 (pvc-2) por inmunohistoquímica en perros domésticos

    OpenAIRE

    Rocío Angélica Ruiz Romero; Eugenia Candanosa Aranda; Félix Sánchez Godoy; Andrés Ducoing Watty

    2007-01-01

    Thirty cases of small intestine with suggestive histopathological lesions of canine parvovirus were evaluated by avidin-biotin-peroxidase complex using a monoclonal antibody developed in mouse against canine parvovirus-2 (CPV-2). Positive immuno-histochemical reactions were obtained in 76.67% (23 cases) of processed samples. The Iymphocytes, macrophages and necrotic cells of the intestinal crypts were the cells that most frequently showed immunopositivity. The intestinal histopathological les...

  1. The Role of Megakaryocytes in Breast Cancer Metastasis to Bone

    Science.gov (United States)

    2014-05-01

    The sections were incubated with 10% donkey serum in PBS for 2 hr, incubated overnight with a primary rabbit antibody (10 μg/ml, ABCAM) to von...Willebrand factor , washed and incubated with a secondary antibody, biotinylated donkey anti-rabbit (1:1000 A. Metastatic Model: SDF-1 B. Metastatic Model...TPO C. Non-metastatic Model: SDF-1 D. Non-metastatic Model: TPO 7 in 10% donkey serum) for 2 hr. Antigens were visualized with avidin

  2. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    Science.gov (United States)

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  3. Half-sandwich ruthenium(II) biotin conjugates as biological vectors to cancer cells.

    Science.gov (United States)

    Babak, Maria V; Plażuk, Damian; Meier, Samuel M; Arabshahi, Homayon John; Reynisson, Jóhannes; Rychlik, Błażej; Błauż, Andrzej; Szulc, Katarzyna; Hanif, Muhammad; Strobl, Sebastian; Roller, Alexander; Keppler, Bernhard K; Hartinger, Christian G

    2015-03-23

    Ruthenium(II)-arene complexes with biotin-containing ligands were prepared so that a novel drug delivery system based on tumor-specific vitamin-receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru(II) -biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012-2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half-sandwich ruthenium(II)-biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed.

  4. Cell penetrating peptide-modified poly(lactic-co-glycolic acid) nanoparticles with enhanced cell internalization.

    Science.gov (United States)

    Steinbach, Jill M; Seo, Young-Eun; Saltzman, W Mark

    2016-01-01

    The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2h, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE compared to Avidin NPs, both formulations resulted in similar internalization levels (48 and 64-fold, respectively) after 24h. Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery.

  5. Detection of molecules and cells using nuclear magnetic resonance with magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Rümenapp, Christine, E-mail: ruemenapp@tum.de [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany); Gleich, Bernhard [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany); Mannherz, Hans Georg [Abteilung für Anatomie und Molekulare Embryologie, Ruhr Universität Bochum, Bochum (Germany); Haase, Axel [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany)

    2015-04-15

    For the detection of small molecules, proteins or even cells in vitro, functionalised magnetic nanoparticles and nuclear magnetic resonance measurements can be applied. In this work, magnetic nanoparticles with the size of 5–7 nm were functionalised with antibodies to detect two model systems of different sizes, the protein avidin and Saccharomyces cerevisiae as the model organism. The synthesised magnetic nanoparticles showed a narrow size distribution, which was determined using transmission electron microscopy and dynamic light scattering. The magnetic nanoparticles were functionalised with the according antibodies via EDC/NHS chemistry. The binding of the antigen to magnetic nanoparticles was detected through the change in the NMR T{sub 2} relaxation time at 0.5 T (≈21.7 MHz). In case of a specific binding the particles cluster and the T{sub 2} relaxation time of the sample changes. The detection limit in buffer for FITC-avidin was determined to be 1.35 nM and 10{sup 7} cells/ml for S. cerevisiae. For fluorescent microscopy the avidin molecules were labelled with FITC and for the detection of S. cerevisiae the magnetic nanoparticles were additionally functionalised with rhodamine. The binding of the particles to S. cerevisiae and the resulting clustering was also seen by transmission electron microscopy.

  6. Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes.

    Science.gov (United States)

    Shimazaki, Youji; Sato, Yuki

    2016-05-15

    The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.

  7. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    Energy Technology Data Exchange (ETDEWEB)

    Geerds, Christina [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Wohlmann, Jens; Haas, Albert [University of Bonn, Ulrich-Haberland Strasse 61a, 53121 Bonn (Germany); Niemann, Hartmut H., E-mail: hartmut.niemann@uni-bielefeld.de [Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany)

    2014-06-18

    The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

  8. PEG Functionalization of Whispering Gallery Mode Optical Microresonator Biosensors to Minimize Non-Specific Adsorption during Targeted, Label-Free Sensing

    Directory of Open Access Journals (Sweden)

    Fanyongjing Wang

    2015-07-01

    Full Text Available Whispering Gallery Mode (WGM optical microresonator biosensors are a powerful tool for targeted detection of analytes at extremely low concentrations. However, in complex environments, non-specific adsorption can significantly reduce their signal to noise ratio, limiting their accuracy. To overcome this, poly(ethylene glycol (PEG can be employed in conjunction with appropriate recognition elements to create a nonfouling surface capable of detecting targeted analytes. This paper investigates a general route for the addition of nonfouling elements to WGM optical biosensors to reduce non-specific adsorption, while also retaining high sensitivity. We use the avidin-biotin analyte-recognition element system, in conjunction with PEG nonfouling elements, as a proof-of-concept, and explore the extent of non-specific adsorption of lysozyme and fibrinogen at multiple concentrations, as well as the ability to detect avidin in a concentration-dependent fashion. Ellipsometry, contact angle measurement, fluorescence microscopy, and optical resonator characterization methods were used to study non-specific adsorption, the quality of the functionalized surface, and the biosensor’s performance. Using a recognition element ratio to nonfouling element ratio of 1:1, we showed that non-specific adsorption could be significantly reduced over the controls, and that high sensitivity could be maintained. Due to the frequent use of biotin-avidin-biotin sandwich complexes in functionalizing sensor surfaces with biotin-labeled recognition elements, this chemistry could provide a common basis for creating a non-fouling surface capable of targeted detection. This should improve the ability of WGM optical biosensors to operate in complex environments, extending their application towards real-world detection.

  9. A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

    Science.gov (United States)

    Casarin, Elisabetta; Lucchese, Laura; Grazioli, Santina; Facchin, Sonia; Realdon, Nicola; Brocchi, Emiliana; Morpurgo, Margherita; Nardelli, Stefano

    2016-01-01

    Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits

  10. Development of a paper-based carbon nanotube sensing microfluidic device for biological detection.

    Science.gov (United States)

    Yang, Shih-I; Lei, Kin Fong; Tsai, Shiao-Wen; Hsu, Hsiao-Ting

    2013-01-01

    Carbon nanotube (CNT) has been utilized for the biological detection due to its extremely sensitive to biological molecules. A paper-based CNT sensing microfluidic device has been developed for the detection of protein, i.e., biotin-avidin, binding. We have developed a fabrication method that allows controlled deposition of bundled CNTs with well-defined dimensions to form sensors on paper. Then, polydimethyl siloxane (PDMS) was used to pattern the hydrophobic boundary on paper to form the reaction sites. The proposed fabrication method is based on vacuum filtration process with a metal mask covering on a filter paper for the definition of the dimension of sensor. The length, width, and thickness of the CNT-based sensors are readily controlled by the metal mask and the weight of the CNT powder used during the filtration process, respectively. Homogeneous deposition of CNTs with well-defined dimensions can be achieved. The CNT-based sensor on paper has been demonstrated on the detection of the protein binding. Biotin was first immobilized on the CNT's sidewall and avidin suspended solution was applied to the site. The result of the biotin-avidin binding was measured by the resistance change of the sensor, which is a label-free detection method. It showed the CNT is sensitive to the biological molecules and the proposed paper-based CNT sensing device is a possible candidate for point-of-care biosensors. Thus, electrical bio-assays on paper-based microfluidics can be realized to develop low cost, sensitive, and specific diagnostic devices.

  11. A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

    Directory of Open Access Journals (Sweden)

    Elisabetta Casarin

    Full Text Available Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR disease. The avidin-nucleic-acid-nanoassembly (ANANAS is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA

  12. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  13. Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue.

    Science.gov (United States)

    Hermes, B; Feldmann-Böddeker, I; Welker, P; Algermissen, B; Steckelings, M U; Grabbe, J; Henz, B M

    2000-01-01

    In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.

  14. Aligned deposition and electrical measurements on single DNA molecules

    DEFF Research Database (Denmark)

    Eidelshtein, Gennady; Kotlyar, Alexander; Hashemi, Mohtadin;

    2015-01-01

    A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)–poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg2+ ions...... bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin–DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements...

  15. Immunohistochemical study on localization of serotonin immunoreactive cells in the gastrointestinal tract of the European catfish (Silurus glanis, L.).

    Science.gov (United States)

    Köprücü, S; Yaman, M

    2015-02-01

    In this study, it was aimed to identify the distribution of serotonin immunoreactive cells within the gastrointestinal tract (GIT) of European catfish (Silurus glanis). For this purpose, the tissue samples were taken from the stomach (cardia, fundus and pylorus region) and intestine (anterior, middle and posterior region). They were examined by applying the avidin-biotin-immunoperoxidase method. The serotonin containing immunoreactive cells are presented in all regions of the GIT. It was determined to be localized generally in different distribution within the stomachs and intestines of S. glanis. It was found that the most intensive regions of immunoreactive cells were the cardia stomach and posterior of intestine.

  16. Study of Immunoassay Methods for Recombinant Human Erythropoietin(rhEPO)Using Competitive ELISA

    Institute of Scientific and Technical Information of China (English)

    Jin YAN; Jie Bo MI; Wen Bao CHANG

    2004-01-01

    Two different immunoassay methods, competitive indirect enzyme-linked immuno- sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO.The linear ranges were 50-20000 ng/mL and 10-50000 ng/mL for CI-ELISA and ACI-ELISA, respectively.The low detection limits of CI-ELISA and ACI-ELISA were 62.8 ng/mL and 8.5 ng/mL, respectively.

  17. Dual functionalized graphene oxide serves as a carrier for delivering oligohistidine- and biotin-tagged biomolecules into cells.

    Science.gov (United States)

    Jana, Batakrishna; Mondal, Goutam; Biswas, Atanu; Chakraborty, Indrani; Saha, Abhijit; Kurkute, Prashant; Ghosh, Surajit

    2013-11-01

    A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell.

  18. Refractometric sensing using propagating versus localized surface plasmons: a direct comparison.

    Science.gov (United States)

    Svedendahl, Mikael; Chen, Si; Dmitriev, Alexandre; Käll, Mikael

    2009-12-01

    We present a direct experimental comparison between the refractive index sensing capabilities of localized surface plasmon resonances (LSPRs) in gold nanodisks and propagating surface plasmon resonances (SPRs) on 50 nm gold films. The comparison is made using identical experimental conditions, and for the same resonance wavelength, lambda(SP) congruent with 700 nm. Biosensing experiments with biotin-avidin coupling reveal that the two sensing platforms have very similar performance, despite a superior bulk refractive index sensing figure of merit for the SPR sensor. The results demonstrate that LSPR sensing based on simple transmission or reflection measurements is a highly competitive technique compared to the traditional SPR sensor.

  19. Identification of monoclonal antibodies against the trypomastigote stage of Trypanosoma cruzi by use of iminobiotinylated surface polypeptides.

    Science.gov (United States)

    Beard, C A; Wrightsman, R A; Manning, J E

    1985-08-01

    The surface polypeptides of epimastigotes and tissue culture-derived trypomastigotes of Trypanosoma cruzi have been isolated free of most cytosolic components by use of the 2-iminobiotin-avidin interaction. Polypeptides of the trypomastigote stage obtained by this technique are recognized by serum antibodies from Chagasic patients and T. cruzi-infected mice. These polypeptides have been used as the detecting antigen for the identification of hybridoma cells producing monoclonal antibodies against the surface proteins of the trypomastigote stage of T. cruzi. These experiments document a practical approach for obtaining T. cruzi surface proteins in sufficient quantity and purity for use in immunological studies.

  20. Immunohistochemical localization of atrial natriuretic factor (ANF) in the excretory system of the rabbit parotid gland.

    Science.gov (United States)

    Valentino, B; Farina Lipari, E; Carini, F; Valenza, V

    1999-01-01

    The immunohistochemical localization of atrial natriuretic factor (ANF) in the rabbit parotid gland was performed using an antibody against rabbit ANF and avidin-biotin or streptoavidin as detector. Results showed positivity in cuboidal and columnar cells of intralobular ducts and in basal cells of extralobular and main excretory duct. These data support the hypothesis that ANF produced by intralobular ducts could act through a paracrine mechanism; ANF produced by extralobular and main ducts may play a role in the regulation of salivary composition.

  1. Activite succino-deshydrogenasique des microsomes et mode d'incorporation du succinate 2,3-(14)C dans les acides gras des microsomes de foie de rat in vitro.

    Science.gov (United States)

    Rous, S; Aubry, L; Bonini, F

    1970-03-16

    The incorporations of 2,3-(14)C-succinate 2-(14)C-acetate into fatty acids of different cellular fractions of rat liver were studied. Acetate was incorporated mainly into supernatant and succinate into microsomal fatty acids. Mitochondria only could intensively decarboxylate pyruvate. Avidine inhibited fatty acid synthesis from succinate mainly in the supernatant. It is suggested that succinate is an important physiological precursor of fatty acids in the liver and that an active succino-dehydrogenase is present in microsomes.

  2. Indium arsenide nanowire field-effect transistors for pH and biological sensing

    Energy Technology Data Exchange (ETDEWEB)

    Upadhyay, S.; Krogstrup, P.; Nygård, J., E-mail: nygard@nbi.dk [Center for Quantum Devices and Nanoscience Center, Niels Bohr Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen (Denmark); Frederiksen, R.; Lloret, N.; Martinez, K. L. [Bio-Nanotechnology and Nanomedicine Laboratory, Department of Chemistry and Nanoscience Center, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen (Denmark); De Vico, L.; Jensen, J. H. [Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen (Denmark)

    2014-05-19

    Indium Arsenide is a high mobility semiconductor with a surface electron accumulation layer that allows ohmic electrical contact to metals. Here, we present nanowire devices based on this material as a platform for chemical and biological sensing. The sensing principle involves the binding of a charged species at the sensor surface transduced via field effect into a change in current flowing through the sensor. We show the sensitivity of the platform to the H{sup +} ion concentration in solution as proof of principle and demonstrate the sensitivity to larger charged protein species. The sensors are highly reproducible and reach a detection limit of 10 pM for Avidin.

  3. Immunohistochemical diagnosis of Neospora caninum in tissue sections.

    Science.gov (United States)

    Lindsay, D S; Dubey, J P

    1989-11-01

    An avidin-biotin-peroxidase complex immunoperoxidase staining method was developed to detect Neospora caninum in formalin-fixed, paraffin-embedded tissue sections. Specific antiserum to N caninum was made in rabbits and used to probe tissues from dogs naturally and experimentally infected with N caninum. The test detected tachyzoites and bradyzoites of N caninum. A reaction was not observed to Toxoplasma gondii, Hammondia hammondi, Sarcocystis cruzi, S capricanis, S tenella, Besnoitia jellisoni, Caryospora bigenetica, Hepatazoon canis, Atoxoplasma sp, or the organism causing canine dermal coccidiosis. When antiserum made in rabbits to T gondii was used in the test, reaction to N caninum was not observed.

  4. Energy landscape investigation by wavelet transform analysis of atomic force spectroscopy data in a biorecognition experiment.

    Science.gov (United States)

    Bizzarri, Anna Rita

    2016-01-01

    Force fluctuations recorded in an atomic force spectroscopy experiment, during the approach of a tip functionalized with biotin towards a substrate charged with avidin, have been analyzed by a wavelet transform. The observation of strong transient changes only when a specific biorecognition process between the partners takes place suggests a drastic modulation of the force fluctuations when biomolecules recognize each other. Such an analysis allows to investigate the peculiar features of a biorecognition process. These results are discussed in connection with the possible role of energy minima explored by biomolecules during the biorecognition process.

  5. Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Deborggraeve, Stijn; Büscher, Philippe;

    2011-01-01

    We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept)avidin-c......We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept...

  6. Use of enzyme label for quantitative evaluation of liposome adhesion on cell surface: studies with J774 macrophage monolayers.

    Science.gov (United States)

    Trubetskoy, V S; Dormeneva, E V; Tsibulsky, V P; Repin, V S; Torchilin, V P

    1988-07-01

    A method for quantitation of cell surface-bound liposomes utilizing J774 macrophage monolayers is developed. Surface-bound biotinyl-containing and 125I-labeled liposomes were quantified with avidin-peroxidase in an ELISA-like assay. Peroxidase substrate absorbance values were recalculated into the absolute amount of liposomal lipid using a special calibration plot. Total liposome uptake by macrophages was determined following the binding of 125I radioactivity. The approach suggested allows quantitative evaluation of the changes in the content of surface-adhered liposomes during their interaction with cells in vitro.

  7. ELISA for evaluating the incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes

    DEFF Research Database (Denmark)

    Zimmermann-Nielsen, E; Svehag, S E; Thorlacius-Ussing, O;

    2001-01-01

    An ELISA that measures plasma derived complement (C) split-products C3b/iC3b deposited on solid-phase immune complexes during C activation is described. Plates are coated with BSA, anti-BSA and plasma is added. Deposited C3b/iC3b is then detected by biotinylated anti-C3c-antibodies, avidin......) or classical pathway (CP) with regard to age or gender was demonstrated. The total coefficient of variation was systemic lupus erythematosus (SLE). There was a weak correlation between...

  8. Oligonucleotide-stabilized fluorescent silver nanoclusters for the specific and sensitive detection of biotin.

    Science.gov (United States)

    Xiong, Xiaoli; Tang, Yan; Zhao, Jingjin; Zhao, Shulin

    2016-02-21

    A novel biotin fluorescent probe based on oligonucleotide-stabilized silver nanoclusters (DNA-AgNCs) was synthesized by employing a biotinylated cytosine-rich sequence as a synthesized template. The fluorescence properties of the DNA-AgNCs are related to the modified position of the DNA. When biotin is linked to the middle thymine base of the DNA sequence, the DNA-AgNCs emit the strongest fluorescence. Moreover, the stability of the DNA-AgNCs was affected by avidin through biotin-avidin binding, quenching the fluorescence of the DNA-AgNCs. In contrast, if free biotin is further introduced into this system, the quenching is apparently weakened by competition, leading to the restoration of fluorescence. This phenomenon can be utilized for the detection of biotin. Under the optimal conditions, the fluorescence recovery is linearly proportional to the concentration of biotin in the range of 10 nM-1.0 μM with a detection limit of 6.0 nM. This DNA-AgNCs probe with excellent fluorescent properties is sensitive and selective for the detection of biotin and has been applied for the determination of biotin in wheat flour.

  9. ABC-ELISA法与快速ELISA试剂盒法检测华支睾吸虫抗体的结果比较

    Institute of Scientific and Technical Information of China (English)

    崔惠儿; 方悦怡; 梁文佳; 张启明; 刘美真; 裴福全

    2001-01-01

    @@ ABC(Avidin-Biotin-Peroxidase complex)技术,即生物素-亲和素-酶结合物技术,80年代开始已广泛应用于各种免疫学检测.这种技术利用生物素(biotin)与亲和素(avidin)之间具有的高度亲和力以及1分子亲和素可与4分子生物素结合的特性,大大增强检测的敏感性.为了进一步地改进目前应用于华支睾吸虫病防治现场的ELISA试剂盒诊断方法,本实验用ABC-ELISA法检测华支睾吸虫感染循环抗体,并与快速ELISA试剂盒法(简称快速ELISA法)进行比较.

  10. Fabrication of Bioactive Surfaces by Functionalization of Electroactive and Surface-Active Block Copolymers

    Directory of Open Access Journals (Sweden)

    Omotunde Olubi

    2014-08-01

    Full Text Available Biofunctional block copolymers are becoming increasingly attractive materials as active components in biosensors and other nanoscale electronic devices. We have described two different classes of block copolymers with biofuctional properties. Biofunctionality for block copolymers is achieved through functionalization with appropriate biospecific ligands. We have synthesized block copolymers of electroactive poly(3-decylthiophene and 2-hydroxyethyl methacrylate by atom transfer radical polymerization. The block copolymers were functionalized with the dinitrophenyl (DNP groups, which are capable of binding to Immunoglobulin E (IgE on cell surfaces. The block copolymers were shown to be redox active. Additionally, the triblock copolymer of α, ω-bi-biotin (poly(ethylene oxide-b-poly (styrene-b-poly(ethylene oxide was also synthesized to study their capacity to bind fluorescently tagged avidin. The surface-active property of the poly(ethylene oxide block improved the availability of the biotin functional groups on the polymer surfaces. Fluorescence microscopy observations confirm the specific binding of biotin with avidin.

  11. Laser-induced heating integrated with a microfluidic platform for real-time DNA replication and detection

    Science.gov (United States)

    Hung, Min-Sheng; Ho, Chia-Chin; Chen, Chih-Pin

    2016-08-01

    This study developed a microfluidic platform for replicating and detecting DNA in real time by integrating a laser and a microfluidic device composed of polydimethylsiloxane. The design of the microchannels consisted of a laser-heating area and a detection area. An infrared laser was used as the heating source for DNA replication, and the laser power was adjusted to heat the solutions directly. In addition, strong biotin-avidin binding was used to capture and detect the replicated products. The biotin on one end was bound to avidin and anchored to the surface of the microchannels, whereas the biotin on the other end was bound to the quantum dots (Qdots). The results showed that the fluorescent intensity of the Qdots bound to the replicated products in the detection area increased with the number of thermal cycles created by the laser. When the number of thermal cycles was ≥10, the fluorescent intensity of the Qdots was directly detectable on the surface of the microchannels. The proposed method is more sensitive than detection methods entailing gel electrophoresis.

  12. Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor.

    Science.gov (United States)

    Mishra, Rupesh K; Hayat, Akhtar; Catanante, Gaëlle; Istamboulie, Georges; Marty, Jean-Louis

    2016-02-01

    In this work, we propose for the first time a sensitive Ochratoxin A (OTA) detection in cocoa beans using competitive aptasensor by differential pulse voltammetry (DPV). In the proposed method, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE), and percentage binding was calculated. The detection was performed after adding avidin-ALP to perform avidin-biotin reaction; the signal was generated through a suitable substrate 1-naphthyl phosphate (1-NP), for alkaline phosphatase (ALP). The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns specifically designed for OTA. The developed aptasensor showed a good linearity in the range 0.15-5 ng/mL with the limit of detection (LOD) 0.07 ng/mL and 3.7% relative standard deviation (RSD). The aptasensor displayed good recovery values in the range 82.1-85% with 3.87% RSD, thus, demonstrated the efficiency of proposed aptasensor for such matrices.

  13. Disordered array of Au covered Silicon nanowires for SERS biosensing combined with electrochemical detection

    Science.gov (United States)

    Convertino, Annalisa; Mussi, Valentina; Maiolo, Luca

    2016-04-01

    We report on highly disordered array of Au coated silicon nanowires (Au/SiNWs) as surface enhanced Raman scattering (SERS) probe combined with electrochemical detection for biosensing applications. SiNWs, few microns long, were grown by plasma enhanced chemical vapor deposition on common microscope slides and covered by Au evaporated film, 150 nm thick. The capability of the resulting composite structure to act as SERS biosensor was studied via the biotin-avidin interaction: the Raman signal obtained from this structure allowed to follow each surface modification step as well as to detect efficiently avidin molecules over a broad range of concentrations from micromolar down to the nanomolar values. The metallic coverage wrapping SiNWs was exploited also to obtain a dual detection of the same bioanalyte by electrochemical impedance spectroscopy (EIS). Indeed, the SERS signal and impedance modifications induced by the biomolecule perturbations on the metalized surface of the NWs were monitored on the very same three-electrode device with the Au/SiNWs acting as both working electrode and SERS probe.

  14. A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

    Science.gov (United States)

    Banasik, Michał; Sachadyn, Paweł

    2016-09-01

    A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

  15. Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing.

    Science.gov (United States)

    Billah, Md Morsaline; Hodges, Christopher S; Hays, Henry C W; Millner, P A

    2010-11-01

    The successful construction of an immunosensor depends on having an effective procedure for immobilising the bio-recognition element to the transducer surface. In the present study, an amino-terminated 4-aminothiophenol (ATP) self-assembled monolayer (SAM) was modified with heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate to couple reduced anti-myoglobin half-antibody fragments. The disulphide groups present in the hinge region of IgG molecules were selectively cleaved by 2-mercaptoethylamine to produce reduced half-antibody fragments with free sulphydryl groups. The maleimide terminated 4-ATP SAM modified surface was coupled to these reduced antibody fragments to produce highly oriented immobilization of the half-antibody via its Fc domain and to allow free access to the Fv bindings sites. This represents an improvement by comparison with biotin/avidin mediated IgG attachment which is essentially randomly oriented. Functional immunosensors were able to detect myoglobin in both phosphate buffered saline and whole serum over the range of concentrations from 10(-13)M to 10(-6)M, and order of magnitude better than avidin/biotin linked immunosensors. In addition, atomic force microscopy (AFM) was carried out to elucidate the nanotopology of the immunosensor surface at different stages of fabrication; the images demonstrate that half antibodies bind as described and show structural changes on subsequent antigen binding.

  16. A simple strategy based on photobiotin irradiation for the photoelectrochemical immobilization of proteins on electrode surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Cosnier, Serge [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France)]. E-mail: Serge.Cosnier@ujf-grenoble.fr; Molins, Carmen [Departamento Quimica Analitica, Universitat de Valencia (Spain); Mousty, Christine [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France); Galland, Bruno [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France); Lepellec, Arielle [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France)

    2006-03-15

    A photoactivable organic polymer was prepared first by electrogeneration of a conductive biotinylated polypyrrole film in acetonitrile electrolyte. The successive anchoring of avidin and photobiotin led to a multilayer configuration. The latter was illuminated with light (wavelength 370-400 nm) in the presence of proteins adsorbed onto its surface. The irradiation allowed the covalent linking of the proteins to the modified electrode. As a result of the photochemical reaction, a monolayer of enzyme (glucose oxidase, GOX or alkaline phosphatase, AP) was covalently bound to the photobiotin-modified surface with retention of their catalytic activities. The surfacic activities were 34 and 1.69 mU cm{sup -2} for GOX and AP photobiotin electrodes, respectively. These enzyme electrodes were compared to similar configurations obtained through the immobilization of biotinylated glucose oxidase or avidin-conjugated alkaline phosphatase on biotinylated polypyrrole film. Our results suggest that both procedures led to the immobilization of the same enzyme amount, namely a protein monolayer. This novel photo-immobilization methodology was also successfully applied to the anchoring of an anti-cholera toxin antibody which was then detected by a secondary antibody labelled with a peroxidase.

  17. [In vitro experimental study of rat cardiomyocyte injury with targeting of perfluorocarbon lipid particles].

    Science.gov (United States)

    He, Baiyong; Li, Zhaohuan; Tang, Hong; Li, Guohua; Chen, Song; Wang, Lian; Song, Haibo; Fang, Hua; Zeng, Jun

    2011-12-01

    The present study was to investigate in vitro the rat cardiomyocyte injury with targeting of home-made perfluorocarbon lipid particles with avidin-biotin interaction. Neonatal rat cardiomyocytes were cultured in vitro and divided into two groups: TNF-alpha activated group and non-activated group. Those in the TNF-alpha activated group were exposed to 200 ng/ml TNF-alpha solution for 6 hours and then cardiomyocytes in both groups were pretargeted with biotinylated ICAM-1 monoclonal antibodies, and were exposed to streptavidin, and then to homemade green fluorescently-labeled biotinylated perfluorocarbon lipid particles. Cardiomyocytes nucleus stained with Hoechst. The results were detected with fluorescence microscope. As a result, in TNF-alpha activated group, around blue fluorescent cardiomyocytes nucleus, a great amount of green fluorescent particles were found, while there were few green fluorescent particles in non-TNF activated group. It has been shown that ICAM-1 is expressed in the surface of cardiomyocytes when they are stimulated by TNF-alpha. Perfluorocarbon lipid particles associated with ICAM-1 monoclonal antibodies can be targeted to injured cardiomyocytes by avidin-biotin interaction.

  18. Application of dual color fluorescence in situ hybridization (D—FISH) to the diagnosis of a 49,XXXXY chromosomal abnormality

    Institute of Scientific and Technical Information of China (English)

    LiuYZ; ZengX

    2002-01-01

    Objective:To study the technique of D-FISH and its application in the diagnosis of a 49.XXXXY chromosomal abnormality.Methods:Biotin-labeled alpha satellite X chromosome DNA(pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus.After washing,the slides were treated with avidin-FITC,rhodamine-FITC and anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.Results:The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal.The chromosome or cytoplasm counter-stained with DAPI showed blue.The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43% and 92.57%,respectively,while that of the Y chromosome hybridization signal was 99.5% and 99.8%,respectively.Conclusion:D-FISH is a valuable technique in diagnosing 49,XXXXY chromosomal abnormality and other sex chromosomal abnormalities.

  19. QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV.

    Science.gov (United States)

    Hong, Sung-Rok; Jeong, Hyun-Do; Hong, Suhee

    2010-08-15

    Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3' end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin-biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016muM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.

  20. Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture.

    Science.gov (United States)

    Gutiérrez-Zúñiga, Gabriela Guadalupe; Hernández-López, José Luis

    2016-01-01

    A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule ((Btn)Ab-AgPSA-(HRP)Ab), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population.

  1. The preparation and property of a poly(lactic acid)biomaterial modified by biotin%一种生物素改性聚乳酸生物材料的制备与性能研究

    Institute of Scientific and Technical Information of China (English)

    严好; 潘君; 江伟民; 张晓喜; 王远亮

    2011-01-01

    研究试图通过本体改性,将生物素接枝到聚乙二醇接枝聚乳酸(PPLA)上,以改善聚乳酸微球在药物缓释应用中血液循环时间短和无主动靶向性的缺点.在本实验室制备的聚乙二醇接枝改性聚乳酸的基础之上,采用N-羟基琥珀酰亚胺活化酯法,将生物素接枝到PPLA上,制备生物素改性聚乳酸(BPLA),通过茚三酮显色、核磁共振(1H-NMR),差示扫描量热(DSC),静态水接触角、荧光蛋白标记法对材料进行表征与检测.结果表明,生物素已经共价接枝到聚乳酸上;与PPLA相比,BPLA明显降低了对牛血清白蛋白(BSA)的吸附,有望提高聚乳酸微球在血液循环系统中的停留时间;同时能与生物素的配体亲和素结合,有望通过生物素和亲和素的高亲和性实现主动靶向,可能使BPLA在药物缓释中有潜在应用价值.%The aim of this research is to overcome the drawbacks of PLA in drug delivery system,which are short blood circulation time and non-active targeting. Based on PEG-graft-PLA (PPLA) developed in our lab, biotin was grafted onto PLA to produce BPLA by NHS-activated method. The ninhydrin coloration,1H-NMR, DSC,water contact angle test, fluorescence labeled protein absorption test were used to characterize BPLA. Results from ninhydrin coioration,1H-NMR,DSC,water contact angle test showed the biotin had been covalently grafted onto PPLA. Fluorescence labeling test demonstrated that the new BPLA had the property of decreasing the nonspecific protein (BSA) absorption and increasing specific protein absorption (Avidin) evidently compared with PPLA. It was predicted that BPLA could increase the blood circulation time,meanwhile can combine with its ligand-avidin through high affinity or combine with the biotionylated protein via avidin as arm, which may have the active targeting in drug delivery system.

  2. Preparation of {sup 166} Dy/{sup 166} Ho DTPA-bis biotin as a system of In vivo generator; Preparacion de {sup 166} Dy/{sup 166} Ho DTPA-bis biotina como un sistema de generador In vivo

    Energy Technology Data Exchange (ETDEWEB)

    Jimenez V, M.R

    2003-07-01

    The objective of this work was to synthesize the complex {sup 166} Dy/{sup 166} Ho - diethylen triamine pentaacetic-bis Biotin ({sup 166} Dy/{sup 166} Ho DTPA-bis Biotin) to evaluate its potential as a new radiopharmaceutical in directed radiotherapy. The Dysprosium-166 was obtained for neutron irradiation of {sup 164} Dy{sub 2}0{sub 3} in the TRIGA Mark III reactor. The labelled was carried out in aqueous solution to p H 8.0 for addition of {sup 166} Dy Cl{sub 3} to the diethylen triamine pentaacetic-{alpha}, {omega}-bis Biotin (DTPA-bis Biotin). The radiochemical purity was determined for HPLC and ITLC. The biological integrity of the marked biotin is evaluated by the biological recognition of the avidin for HPLC - molecular exclusion with and without avidin addition. The studies of stability in vitro were made in dilutions of saline solution to 0.9% and with human serum at 37 C incubated 1 and 24 hours. The complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin was obtained with a radiochemical purity of 99.1 {+-} 0.6%. The biological recognition of the complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin for the avidin it doesn't affect the labelling procedure. The studies in vitro demonstrated that the {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin is stable after the dilution in saline solution and in human serum that there is not translocation of the one radionuclide subsequent son to the beta decay of the {sup 166} Dy that could produce the {sup 166} Ho{sup 3+} liberation. The studies of Biodistribution in healthy mice demonstrated that the one complex {sup 166} Dy/{sup 166} Ho DTPA-bis Biotin have a high renal distribution. In conclusion the radiolabelled biotin in this investigation has the appropriate properties to be used as an In vivo generator system stable for directed radiotherapy. (Author)

  3. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer

  4. Simple solutions to false results with plate/slide agglutination tests in diagnosis of infectious diseases of man and animals.

    Science.gov (United States)

    Saxena, Hari Mohan; Chothe, Shubhada; Kaur, Paviter

    2015-01-01

    We have developed a new Superagglutination test for serodiagnosis of infectious diseases. It differs from conventional plate/slide agglutination tests (PAT/SAT) by three additional steps: prior staining of serum antibody by adding a dye and addition of diluted biotinylated antiglobulin and avidin in sequence after mixing the antigen with the test serum. The new steps circumvent the problems of false positive and false negative results of PAT/SAT. In serodiagnosis of brucellosis, Superagglutination test had higher positive predictive value and specificity than Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) and higher negative predictive value and sensitivity than RBPT, STAT, ELISA and Complement Fixation Test (CFT).•Superagglutination is a simple, accurate and economic screening test for infections.•More specificity, sensitivity, positive & negative predictive value than RBPT, STAT.•More sensitivity, negative predictive value than ELISA and Complement Fixation Test.

  5. Biotin nutritional status of vegans, lactoovovegetarians, and nonvegetarians.

    Science.gov (United States)

    Lombard, K A; Mock, D M

    1989-09-01

    Urinary excretion of biotin (total avidin-binding substances) was measured in adults and children who were adhering to one of the following self-selected diets: strict vegetarian (vegan), lactoovovegetarian, or mixed (containing meat and dairy products as well as plant-derived foods). In a subset of subjects, plasma biotin concentrations were also measured. In adults the biotin excretion rate was significantly greater in the vegan group than in either the lactoovovegetarian or the mixed-diet groups; the latter were not significantly different from one another. In children the biotin excretion rates in both the vegan group and the lactoovovegetarin group were significantly greater than in the mixed-diet group. A similar trend (vegan greater than lactoovovegetarian greater than mixed) was detected in the plasma concentrations of biotin of adults and children but differences were not generally statistically significant. These observations provide evidence that the biotin nutritional status of vegans is not impaired.

  6. Immunolocalization of nerve growth factor (NGF) and its receptors (TrkA and p75LNGFR) in the reproductive organs of Shiba goats.

    Science.gov (United States)

    Ren, LongQuan; Medan, Mohamed S; Weng, Qiang; Jin, Wanzhu; Li, ChunMei; Watanabe, Gen; Taya, Kazuyoshi

    2005-06-01

    The objective of this study was to determine the immunolocalization of NGF and its receptors (TrkA and p75LNGFR) in the reproductive tract of the Japanese Shiba goats. Five adult goats were used in this study and sections of ovaries, uteri and oviducts were immunostained by the avidin-biotin-peroxidase complex method (ABC). The results showed that NGF and its receptors (TrkA and p75LNGFR) were expressed in granulosa cells, theca cells, interstitial cells and lutein cells in ovaries. Immunoreactions for NGF, TrkA and p75LNGFR were also detectable in epithelial cells and muscle cells of the ampulla and isthmus of the oviduct, and in epithelial cells and uterine glands of the uterus. These results strongly suggest autocrine and paracrine regulation of reproductive function by NGF in the reproductive tract of female Shiba goats.

  7. Analysis of the Protein Electrophoresis Patterns of Curvularia Lunata in Maize and Immunological Detection of the Pathogenic Culture

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Twelve isolates of Curvularia lunata and six related species from maize or other host plants are examined for the analysis of polyacrylamide gel electrophores is(SDS-PAGE)patterns of their soluble proteins. Differences exist significantly among different species and the same species.There is a special protein band for Curvularia at relative mobility value 0.177 (Rf 0.177)and a special protein band for C.lunata at Rf 0.225.Two kinds of primary antibodies to C.lunata are used to indirect ELISA and Avidin Biotinperoxidase Complex (ABC) immun oassay to evaluate their detecting sensitivity and specificity.The sensitivity of ABC immuno-assay is higher than indirectELISA.APAbs to C.lunata is special to C.lunata.

  8. Toxoplasmosis in wild turkeys: a case report and serologic survey.

    Science.gov (United States)

    Quist, C F; Dubey, J P; Luttrell, M P; Davidson, W R

    1995-04-01

    Toxoplasmosis was diagnosed in a free-ranging wild turkey (Meleagris gallopavo) from West Virginia (USA) in June 1993. Gross findings included emaciation, splenomegaly, multifocal necrotizing hepatitis and splenitis, and crusting dermatitis on the head and neck. Histologically, multifocal necrosis with mononuclear inflammation was present in kidney, liver, spleen, heart, lungs, and pancreas. Toxoplasma gondii was confirmed in sections of liver by avidin-biotin immunohistochemical analysis. Subsequently, a retrospective serosurvey of wild turkeys for T. gondii antibodies was conducted using turkey sera collected between 1984 and 1989. An antibody prevalence of 10% was detected in 130 birds from 21 locations in the southeastern United States. While wild turkeys in the Southeast have T. gondii antibodies, this is only the second natural case of fatal toxoplasmosis reported; it appears that wild turkeys infrequently develop clinical disease when infected with T. gondii.

  9. Novel compact architecture for high-resolution sensing with plasmonic gratings in conical mounting

    Science.gov (United States)

    Ruffato, Gianluca; Pasqualotto, Elisabetta; Sonato, Agnese; Zacco, Gabriele; Silvestri, Davide; Dettin, Monica; Morpurgo, Margherita; De Toni, Alessandro; Romanato, Filippo

    2013-05-01

    A novel compact architecture implementing grating-coupled surface plasmon resonance (GCSPR) based on polarization modulation in conical mounting is presented. In this system a plasmonic grating is azimuthally rotated in order to support the excitation of high-sensitivity surface plasmon polaritons (SPPs). At SPP resonance, a scan of the incident polarization is performed before and after the binding event and the phase term of the output trend is exploited as sensing parameter. The mechanical complexity of the SPR system is significantly reduced and a resolution down to 10-7 refractive index units is assured. In this work a numerical study of the polarization-based grating-coupled SPR technique is performed and analyzed with Chandezon's method. Therefore an experimental test on an assembled prototype is presented and applied to the detection of binding events on the grating surface (avidin/biotin reaction, DNA/PNA probes).

  10. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylaminotoluic Acid] (PATA for Efficient Biotinylation of Peptides and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Martina Jezowska

    2012-11-01

    Full Text Available Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylaminotoluic acid (PATA gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

  11. Esmond E. Snell--the pathfinder of B vitamins and cofactors.

    Science.gov (United States)

    Hayashi, Hideyuki; Tanase, Sumio; Yagi, Toshiharu

    2010-04-01

    Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

  12. Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis.

    Science.gov (United States)

    Mei, Jie; Tian, Yan-Ping; He, Wen; Xiao, Yu-Xiu; Wei, Juan; Feng, Yu-Qi

    2010-10-29

    The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin-biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.

  13. Direct biomolecule binding on nonfouling surfaces via newly discovered supramolecular self-assembly of lysozyme under physiological conditions.

    Science.gov (United States)

    Yang, Peng

    2012-08-01

    When lysozyme is dissolved in a neutral HEPES buffer solution (pH = 7.4) with 0.001-0.050 M TCEP added, a fast phase transition process occurs and the resulting novel fiber-like hierarchical supramolecular assemblies made by primary spherical-particle aggregation can function as a "superglue" that binds strongly and quickly onto non-fouling coatings. This binding is highly selective towards lysozyme, and excludes synthetic, chemical/physical activation/deactivation (blocking) steps. By using biotinylated lysozyme, such a phase transition quickly creates a perfect biotinylated surface on non-fouling surfaces for avidin binding, showing great potential for the development of low-cost and practical biochips.

  14. Gold Nanoparticles With Special Shapes: Controlled Synthesis, Surface-enhanced Raman Scattering, and The Application in Biodetection

    Directory of Open Access Journals (Sweden)

    Jinghong Li

    2007-12-01

    Full Text Available Specially shaped gold nanoparticles have intrigued considerable attention becausethey usually possess high-sensitivity surface-enhanced Raman scattering (SERS and thusresult in large advantages in trace biodetermination. In this article, starch-capped goldnanoparticles with hexagon and boot shapes were prepared through using a nontoxic andbiologically benign aqueous-phase synthetic route. Shape effects of gold nanoparticles onSERS properties were mainly investigated, and found that different-shaped goldnanoparticles possess different SERS properties. Especially, the boot-shaped nanoparticlescould induce more 100-fold SERS enhancements in sensitivity as compared with those fromgold nanospheres. The extremely strong SERS properties of gold nanoboots have beensuccessfully applied to the detection of avidin. The unique nanoboots with high-sensitivitySERS properties are also expected to find use in many other fields such as biolabel,bioassay, biodiagnosis, and even clinical diagnosis and therapy.

  15. Histochemical analysis of carbohydrate moieties and sugar-specific acceptors in the kidneys of the laboratory mouse and the golden spiny mouse (Acomys russatus).

    Science.gov (United States)

    Coppee, I; Gabius, H J; Danguy, A

    1993-10-01

    The aims of this work were to histochemically compare the pattern of lectin binding and endolectin expression in different portions of nephrons of two rodent species producing either normal hyperosmotic urine (the laboratory mouse) or highly concentrated urine (Acomys russatus, the golden spiny mouse). A panel of biotinylated lectins and neoglycoproteins and the avidin-biotin-peroxidase complex technique were used on Bouin's fixed, paraffin-embedded sections. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins and neoglycoproteins. Significant differences were also evident between comparable tubular segments in laboratory and golden spiny mouse kidneys. Whether the histochemical expression of sugar moieties of glycoconjugates as well as endolectins, thus both sides of presumed protein-carbohydrate interactions, may be correlated to the various glycoproteins which would include constituents of the glycocalyx and domains of a variety of transport enzymes deserves further studies.

  16. MicroRNA Sensor Based on Magnetic Beads and Enzymatic Probes

    Science.gov (United States)

    Zhang, Yue; Zhou, Dejian; He, Junhui

    2014-12-01

    MicroRNAs are associated with multiple cellular processes and diseases. Here, we designed a highly sensitive, magnetically retrievable biosensor using magnetic beads (MBs) as a model RNA sensor. The assay utilized two biotinylated probes, which were hybridized to the complementary target miRNA in a sandwich assay format. One of the biotinylated ends of the hybridization complex was immobilized onto the surface of a NeutrAvidin (NAV) coated MB and the other biotinylated end was conjugated to HRP via NAV-biotin interaction. The results were presented by colorimetric absorbance of the resorufin product from amplex red oxidation. We show that by combining the use of MBs as well as bio-specific immobilization, the sensitivity of miRNA detection is down to 100 pM. This model HRP-MBs system can be used for simple, rapid colorimetric quantification of low level DNA/RNA or other small molecules.

  17. Recovery of Lyme disease spirochetes from patients.

    Science.gov (United States)

    Steere, A. C.; Grodzicki, R. L.; Craft, J. E.; Shrestha, M.; Kornblatt, A. N.; Malawista, S. E.

    1984-01-01

    Since the summer of 1982, we have cultured patient specimens for Lyme disease spirochetes. Of 118 patients cultured, four specimens yielded spirochetes: two from blood, one from a skin biopsy specimen of erythema chronicum migrans (ECM), and one from cerebrospinal fluid. All four isolates appeared identical when examined with a monoclonal antibody. However, attempts to recover the spirochete from synovium or synovial fluid were unsuccessful. In addition, the organism could not be visualized in skin or synovial biopsy specimens using the avidin-biotin peroxidase complex detection system. Thus, the current yield in culturing spirochetes from patients is quite low, and it is not yet known whether the organism is still alive later in the disease when arthritis is present. PMID:6393606

  18. Metallothionein expression in placental tissue in Menkes' disease

    DEFF Research Database (Denmark)

    Hærslev, T.; Krag Jacobsen, G.; Horn, N.;

    1995-01-01

    . The avidin-biotin-complex (ABC)-technique was used. The copper content was measured by neutron activation analysis (NAA). In all placental tissue sections positive MT immunostaining appeared only in the trophoblast and only in proliferating cells. In placental tissue sections obtained from foetuses......Menkes' disease is a recessive X-linked disturbance of copper metabolism, resulting in accumulation of copper in several extra-hepatic tissues including the placenta. Metallothionein (MT) is a low-molecular weight protein with a high affinity for group II metal ions, such as copper. Its synthesis...... is induced by the presence of the ions. The aim of this study was to investigate the pattern of the MT immunoreactivity in placental tissue obtained from women at-risk of Menkes' disease in order to examine whether the MT occurrence and distribution may reflect the copper content. Placental tissue from six...

  19. Inhibition of hepatic fibrosis with artificial microRNA using ultrasound and cationic liposome-bearing microbubbles.

    Science.gov (United States)

    Yang, D; Gao, Y-H; Tan, K-B; Zuo, Z-X; Yang, W-X; Hua, X; Li, P-J; Zhang, Y; Wang, G

    2013-12-01

    We sought to investigate the antifibrotic effects of an artificial microRNA (miRNA) targeting connective tissue growth factor (CTGF) using the ultrasound-targeted cationic liposome-bearing microbubble destruction gene delivery system. Cationic liposomes were conjugated with microbubbles using a biotin-avidin system. Plasmids carrying the most effective artificial miRNA sequences were delivered by ultrasound-targeted cationic liposome-bearing microbubble destruction gene delivery system to rats with hepatic fibrosis. The results show that this method of gene delivery effectively transported the plasmids to the rat liver. The artificial miRNA reduced hepatic fibrosis pathological alterations as well as the protein and mRNA expressions of CTGF and transforming growth factor β1. Furthermore, the CTGF gene silencing decreased the levels of type I collagen and α-smooth muscle actin (Pliposome-bearing microbubble destruction may be an efficacious therapeutic method to ameliorate hepatic fibrosis.

  20. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    glycoprotein glycoforms to their receptors is inhibited by steroids. Testosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoform A to its receptor. Cortisone, aldosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoforms B......A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...... and C to their receptor. A difference in the cellular content of alpha 1-acid glycoprotein glycoforms and alpha 1-acid glycoprotein receptors separates the spermatocytes and the early spermatids from the late spermatids. The difference in receptor composition implies a difference in the effect...

  1. Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

    Science.gov (United States)

    Azam, Md Golam; Yamasuji, Mutsumi; Krawczyk, Tomasz; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2015-07-01

    We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe.

  2. Immunohistochemical screening for viral agents in cheetahs (Acinonyx jubatus) with myelopathy.

    Science.gov (United States)

    Shibly, S; Schmidt, P; Robert, N; Walzer, C; Url, A

    2006-10-21

    Numerous cases of acute-onset progressive ataxia, hindlimb paresis and paralysis of unknown aetiology occurred during 1993 to 2003 in cheetahs (Acinonyx jubatus) within the European Endangered Species Programme (eep). This study describes the immunohistochemical investigation of a possible viral aetiology of the "cheetah myelopathy". Antibodies to feline herpesvirus type 1, canine distemper virus, canine parvovirus and Borna disease virus were applied to formalin-fixed and paraffin-embedded brain and spinal cord sections from 25 affected cheetahs aged between three-and-a-half months and 13 years. Using the avidin-biotin complex technique, none of the antibodies gave positive immunosignals in either the brain or the spinal cord tissue.

  3. Programming A Molecular Relay for Ultrasensitive Biodetection through (129)Xe NMR.

    Science.gov (United States)

    Wang, Yanfei; Roose, Benjamin W; Philbin, John P; Doman, Jordan L; Dmochowski, Ivan J

    2016-01-26

    A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized (129) Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]-Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn(2+) in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive (129) Xe NMR spectroscopy.

  4. Monosynaptic connections between primary afferents and giant neurons in the turtle spinal dorsal horn

    DEFF Research Database (Denmark)

    Fernández, A; Radmilovich, M; Russo, R E

    1996-01-01

    This paper reports the occurrence of monosynaptic connections between dorsal root afferents and a distinct cell type-the giant neuron-deep in the dorsal horn of the turtle spinal cord. Light microscope studies combining Nissl stain and transganglionic HRP-labeling of the primary afferents have...... revealed the occurrence of axosomatic and axodendritic contacts between labeled boutons and giant neurons. The synaptic nature of these contacts has been confirmed by use of electron microscope procedures involving the partial three-dimensional reconstruction of identified giant neurons. Intracellular...... recording in spinal cord slices provided functional evidence indicating the monosynaptic connections between dorsal root afferents and giant neurons. The recorded neurons were morphologically identified by means of biocytin injection and with avidin conjugates. Electrical stimulation of the ipsilateral...

  5. Analysis of ultra-high sensitivity configuration in chip-integrated photonic crystal microcavity bio-sensors

    Energy Technology Data Exchange (ETDEWEB)

    Chakravarty, Swapnajit, E-mail: swapnajit.chakravarty@omegaoptics.com; Hosseini, Amir; Xu, Xiaochuan [Omega Optics, Inc., Austin, Texas 78757 (United States); Zhu, Liang; Zou, Yi [Department of Electrical and Computer Engineering, University of Texas at Austin, Austin, Texas 78758 (United States); Chen, Ray T., E-mail: raychen@uts.cc.utexas.edu [Omega Optics, Inc., Austin, Texas 78757 (United States); Department of Electrical and Computer Engineering, University of Texas at Austin, Austin, Texas 78758 (United States)

    2014-05-12

    We analyze the contributions of quality factor, fill fraction, and group index of chip-integrated resonance microcavity devices, to the detection limit for bulk chemical sensing and the minimum detectable biomolecule concentration in biosensing. We analyze the contributions from analyte absorbance, as well as from temperature and spectral noise. Slow light in two-dimensional photonic crystals provide opportunities for significant reduction of the detection limit below 1 × 10{sup −7} RIU (refractive index unit) which can enable highly sensitive sensors in diverse application areas. We demonstrate experimentally detected concentration of 1 fM (67 fg/ml) for the binding between biotin and avidin, the lowest reported till date.

  6. Affinity cytochemistry analysis of mast cells in skin lesions: a possible tool to assess the timing of lesions after death.

    Science.gov (United States)

    Bonelli, A; Bacci, S; Norelli, G A

    2003-12-01

    The histamine content in vital wounds is known to increase, with a zenith after 3 h, and then decrease until 24 h after wounding. We addressed whether this biochemical alteration has a morphological counterpart. Since the main source of skin histamine are mast cells, the distribution and number of these cells was assessed upon labeling with fluorescent avidin and with antibodies to the mast cell specific enzymes, chymase and tryptase. Analyses were performed on skin from 15 healthy controls (from surgical biopsies), from 15 post-mortem lesions and 75 vital lesions, obtained at autopsy from subjects who had survived from a few seconds to 24 h. The number of mast cells per unit area of section surface increased progressively with survival time, up to a maximum in subjects who survived 1-3 h ( pdeath ( paffinity cytochemistry can help to discriminate vital from post-mortem lesions and to estimate survival time after lesions.

  7. Advantage of affinity histochemistry combined with histology to investigate death causes: indications from sample cases.

    Science.gov (United States)

    Bacci, Stefano; DeFraia, Beatrice; Romagnoli, Paolo; Bonelli, Aurelio

    2011-11-01

    Mast cell histochemistry has been proposed in addition to classic histological methods to estimate the course of traumatic events before and after death. We have addressed the utility of this approach on nine victims of different types of trauma. Sections of wounded skin were stained with hematoxylin and eosin and with fluorescent avidin to tag mast cells. Mast cell numbers were evaluated by both direct and digitalized counts. Intact skin was used as control. The results on mast cells implemented the findings upon hematoxylin and eosin stain and helped to put the wounds and death in chronological sequence. Digitalized morphometry allowed to reduce intra- and inter-observer variation. We conclude that combined histological and histochemical analyses can be of practical use in forensic pathology, that a preliminary setting of the reference values is needed for each laboratory, and that image analysis can be of help for the quantification of the results.

  8. THE EFFECT OF MICROWAVE AND BANDAGING TREATMENT ON SKIN IMMUNOLOGICAL CELLS IN CHRONIC LIMB LYMPHEDEMA

    Institute of Scientific and Technical Information of China (English)

    曹卫刚; 张涤生; 干季良

    2000-01-01

    Objective To clarify the characteristics of immunological reactions in skin tissues of non - filarial lymphedema patients with or without skin bacterial infection. Methods Avidin-biotin peroxidase (ABC)immunohistochemical method was used to examine the local skin tissue infiltrating inflammatory cells in 16chronic limb lymphedema patients before and after two courses of microwave and bandaging treatment. Results There was a significant increase of T lymphocyte infiltration in lymphedematous skin tissues; after two courses of microwave treatment, T lymphocyte infiltration was greatly resolved whereas the number of macrophages, which can lyse the stagnant proteins in lymphedematous tissues through proteolysis increased. Conclusion Microwave and bandaging treatment can promote regression of extremity edema by reducing chronic inflammation and enhancing the stagnant protein lysis capability in lymphedematous skin tissues.

  9. Immunoradiometric assay for carcinoembryonic antigen using avidin—biotin separation technique

    Institute of Scientific and Technical Information of China (English)

    SONGShiping; TANGGuozhong; 等

    1999-01-01

    A sensitive,specific,noncompetitive,sandwich-type radioimmunoassay for carcinoembryonic antigen(CEA) has been developed in our laboratory,which can be performed conveniently.The assay involves two monoclonal antibodies,selected for high affinity and specificity and also for reaction against antigenic sites on CEA that are distal from each other.One of these antibodies was labeled with 125I and the other was conjugated covalently to biotin.Polystyrene tubes were conjugated covalently to avidin.These tubes represent a rapid,simple method for separating the CEA-bound antibody from the free antibody.The biotin-antibody-CEA-125I-labeled antibody complexes bind to the tubes and CEA concentration is driectly related to counts per minute.This assay can detect the CEA at a concentrastion of 0.22μg/L in serum.

  10. New one step functionalization of polycrystalline diamond films using amine derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Agnes, Charles; Ruffinatto, Sebastien; Delbarre, Emma; Roget, Andre; Arnault, Jean-Charles; Omnes, Franck; Mailley, Pascal, E-mail: pascal.mailley@cea.fr, E-mail: franck.omnes@grenoble.cnrs.fr

    2010-11-15

    Diamond received tremendous interest for analytical sciences due to its intrinsic properties. However, the analytical perception of chemical environment requires surface functionalization that brings selectivity to the detection event. Thereby, many works focused on diamond modification using chemical or biochemical entities. We proposed here, a new and straightforward methodology for diamond (bio)functionalization. This method involves the chemical reaction between (bio)chemical entities presenting a primary amine moiety, used as grafting site, and hydrogenated diamond surface. This reaction allows in one step to modify diamond surface whatever its doping level and its crystalline quality. The effectiveness of this new method is exposed here through the grafting of one redox species, ferrocene, and of one biochemical, biotin. The impacts of both functionalization duration and pH are investigated and the robustness of the formed bond is demonstrated owing to biotin-avidin coupling.

  11. Methods to immobilize GPCR on the surface of SPR sensors.

    Science.gov (United States)

    Martínez-Muñoz, Laura; Barroso, Rubén; Paredes, Anabel Guedán; Mellado, Mario; Rodríguez-Frade, José Miguel

    2015-01-01

    The G protein-coupled receptors (GPCRs) form one of the largest membrane receptor families. The nature of the ligands that interact with these receptors is highly diverse; they include light, peptides and hormones, neurotransmitters, and small molecular weight compounds. The GPCRs are involved in a wide variety of physiological processes and thus hold considerable therapeutic potential.GPCR function is usually determined in cell-based assays, whose complexity nonetheless limits their use. The use of alternative, cell-free assays is hampered by the difficulties in purifying these seven-transmembrane domain receptors without altering their functional properties. Several methods have been proposed to immobilize GPCR on biosensor surfaces which use antibodies or avidin-/biotin-based capture procedures, alone or with reconstitution of the GPCR physiological microenvironment. Here we propose a method for GPCR immobilization in their native membrane microenvironment that requires no manipulation of the target receptor and maintains the many conformations GPCR can adopt in the cell membrane.

  12. Metallothionein:An overview

    Institute of Scientific and Technical Information of China (English)

    N Thirumoorthy; KT Manisenthil Kumar; A Shyam Sundar; L Panayappan; Malay Chatterjee

    2007-01-01

    Metallothioneins(MTs)were discovered in 1957 by Margoshes and Vallee and identified as low-molecular weight and sulphydryl rich proteins.It is not surprising that most mammalian tissues contain age related basal levels of MTs since they are involved in metalloregulatory processes that include cell growth and multiplication.In an effort to understand the biology of this intriguing tumor,various biomarkers such as oncogenes,p53 tumor suppressor gene,waf 1 protein,proliferating cell nuclear antigen,telomerase,microsatellite markers and cytogenetic changes have been examined.One biomarker which has recently shown to be expressed in various human tumors but still less reported in carcinoma is MT.Immunohistochemical detection of MT proteins in cold acetone-fixed paraffin embedded liver sections was performed by the streptavidin-avidin-biotin immunoperoxidase complex method.

  13. Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei.

    Science.gov (United States)

    Johnson, M D; Fresco, J R

    1999-07-01

    A methodology has been developed for binding oligodeoxyribonucleotide 'third strands' to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 alpha-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.

  14. 肝癌靶向液态氟碳脂质微球造影剂的体外寻靶实验研究%Targeting study in vitro of human hepatocellular carcinoma-targeted perfluorocarbon lipid particles

    Institute of Scientific and Technical Information of China (English)

    陈松; 王志刚; 康娟; 李攀; 郑元义; 伍星; 杨春江; 许川山

    2008-01-01

    目的 制备液态氟碳脂质微球造影剂并研究其体外基本特性,再引入抗人肝癌细胞单克隆抗体,采用生物素亲和素法,进行体外靶向结合的研究.方法 采用旋转蒸发法和高压均质法制备一种纳米液态氟碳脂质微球造影剂,观察其外观、形态、大小及分布情况,计算其浓度,并测定其粒径、电位;制备生物素的抗人肝癌细胞单克隆抗体HAb18,用HABA/Avidin试剂盒检测抗体的生物素化程度;制备NBD标记的生物素化的微球,采用生物素亲和素法研究其体外靶向作用.结果 所制备的液态氟碳脂质微球造影剂外观呈乳白色的混悬液,镜下观察,微球形态圆整,大小均一,性质稳定,平均粒径171.9 nm;采用生物素亲和素法制备的NBD标记的生物素化的微球可靶向结合到靶细胞上.结论 成功制备出稳定的纳米液态氟碳脂质微球造影剂,并引入抗人肝癌细胞单克隆抗体,通过生物素亲和素系统可将其特异结合到靶细胞上.%Objective To develop perfluorocarbon lipid particles and investigate their basic properties,and target them to human hepatocellular carcinoma cells in vitro by hepatoma monocolonal antibody HAb18 with avidin-biotin interaction.Methods Rotary evaporation and high pressure homogen were used to prepare perfluorocarbon lipid particles, and the appearance and distribution of them were investigated by microscope and electron microscope, the concentration and the size and electric potential were detected.The biotinylated monoclonal antibody HAbl8 was prepared, then the biotinylated degree of the antibody was determined.The biotinylated perfluoroearbon lipid particles labelled with NBD were prepared and targeted to human hepatocellular carcinoma cells in vitro with avidin-biotin interaction.Results These perfluorocarbon lipid nanoparticles were uniform and stable,and the mean diameter of them was 171.9 nm.Hepatocellular carcinoma cells were surrounded by the

  15. Immunohistochemical localization of glutamate transporter EAAC1 in the brainstem of adult rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fu-xing; LIU Tao; ZHAO Jing-wei; LI Jin-lian; DONG Yu-lin; LI Ji-shuo

    2001-01-01

    Objective: To observe the distribution of EAAC1, a subtype of glutamate transporters, in the brainstem of adult rat. Methods: Immunocytochemical staining with avidin-biotin complex (ABC) method was employed. Results:EAAC1 was widely distributed throughout the brainstem. In many regions, the EAAC1-like immunoreactivity was primarily distributed in the neuropil. Cell body staining was observed in the prepositus hypoglossal nucleus, external cortex of the inferior colliculus, red nucleus, substantia nigra, mesencephalic raphe nuclei, ventral tegmental nucleus, superior olivary complex, nucleus of the trapezoid body, cochlear nucleus, sensory trigeminal complex, Barrington's nucleus,trigeminal motor nucleus, parabrachial nuclei, dorsal nucleus of vagus, hypoglossal nucleus, locus coeruleus, lateral and superior vestibular nuclei, lateral paragigantocellular nucleus and dorsal paragigantocellular nucleus. Conclusion: Glutamate transporter EAAC 1 is widely distributed throughout the brainstem of adult rat, which may play an important role in excitatory activities of the neurons induced by glutamate.

  16. Phenotypic detection ofT lymphocyte subsets in Bali cattle lymph nodes with malignant catarrhal fever by immunohistochemical techniques

    Directory of Open Access Journals (Sweden)

    Rini Damayanti

    1996-06-01

    Full Text Available A study was carried out to detect the phenotype ofT cell subsets in six Bali cattle affected with malignant catarrhal fever (MCF. This was performed by means of immunohistochemical technique using avidin-biotin-peroxidase complex methods. Seven monoclonal antibodies against lymphocyte surface antigen were used to detect T cell subsets ie . CD1, CD2, CD4, CD5, CD8, CD45, and WC 1. The results showed that the subsets were all detected either in the MCFor non-MCF infected Bali cattle lymph nodes. However, CD8 was more predominantly occupied in the MCF Bali cattle . This indicated that CD8 was a cytotouic Tlymphocytes and acted as potential mediators for immunopathological process in MCF.

  17. Slotted photonic crystal cavities with integrated microfluidics for biosensing applications.

    Science.gov (United States)

    Scullion, M G; Di Falco, A; Krauss, T F

    2011-09-15

    We demonstrate the detection of dissolved avidin concentrations as low as 15 nM or 1 μg/ml using functionalized slotted photonic crystal cavities with integrated microfluidics. With a cavity sensing surface area of approximately 2.2 μm(2), we are able to detect surface mass densities of order 60 pg/mm(2) corresponding to a bound mass of approximately 100 ag. The ultra-compact size of the sensors makes them attractive for lab-on-a-chip applications where high densities of independent sensing elements are desired within a small area. The high sensitivity over an extremely small area is due to the strong modal overlap with the analyte enabled by the slotted waveguide cavity geometry that we employ. This strong overlap results in larger shifts in the cavity peak wavelength when compared to competing approaches.

  18. Amylin Detection with a Miniature Optical-Fiber Based Sensor

    Science.gov (United States)

    Liu, Zhaowen; Ann, Matsko; Hughes, Adam; Reeves, Mark

    We present results of a biosensor based on shifts in the localized surface plasmon resonance of gold nanoparticles self-assembled on the end of an optical fiber. This system allows for detection of protein expression in low sensing volumes and for scanning in cell cultures and tissue samples. Positive and negative controls were done using biotin/avidin and the BSA/Anti-BSA system. These demonstrate that detection is specific and sensitive to nanomolar levels. Sensing of amylin, an important protein for pancreatic function, was performed with polyclonal and monoclonal antibodies. The measured data demonstrates the difference in sensitivity to the two types of antibodies, and titration experiments establish the sensitivity of the sensor. Further experiments demonstrate that the sensor can be regenerated and then reused.

  19. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  20. Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

    Science.gov (United States)

    Paba, Jaime; Ricart, Carlos A O; Fontes, Wagner; Santana, Jaime M; Teixeira, Antonio R L; Marchese, Jason; Williamson, Brian; Hunt, Tony; Karger, Barry L; Sousa, Marcelo V

    2004-01-01

    Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

  1. Programming A Molecular Relay for Ultrasensitive Biodetection through 129 Xe NMR

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yanfei [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Roose, Benjamin W. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Philbin, John P. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Doman, Jordan L. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA; Dmochowski, Ivan J. [Department of Chemistry, University of Pennsylvania, 231 South 34th Street Philadelphia PA 19104-6323 USA

    2015-12-21

    We reported a supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized 129Xe NMR. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. Moreover, the protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn2+ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive 129Xe NMR spectroscopy.

  2. Multilayers Assembly of DNA Probe for Biosensor

    Institute of Scientific and Technical Information of China (English)

    谢文章; 路英杰; 隋森芳

    2002-01-01

    Surface plasmon resonance (SPR) was a sensitive method to study molecular interactions. Based on the specific binding, this paper presented the molecular assembly of protein-nucleic acid multilayers on the surface of a gold film. The first layer was a biotin-lipid (B-DMPE/DMPE) containing a monolayer prepared using the Langmuir-Blodgett (LB) technique. The second and third layers were avidin and DNA labeled biotin, respectively. The fourth layer was anti-DNA antibody extracted from the serum of patients with systemic lupus erythematosus (SLE). These interactions provide stability in the multilayer films of the complexes. The multilayer formation process was detected by SPR spectroscopy. The results show that the chip-based sensor system can be used for functional characterization of protein-protein and protein-DNA interactions.

  3. Comparative analysis of toxin detection in biological and enviromental samples

    Science.gov (United States)

    Ogert, Robert A.; Burans, James; O'Brien, Tom; Ligler, Frances S.

    1994-03-01

    The basic recognition schemes underlying the principles of standard enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) protocols are increasingly being adapted for use with new detection devices. A direct comparison was made using a fiber optic biosensor that employs evanescent wave detection and an ELISA using avidin-biotin. The assays were developed for the detection of Ricinus communis agglutinin II, also known as ricin or RCA60. Detection limits between the two methods were comparable for ricin in phosphate buffered saline (PBS), however results in complex samples differed slightly. In PBS, sensitivity for ricin was 1 ng/ml using the fiber optic device and 500 pg/ml using the ELISA. The fiber optic sensor could not detect ricin directly in urine or serum spiked with 5 ng/ml ricin, however, the ELISA showed detection but at reduced levels to the PBS control.

  4. Spectroscopy on the wing: naturally inspired SERS substrates for biochemical analysis.

    Science.gov (United States)

    Garrett, Natalie L; Vukusic, Peter; Ogrin, Feodor; Sirotkin, Evgeny; Winlove, C Peter; Moger, Julian

    2009-03-01

    We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface-enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface-enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme-linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large-scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS-based bioassays.

  5. Stabilization of human prostatic acid phosphatase by coupling with chondroitin sulfate.

    Science.gov (United States)

    Luchter-Wasylewska, E; Dulińska, J; Ostrowski, W S; Torchilin, V P; Trubetskoy, V S

    1991-02-01

    Human prostatic acid phosphatase (PAP) (EC 3.1.3.2) was covalently linked to chondroitin sulfate A from whale cartilage. In order to bind the protein amino groups with the preactivated carboxyl groups of chondroitin sulfate, 1-ethyl-3-(3'-dimethylaminepropyl)carbodiimide and N-hydroxysulfosuccinimide were used as coupling agents. The product was soluble and enzymatically active. The activity was on average 25% higher than that of the free enzyme. The product was heterogeneous in respect to charge and Mr (50-1500) kDa, as determined by chromatography on Sephacryl S 300 and polyacrylamide gel electrophoresis. The resulting polymers contained covalently bound chondroitin sulfate, as shown by the biotin-avidin test. The modified enzyme is more resistant against various denaturing agents, e.g., urea, ethanol, and heat. Thus covalent modification of PAP by cross-linking to chondroitin sulfate could be the preferred method for stabilization of its biological activity.

  6. Principles of connectivity among morphologically defined cell types in adult neocortex.

    Science.gov (United States)

    Jiang, Xiaolong; Shen, Shan; Cadwell, Cathryn R; Berens, Philipp; Sinz, Fabian; Ecker, Alexander S; Patel, Saumil; Tolias, Andreas S

    2015-11-27

    Since the work of Ramón y Cajal in the late 19th and early 20th centuries, neuroscientists have speculated that a complete understanding of neuronal cell types and their connections is key to explaining complex brain functions. However, a complete census of the constituent cell types and their wiring diagram in mature neocortex remains elusive. By combining octuple whole-cell recordings with an optimized avidin-biotin-peroxidase staining technique, we carried out a morphological and electrophysiological census of neuronal types in layers 1, 2/3, and 5 of mature neocortex and mapped the connectivity between more than 11,000 pairs of identified neurons. We categorized 15 types of interneurons, and each exhibited a characteristic pattern of connectivity with other interneuron types and pyramidal cells. The essential connectivity structure of the neocortical microcircuit could be captured by only a few connectivity motifs.

  7. Cystathionase: high-performance liquid chromatography. Molecular cloning in lambda gt11. Nonradioactive immunodetection of fusion protein.

    Science.gov (United States)

    Shuster, A M; Kvashuk, O A; Chumakov, I; Prassolov, V S; Gabibov, A G

    1989-04-01

    A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.

  8. Serial analysis of gene expression in turkey sperm storage tubules in the presence and absence of resident sperm.

    Science.gov (United States)

    Long, Ezhou L; Sonstegard, Tad S; Long, Julie A; Van Tassell, Curtis P; Zuelke, Kurt A

    2003-08-01

    Turkey sperm lose viability within 8-18 h when stored as liquid semen using current methods and extenders. In contrast, turkey hens maintain viable, fertile sperm in their sperm storage tubules (SST) for 45 or more days following a single insemination. Our long-term objectives are to identify and characterize differentially expressed genes that may underlie this prolonged sperm storage and then use this information to develop improved methods for storing liquid turkey semen. We employed serial analysis of gene expression (SAGE) to compare gene expression patterns in turkey SST recovered from hens after artificial insemination (AI) with extended semen (sperm AI) or extender alone (control AI). We constructed two separate SAGE libraries with SST RNA obtained from sperm and control AI hens. We used these libraries to generate 95,325 ten-base pair SAGE tags. These 95,325 tags represented 27,430 unique genes. The sperm and control AI libraries contained 47,663 and 47,662 tags representing 18,030 and 19,101 putative unique transcripts, respectively. Approximately 1% of these putative unique genes were differentially expressed (P<0.05) between treatments. Tentative annotations were ascribed to the SAGE tag nucleotide sequences by comparing them against publicly available SAGE tag and cDNA sequence databases. Based on its SAGE tag nucleotide sequence, we cloned a partial turkey avidin cDNA and confirmed its up-regulation in the sperm AI SST. The bioinformatics and experimental procedures employed to clone turkey avidin and confirm its differential expression represent a useful paradigm for analyzing SAGE tag data from relatively uncharacterized model systems.

  9. Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

    Science.gov (United States)

    Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

    2014-11-01

    Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces.

  10. Chemical functionalization of surfaces for building three-dimensional engineered biosensors

    Science.gov (United States)

    Marques, Marco E.; Mansur, Alexandra A. P.; Mansur, Herman S.

    2013-06-01

    This study presents a new approach for developing biosensors based on enzymatic systems with designed three-dimensional structures. Silica glass slides were chemically functionalized at surfaces by reacting with organosilanes, 3-mercaptopropyltriethoxysilane (MPTES), and 3-aminopropyltriethoxysilane (APTES), using sol-gel process at room temperature. The functionalization of the supports was characterized by contact angle measurements and FTIR spectroscopy. The first enzyme layer was covalently immobilized to the support by a bi-functional linker (glutaraldehyde). The second enzyme layer was deposited using the protein conjugation method based on the high affinity "avidin-biotin" interactions. Each enzyme was biotinylated before being added to the nanostructured system and avidin was used as the binder between consecutive enzyme layers. The biochemical response was assayed at all stages to certify that the enzymatic bioactivity was retained throughout the entire layer-by-layer (LBL) process. The model of building 3D-enzymatic systems was evaluated using the enzymatic structure with glucose oxidase (GOx) and horseradish peroxidase (HRP). It was verified that the amino-modified support presented the highest bioactivity response compared to the other chemical functionalities. Moreover, the bienzyme nanostructure demonstrated relevant biochemical activity upon injecting the glucose substrate into the system. Finally, as a proof of concept, the bienzyme systems were assayed using real samples of regular and sugar-free soft drinks where they effectively behaved as structured biosensor for glucose with the built-in 3D hybrid architecture. Based on the results, it can be foreseen the development of promising new nanomaterials for several analytical applications such as monitoring the quality of food and beverages for nutrition purposes.

  11. A palm-size μNMR relaxometer using a digital microfluidic device and a semiconductor transceiver for chemical/biological diagnosis.

    Science.gov (United States)

    Lei, Ka-Meng; Mak, Pui-In; Law, Man-Kay; Martins, Rui P

    2015-08-07

    Herein, we describe a micro-nuclear magnetic resonance (μNMR) relaxometer miniaturized to palm-size and electronically automated for multi-step and multi-sample chemical/biological diagnosis. The co-integration of microfluidic and microelectronic technologies enables an association between the droplet managements and μNMR assays inside a portable sub-Tesla magnet (1.2 kg, 0.46 Tesla). Targets in unprocessed biological samples, captured by specific probe-decorated magnetic nanoparticles (NPs), can be sequentially quantified by their spin-spin relaxation time (T2) via multiplexed μNMR screening. Distinct droplet samples are operated by a digital microfluidic device that electronically manages the electrowetting-on-dielectric effects over an electrode array. Each electrode (3.5 × 3.5 mm(2)) is scanned with capacitive sensing to locate the distinct droplet samples in real time. A cross-domain-optimized butterfly-coil-input semiconductor transceiver transduces between magnetic and electrical signals to/from a sub-10 μL droplet sample for high-sensitivity μNMR screening. A temperature logger senses the ambient temperature (0 to 40 °C) and a backend processor calibrates the working frequency for the transmitter to precisely excite the protons. In our experiments, the μNMR relaxometer quantifies avidin using biotinylated Iron NPs (Φ: 30 nm, [Fe]: 0.5 mM) with a sensitivity of 0.2 μM. Auto-handling and identification of two targets (avidin and water) are demonstrated and completed within 2.2 min. This μNMR relaxometer holds promise for combinatorial chemical/biological diagnostic protocols using closed-loop electronic automation.

  12. Influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein in primary hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Qinghua Wu; Qiang Fu; Xinhua Wang; Jianhua Zhao; Liwei Liu; Shirong Tang

    2008-01-01

    BACKGROUND: The pharmacological action of opioid drugs is related to signal transduction of inhibitory guanine nucleotide binding protein.OBJECTIVE: To quantitatively and qualitatively analyze the influence of morphine on levels of type Ⅱ inhibitory guanine nucleotide binding protein (Gi2 protein) in primary cultured hippocampal neurons at different time points.DESIGN, TIME AND SETTING: A randomized controlled study, which was performed at the Department of Neurobiology, Changzheng Hospital, Second Military Medical University of Chinese PLA between September 2002 and March 2004.MATERIALS: Cerebral hippocampal neurons were obtained from newborn SD rats at 1-2 days of age. Biotin-antibody Ⅱ-avidin fluorescein isothiocyanate (Avidin-FITC) was purchased from Sigma Company (USA) and the Gi2 protein polyclonal antibody from Santa Cruz Biochemistry Company (USA).METHODS: Seven days after culture, mature hippocampal neurons were randomly divided into six groups: 4-, 8-, 16-, 24-, and 48-hour morphine groups, and a blank control group. Neurons in the morphine groups Received morphine (10μmol/L), which could cause alterations of G-protein mRNA and cAMP expression in the prefrontal cortex. Neurons in the blank control group were given the same volume of saline.MAIN OUTCOME MEASURES: Gi2 protein levels were detected by an immunofluorescence technique, and were analyzed by the image analytic system with the use of green fluorescence intensity.RESULTS: Gi2 protein levels in hippocampal neurons gradually decreased in the 4-, 8-, 16-, 24-, and 48-hour morphine groups. In particular, Gi2 protein levels in the 16-, 24-, and 48-hour morphine groups were significantly lower than that in the blank control group (P<0.05-0.01).CONCLUSION: Morphine may decrease Gi2 protein level in primary hippocampal neurons, and the decreasing trend is positively related to morphine-induced time.

  13. Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

    Science.gov (United States)

    Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

    2011-11-15

    Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

  14. 以短肽K237为配体的靶向脂质体超声造影剂构建方法%Preparation of liposome ultrasonic contrast agent with ligand peptide K237

    Institute of Scientific and Technical Information of China (English)

    何洁; 杨莉; 李颖嘉; 孙学刚; 刁建新; 李传刚; 宾建平; 龚渭冰

    2011-01-01

    目的 探讨以与血管内皮生长因子(VEGF)的主要受体KDR特异性结合的短肽K237(P)为配体制备靶向脂质体超声造影剂(P-Bio-Av-Bio-Mbs)的方法. 方法 采用生物素-亲和素桥接法构建P-Bio-Av-Bio-Mbs,流式细胞术筛选最佳配体适配剂量,光镜及荧光显微镜观察靶向微泡与KDR强阳性表达的人大肠癌LOVO细胞结合情况,计算花环形成率.分别以5、50、99 ml/h速率水流冲刷,光镜观察靶向微泡与LOVO细胞结合情况. 结果 不同亲和素剂量下(0、2、6、10、30 μg),微泡表面亲和素携带率差异有统计学意义( P<0.05).Mavidin=6 μg时,携带率增长达平台期;不同短肽剂量下(0、30、40、50、60、70、100 μg),微泡表面短肽携带率差异有统计学意义( P<0.05),当MK237=50 μg时,微泡表面短肽携带率增长达平台期.光镜下KDR强阳性表达的LOVO细胞周围花环形成率高达90.52%,荧光显微镜下微泡外壳发出明亮绿色荧光.随冲刷速度增加,靶细胞周围黏附的靶向微泡减少,在99 ml/h冲刷速度下,靶细胞周围仍可见花环结构. 结论 通过生物素-亲和素桥连作用,短肽K237被有效装配在P-Bio-Av-Bio-Mbs表面,体外具有靶向特异性及一定稳定性.流式细胞术是筛选靶向微泡配体适配剂量的可靠方法.%Objective To assess preparation method of a new kind of targeted liposome ultrasonic contrast agent with small peptide K237 as the ligand which can combine specifically with KDR as the main receptor of VEGF. Methods Targeted bubbles (P-Bio-Ay-Bio-Mbs) were formed through "biotin-avidin" bridge grafting. Flow cytometry screening was performed to explore the best dose of the ligands, then targeted-bubbles were incubated respectively with LOVO and LS174T which were KDR expressed in different cells. Meanwhile, rosette formation rate was calculated. Results The bubble surface's avidin-carrying rates were significant different (P<0. 05) on different dosages of avidin(0, 2

  15. 聚(甲基丙烯酸-2-羟乙酯)改性金表面用于构建生物检测基材%POLY ( 2-HYDROXYETHYL METHACRYLATE) MODIFIED GOLD SURFACES AS BIO-DETECTION SUBSTRATES

    Institute of Scientific and Technical Information of China (English)

    李丹; 王莎莎; 郑军; 陈红

    2011-01-01

    采用电子活化再生原子转移自由基聚合(AGET ATRP)的方法将聚(甲基丙烯酸-2-羟乙酯)( PHEMA)接枝在金表面,对经修饰的金表面的生物惰性做了系统的研究,并利用PHEMA的羟基末端固定生物素(biotin)分子,以biotin对抗生物素蛋白(avidin)的识别为模型,研究了不同厚度的PHEMA对结合avidin的影响,以及该表面作为生物检测基材的可行性.生物惰性研究表明,PHEMA修饰的金表面不但能够有效的排斥纤维蛋白原(Fg)、人血清白蛋白(HSA)和溶菌酶(Lys)的非特异性吸附,还能够抑制3种细胞(L02、L929和EC)的黏附,是一种良好的抗污表面.通过控制聚合时间制备了不同厚度的PHEMA-biotin修饰的表面,同位素125I标记HSA吸附结果表明这几种表面均能够有效排斥非特异性蛋白质吸附,特异性FITC-avidin吸附结果表明,厚度较小时( 16 nm)由于荧光淬灭而难以检测到荧光信号,厚度在16 nm和49 nm之间,荧光信号随厚度增加而增强,通过比较信噪比,认为厚度在49 nm以上时比较理想.该表面在应用于QCM与荧光检测中均表现出良好的检测性能.%Poly(2-hydroxyethyl methacrylate) (PHEMA) was grafted from initiator-immobilized gold surfaces via activator generated by electron transfer atom transfer radical polymerization ( AGET ATRP) to repel nonspecific protein adsorption and immobilize biotin for specific avidin binding. The surface polymerization of HEMA was demonstrated by FTIR and contact angle measurements. The bioinertness of the PHEMA-modified surfaces was systematically studied.125 I-labeled protein (Fg,HAS and Lys) adsorption experiments showed that modification with PHEMA could decrease nonspecific protein adsorption by 82% at least. Cell adhesion results indicated that the Au-PHEM A surface could, to a great extent, resist the adhesion of L929., L02 and endothelium cell (EC). L929 showed slight adhesion while L02 and EC could not adhere at all. PHEMA

  16. Assembly of Colloidal Materials Using Bioadhesive Interactions

    Science.gov (United States)

    Hammer, Daniel A.; Hiddessen, Amy L.; Tohver, Valeria; Crocker, John C.; Weitz, David A.

    2002-01-01

    We have pursued the use of biological crosslinking molecules of several types to make colloidal materials at relatively low volume fraction of colloidal particles. The objective is to make binary alloys of colloidal particles, made of two different colloidal particles coated with complementary biological lock-and-key binding molecules, which assemble due to the biological specificity. The long-term goal is to use low affinity lock-and-key biological interactions, so that the can anneal to form crystalline states. We have used a variety of different surface chemistries in order to make colloidal materials. Our first system involved using selectin-carbohydrate (sialyl-Lewis) interactions; this chemistry is derived from immune system. This chemical interaction is of relatively low affinity, with timescales for dissociation of several seconds. Furthermore, the adhesion mediated by these molecules can be reversed by the chelation of calcium atoms; thus assembled structures can be disassembled reversibly. Our second system employed avidin-biotin chemistry. This well-studied system is of high affinity, and is generally irreversible on a laboratory time-scale. Thus, we would expect selectin-carbohydrate interactions at high molecular density and avidin-biotin interactions to give kinetically-trapped structures; however, at low densities, we would expect significant differences in the structure and dynamics of the two materials, owing to their very different release rates. We have also begun to use a third chemistry - DNA hybridization. By attaching single stranded DNA oligonucleotide chains to beads, we can drive the assembly of colloidal materials by hybridization of complementary DNA chains. It is well known that DNA adenosine-thymine (A-T) and guanine-cytosine (G-C) bases hybridize pairwise with a Gibbs free energy change of 1.7 kcal/mol per base; thus, the energy of the assembly can be modulated by altering the number of complementary bases in the DNA chains. Using

  17. Ion channel mimetic membranes and silica nanotubes prepared from porous aluminum oxide templates

    Science.gov (United States)

    Mitchell, David Tanner

    Chapter 1 provides background information on the template synthesis of nanomaterials. The template synthesis method is examined with special attention to the use of membranes containing monodisperse cylindrical pores as templates. Several examples of the utility of template-synthesized nanomaterials are given. The production of one type of template membrane, nanopore alumina, is reviewed. Reviews of sol-gel and silane chemistry are also provided. In Chapter 2, a sol-gel template synthesis process is used to produce silica nanotubes within the pores of alumina templates. The nanotubes can be modified using a variety of chemistries, typically via a silanization process. Because the nanotubes are formed in a template, the interior and exterior surface can be modified independently. Modified nanotubes can be used for drug detoxification or as extractants for the removal of metal ions. The nanotube surface can also be biotinylated, which causes binding to avidinated surfaces. Composite microtubes of silica and various polymers are also prepared. Additionally, Au nanowires are shown to assemble with colloidal Au particles using dithiols as linkers. Chapter 3 describes the attachment of proteins onto template-synthesized silica nanotubes. The proteins are covalently linked via an aldehyde silane bridge that binds to pendant primary amino moieties on the protein. Protein-modified nanotubes function as highly specific extractants. Avidin-modified nanotubes extract biotin-coated Au nanoparticles from solution with high extraction efficiency. Immunoprotein-modified nanotubes extract the corresponding antibody from solution with high specificity. Antibody-modified nanotubes extract one enantiomer from a racemic mix. Enzymes, including drug detoxification enzymes, were also attached to the nanotubes and were shown to retain their catalytic activity. Immunoproteins on the outside of nanotubes can be used to direct nanotube binding, creating specific labeling agents. Chapter 4

  18. 基于核酸适配子识别的荧光法检测可卡因%Aptamer recognition based fluorescent detection of cocaine

    Institute of Scientific and Technical Information of China (English)

    丁晓莹; 李双; 王周平

    2011-01-01

    基于荧光标记和核酸适配子识别可卡因,建立了简单、灵敏的可卡因新型荧光分析法.在微孔板表面组装亲和素-生物素化可卡因适配子-FAM标记可卡因适配子互补短链复合物,根据加入可卡因前后荧光强度的变化来定量可卡因.实验考察了微孔板包被亲和素浓度、生物素标记适配子用量、FAM标记可卡因适配子互补短链用量、反应温度、反应时间等因素对体系组装和可卡因识别的影响.结果显示,实验选定条件下,可卡因浓度在5~1600 nmol/L范围内与荧光强度呈线性关系,检出限为3.0 nmol/L (3δ);尿样加标回收率为89.5%~98.6%,相对标准偏差小于5%.%A simple, sensitive, and rapid fluorescent analytical method was developed based upon fluorescent labeling and aptamer recognition. The complex of avidin-biotinylated eocaine aptamer-FAM labeled partcomplementary strand of cocaine aptamer was assembled onto the microplate wells. Cocaine was quantified according to the change of the fluorescent intensity induced by the dissociated FAM labeled part-complementary strand of cocaine aptamer. Factors including the amount of avidin coated onto microplate wells, biotinylated cocaine aptamer,FAM labeled part-complementary strand of cocaine aptamer, reaction temperature and time, that affect the assembly and recognition process were investigated. The results show that under the selected conditions, the concentration of cocaine is linear to the fluorescent intensity ranged from 5 to 1600 nmol/L with a detection limit 3. 0 nmol/L (3S/N).The standard recoveries were 89. 5% ~98. 6% for urine samples and the RSD is below 5%.

  19. Development of the Novel PEG-PE-Based Polymer for the Reversible Attachment of Specific Ligands to Liposomes: Synthesis and in vitro Characterization

    Science.gov (United States)

    Biswas, Swati; Dodwadkar, Namita S.; Sawant, Rupa R.; Torchilin, Vladimir P.

    2011-01-01

    Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized polyethylene glycol-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, where after successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal mAb 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound

  20. Cellular membrane trafficking of mesoporous silica nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fang, I-Ju [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulf some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine

  1. High-resolution, high-throughput, positive-tone patterning of poly(ethylene glycol by helium beam exposure through stencil masks.

    Directory of Open Access Journals (Sweden)

    Eliedonna E Cacao

    Full Text Available In this work, a collimated helium beam was used to activate a thiol-poly(ethylene glycol (SH-PEG monolayer on gold to selectively capture proteins in the exposed regions. Protein patterns were formed at high throughput by exposing a stencil mask placed in proximity to the PEG-coated surface to a broad beam of helium particles, followed by incubation in a protein solution. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR spectra showed that SH-PEG molecules remain attached to gold after exposure to beam doses of 1.5-60 µC/cm(2 and incubation in PBS buffer for one hour, as evidenced by the presence of characteristic ether and methoxy peaks at 1120 cm(-1 and 2870 cm(-1, respectively. X-ray Photoelectron Spectroscopy (XPS spectra showed that increasing beam doses destroy ether (C-O bonds in PEG molecules as evidenced by the decrease in carbon C1s peak at 286.6 eV and increased alkyl (C-C signal at 284.6 eV. XPS spectra also demonstrated protein capture on beam-exposed PEG regions through the appearance of a nitrogen N1s peak at 400 eV and carbon C1s peak at 288 eV binding energies, while the unexposed PEG areas remained protein-free. The characteristic activities of avidin and horseradish peroxidase were preserved after attachment on beam-exposed regions. Protein patterns created using a 35 µm mesh mask were visualized by localized formation of insoluble diformazan precipitates by alkaline phosphatase conversion of its substrate bromochloroindoyl phosphate-nitroblue tetrazolium (BCIP-NBT and by avidin binding of biotinylated antibodies conjugated on 100 nm gold nanoparticles (AuNP. Patterns created using a mask with smaller 300 nm openings were detected by specific binding of 40 nm AuNP probes and by localized HRP-mediated deposition of silver nanoparticles. Corresponding BSA-passivated negative controls showed very few bound AuNP probes and little to no enzymatic formation of diformazan precipitates or silver

  2. Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor.

    Science.gov (United States)

    Silin, Vitalii I; Karlik, Evan A; Ridge, Kevin D; Vanderah, David J

    2006-02-15

    A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1-5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F(c) portion of the rho 1D4 antibody through its F(ab) region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin-integrin antibody interaction after activation of the initial template with a goat anti-human integrin beta1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to

  3. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    Directory of Open Access Journals (Sweden)

    He Junkun

    2012-06-01

    Full Text Available Abstract Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to

  4. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

    Science.gov (United States)

    Daniels-Wells, Tracy R.; Helguera, Gustavo; Rodríguez, José A.; Leoh, Lai Sum; Erb, Michael A.; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L.

    2012-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin. PMID:23085102

  5. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

    Science.gov (United States)

    Daniels-Wells, Tracy R; Helguera, Gustavo; Rodríguez, José A; Leoh, Lai Sum; Erb, Michael A; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L

    2013-02-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

  6. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles.

    Science.gov (United States)

    Shen, Zhiqiang; Hou, Nannan; Jin, Min; Qiu, Zhigang; Wang, Jingfeng; Zhang, Bin; Wang, Xinwei; Wang, Jie; Zhou, Dongsheng; Li, Junwen

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL(-1) in PBS and 6.8 × 10(2) to 6.8 × 10(3) CFU mL(-1) in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method.

  7. Immunocytochemical localization of neuropeptide Y, serotonin, substance P and β-endorphin in optic ganglia and brain of Metapenaeus ensis

    Science.gov (United States)

    Ye, Haihui; Wang, Guizhong; Jin, Zhuxing; Huang, Huiyang; Li, Shaojing

    2006-12-01

    By using immunocytochemistry method of Strept Avidin-Biotin-Complex, four kinds of antisera raised against rabbits were applied to observe the immunoreactive neurons and neuropils of serotonin (5-HT), neuropeptide Y (NPY), substance P (SP) and β-Endorphin (β-Ep) in optic ganglia and brain of Metapenaeus ensis. The results showed that, the 5-HT-immunoreactive cells were located in all the four neuropils of optic ganglia. Immunoreactivity of 5-HT was detected in anterior medial protocerebrum neuropils (AMPN), and the inner and outer lateral beside olfactory lobe (OL) of deutocerebrum. The presence of NPY-immunoreactive cells was found in all the four neuropils of the optic ganglia. NPY-immunoreactivity occurred in the anterior median cell cluster, lateral cell cluster of protocerebrum, and cell cluster beside OL and AMPN. SP-immunoreactivity was found in medulla terminalis (MT) of optic ganglia, and lateral cell cluster of protocerebrum and posterior lateral cell cluster of tritocerebrum. β-Ep-immunoreactive cells were in MT only. In conclusion, these specific distribution patterns of the four immunoreactive substances can be used as morphological clues for understanding their different neurophysiological functions.

  8. Comparison of Nuclear Accumulation of p53 Protein with Mutations in the p53 Gene of Human Breast Cancer Tissues

    Institute of Scientific and Technical Information of China (English)

    王萱仪; 查小明; 武正炎; 范萍

    2001-01-01

    Objective The objective was to compare nuclear accumulation of p53 protein with mutations in the p53 gene on the tissues of human breast cancer. Methods Fifty-four invasive ductal carcinomas of breast were analyzed by the method of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) silver stain and strep-avidin-biotin-peroxidase complex (SABC) immunohistochemistry. Results A statistically significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (P<0.01). 22 tumors that demonstrated p53 gene mutations showed nuclear accumulation of p53 protein, while only 9 (28%) showed nuclear accumulation of p53 protein in 32 tumors without p53 gene mutations. Both p53 mutation protein and p53 gene mutations were prevalent in steroid and progesterone receptors negative tumors (P<0.05). A statistically significant association was found between the nuclear accumulation of p53 protein and lymph node invasion (P<0.05), and between p53 gene mutations and lymph node invasion (P<0.05). p53 abnormalities might be associated with an aggressive phenotype in breast cancer. Conclusion The immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in breast cancer tissues, and that this method is useful for rapid screening of p53 abnormalities. However, in order to avoid false positive reaction, the p53 gene mutations should be determined in cases slightly positive for p53 nuclear protein.

  9. Light microscopic immunocytochemical localization of hepatic and intestinal types of fatty acid-binding proteins in rat small intestine.

    Science.gov (United States)

    Shields, H M; Bates, M L; Bass, N M; Best, C J; Alpers, D H; Ockner, R K

    1986-05-01

    Monospecific antisera to purified hepatic fatty acid-binding protein (hFABP) and gut fatty acid-binding protein (gFABP) have been used to localize these two proteins in the small intestine of fed rats at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 4-, 10-, 20-, 22-, and 60-day-old Sprague-Dawley rats. Both cryostat and paraffin sections were studied for the presence of hFABP or gFABP by the avidin-biotin immunoperoxidase method. Slides were graded blind for the intensity of staining. Despite the structural and immunological differences between these two proteins, we showed no major differences between their staining patterns or their staining intensity throughout the intestine during postnatal development. The staining for both fatty acid-binding proteins was cytoplasmic. No brush border staining was found. Staining was more intense in the proximal rather than distal intestine, in the villus rather than crypt cells, and in the apex rather than the base of intestinal cells. Shifts in staining patterns, and staining intensity occurring during development may be related to variations in dietary fat intake, rates of cell proliferation, intestinal anatomy, and mechanisms for fat absorption.

  10. Engineering and Characterization of Collagen Networks Using Wet Atomic Force Microscopy and Environmental Scanning Electron Microscopy

    Science.gov (United States)

    Osborn, Jenna; Coffey, Tonya; Conrad, Brad; Burris, Jennifer; Hester, Brooke

    2014-03-01

    Collagen is an abundant protein and its monomers covalently crosslink to form fibrils which form fibers which contribute to forming macrostructures like tendon or bone. While the contribution is well understood at the macroscopic level, it is not well known at the fibril level. We wish to study the mechanical properties of collagen for networks of collagen fibers that vary in size and density. We present here a method to synthesize collagen networks from monomers and that allows us to vary the density of the networks. By using biotynilated collagen and a surface that is functionalized with avidin, we generate two-dimensional collagen networks across the surface of a silicon wafer. During network synthesis, the incubation time is varied from 30 minutes to 3 hours or temperature is varied from 25°C to 45°C. The two-dimensional collagen network created in the process is characterized using environmental atomic force microscopy (AFM) and scanning electron microscopy (SEM). The network density is measured by the number of strands in one frame using SPIP software. We expect that at body temperature (37°C) and with longer incubation times, the network density should increase.

  11. Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

    Institute of Scientific and Technical Information of China (English)

    Rongzhi Wang; Jiadong Huang; Wei Zhang; Guangmei Lin; Junwei Lian; Libin Jiang; Hongcong Lin; Songfa Wang; Shihua Wang

    2011-01-01

    In this paper,we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains,using multiplex PCR and DNA-DNA hybridization.Multiplex PCR was used to simultaneously amplify three diagnostic genes(tlh,tdh and fia)that serve as molecular markers of V.parahaemolyticus.Biotinylated PCR products were hybridized to primers immobilized on a microarray,and detected by chemiluminesce with avidin-conjugated alkaline phosphatase.With this method,forty-five samples were tested.Eight known virulent strains (tlh+/tdh+/fia+)and four known avirulent strains(tlh+/tdh-/fla+)of the V.parahaemolyttcus were successtuny aetectea,ana no non-spectnc hybridization and cross-hybridization reaction were found from fifteen closely-related strains(tin-/tdh-/fta+)or the Vibrio spp.In addition,all the other eighteen strains of non-Vibrio bacteria(tlh-/tdh-/fla-)gave negative results.The DNA microarray successfully distinguished V.parahaemolyticus from other Vibrio spp.The results demonstrated that this was an efficient and robust method for identifying virulent strains of V.parahaemolyticus.

  12. Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria.

    Science.gov (United States)

    Torun, Ozlem; Hakkı Boyacı, Ismail; Temür, Erhan; Tamer, Uğur

    2012-01-01

    Rapid and sensitive detections of microorganisms are very important for biodefence, food safety, medical diagnosis and pharmaceutics. The present study aims to find out the most proper bioactive surface preparation method to develop rapid, sensitive and selective bacteria biosensor, based on surface plasmon resonance (SPR) spectroscopy. Escherichia coli (E. coli) was used as a model bacterium and four sensing strategies in SPR were tested. Three of these strategies are antibody immobilization methods that are non-specific adsorption, specific adsorption via the avidin-biotin interaction, and immobilization of antibodies via self-assembled monolayer formation. The fourth strategy is a novel method for bacteria enumeration based on the combination of the SPR spectroscopy and immunomagnetic separation with using gold-coated magnetic nanoparticles. According to results, the most efficient SPR method is the one based on gold-coated magnetic nanoparticles. This method allows to specifically separate E. coli from the environment and to quantify rapidly without any labeling procedure. The developed method has a linear range between 30 and 3.0 × 10(4)cfu/ml, and a detection limit of 3 cfu/ml. The selectivity of the method was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the method to detect E. coli in real water samples was also investigated, and the results were compared with the results from plate-counting method. There was no significant difference between the methods (p>0.05).

  13. Protease-mediated release of chemotherapeutics from mesoporous silica nanoparticles to ex vivo human and mouse lung tumors.

    Science.gov (United States)

    van Rijt, Sabine H; Bölükbas, Deniz A; Argyo, Christian; Datz, Stefan; Lindner, Michael; Eickelberg, Oliver; Königshoff, Melanie; Bein, Thomas; Meiners, Silke

    2015-03-24

    Nanoparticles allow for controlled and targeted drug delivery to diseased tissues and therefore bypass systemic side effects. Spatiotemporal control of drug release can be achieved by nanocarriers that respond to elevated levels of disease-specific enzymes. For example, matrix metalloproteinase 9 (MMP9) is overexpressed in tumors, is known to enhance the metastatic potency of malignant cells, and has been associated with poor prognosis of lung cancer. Here, we report the synthesis of mesoporous silica nanoparticles (MSNs) tightly capped by avidin molecules via MMP9 sequence-specific linkers to allow for site-selective drug delivery in high-expressing MMP9 tumor areas. We provide proof-of-concept evidence for successful MMP9-triggered drug release from MSNs in human tumor cells and in mouse and human lung tumors using the novel technology of ex vivo 3D lung tissue cultures. This technique allows for translational testing of drug delivery strategies in diseased mouse and human tissue. Using this method we show MMP9-mediated release of cisplatin, which induced apoptotic cell death only in lung tumor regions of Kras mutant mice, without causing toxicity in tumor-free areas or in healthy mice. The MMP9-responsive nanoparticles also allowed for effective combinatorial drug delivery of cisplatin and proteasome inhibitor bortezomib, which had a synergistic effect on the (therapeutic) efficiency. Importantly, we demonstrate the feasibility of MMP9-controlled drug release in human lung tumors.

  14. Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase.

    Science.gov (United States)

    Walls, A F; Jones, D B; Williams, J H; Church, M K; Holgate, S T

    1990-10-01

    An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.

  15. Controllable microgels from multifunctional molecules: structure control and size distribution

    Science.gov (United States)

    Gu, Zhenyu; Patterson, Gary; Cao, Rong; Armitage, Bruce

    2004-03-01

    Supramolecular microgels with fractal structures were produced by engineered multifunctional molecules. The combination of static and dynamic light scattering was utilized to characterize the fractal dimension (Df) of the microgels and analyze the aggregation process of the microgels. The microgels are assembled from (1) a tetrafunctional protein (avidin), (2) a trifunctional DNA construct known as a three-way junction, and (3) a biotinylated peptide nucleic acid (PNA) that acts as a crosslinker by binding irreversibly to four equivalent binding sites on the protein and thermoreversibly to three identical binding sites on the DNA. The structure of microgels can be controlled through different aggregation mechanisms. The initial microgels formed by titration have a compact structure with Df ˜2.6; while the reversible microgels formed from melted aggregates have an open structure with Df ˜1.8. The values are consistent with the point-cluster and the cluster-cluster aggregation mechanisms, respectively. A narrow size distribution of microgels was observed and explained in terms of the Flory theory of reversible self-assembly.

  16. Influence of Adsorption on Proteins and Amyloid Detection by Silicon Nitride Nanopore.

    Science.gov (United States)

    Balme, Sébastien; Coulon, Pierre Eugène; Lepoitevin, Mathilde; Charlot, Benoît; Yandrapalli, Naresh; Favard, Cyril; Muriaux, Delphine; Bechelany, Mikhael; Janot, Jean-Marc

    2016-09-01

    For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.

  17. Interface of covalently bonded phospholipids with a phosphorylcholine head: characterization, protein nonadsorption, and further functionalization.

    Science.gov (United States)

    Ferez, Lynda; Thami, Thierry; Akpalo, Edefia; Flaud, Valérie; Tauk, Lara; Janot, Jean-Marc; Déjardin, Philippe

    2011-09-20

    Surface anchored poly(methylhydrosiloxane) (PMHS) thin films on oxidized silicon wafers or glass substrates were functionalized via the SiH hydrosilylation reaction with the internal double bonds of 1,2-dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2 Cis). The surface was characterized by X-ray photoelectron spectroscopy, contact angle measurements, atomic force microscopy, and scanning electron microscopy. These studies showed that the PMHS top layer could be efficiently modified resulting in an interfacial high density of phospholipids. Grafted phospholipids made the initially hydrophobic surface (θ = 106°) very hydrophilic and repellent toward avidin, bovine serum albumin, bovine fibrinogen, lysozyme, and α-chymotrypsin adsorption in phosphate saline buffer pH 7.4. The surface may constitute a new background-stable support with increased biocompatibility. Further possibilities of functionalization on the surface remain available owing to the formation of interfacial SiOH groups by Karstedt-catalyzed side reactions of SiH groups with water. The presence of interfacial SiOH groups was shown by zeta potential measurements. The reactivity and surface density of SiOH groups were checked by fluorescence after reaction of a monoethoxy silane coupling agent bearing Alexa as fluorescent probe.

  18. Immunocytochemical localization of neuropeptide Y,serotonin, substance P and β-endorphin in optic ganglia and brain of Metapenaeus ensis

    Institute of Scientific and Technical Information of China (English)

    YE Haihui; WANG Guizhong; JIN Zhuxing; HUANG Huiyang; LI Shaojing

    2006-01-01

    s By using immunocytochemistry method of Strept Avidin-Biotin-Complex, four kinds of antisera raised against rabbits were applied to observe the immunoreactive neurons and neuropils of serotonin (5-HT), neuropeptide Y (NPY), substance P(SP) and β-Endorphin (β-Ep) in optic ganglia and brain of Metapenaeus ensis. The results showed that, the 5-HT-immunoreactive cells were located in all the four neuropils of optic ganglia. Immunoreactivity of 5-HT was detected in anterior medial protocerebrum neuropils (AMPN), and the inner and outer lateral beside olfactory lobe (OL) of deutocerebrum. The presence of NPY-immunoreactive cells was found in all the four neuropils of the optic ganglia.NPY-immunoreactivity occurred in the anterior median cell cluster, lateral cell cluster of protocerebrum,and cell cluster beside OL and AMPN. SP-immunoreactivity was found in medulla terminalis (MT) of optic ganglia, and lateral cell cluster of protocerebrum and posterior lateral cell cluster of tritocerebrum.β-Ep-immunoreactive cells were in MT only. In conclusion, these specific distribution patterns of the four immunoreactive substances can be used as morphological clues for understanding their different neurophysiological functions.

  19. Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers

    Energy Technology Data Exchange (ETDEWEB)

    Ebner, Andreas [Institute of Biophysics, University of Linz, 4040 Linz (Austria)], E-mail: andreas.ebner@jku.at; Hinterdorfer, Peter; Gruber, Hermann J. [Institute of Biophysics, University of Linz, 4040 Linz (Austria)

    2007-10-15

    Atomic force microscopy (AFM) has developed into a key technique for elucidation of biological systems on the single molecular level. In particular, molecular recognition force microscopy has proven to be a powerful tool for the investigation of biological interactions under near physiological conditions. For this purpose, ligands are tethered to AFM tips and the interaction forces with cognate receptors on the sample surface are measured with pico-Newton accuracy. In the first step of tip functionalization, amino groups are typically introduced on the initially inert AFM tip. Several methods have been developed to reproducibly adjust the desired low density of amino groups on the tip surface, i.e. esterification with ethanolamine, gas-phase silanization with aminopropyl-triethoxysilane (APTES), or treatment with aminophenyl-trimethoxysilane (APhS) in toluene solution. In the present study, the usefulness of these methods for attachments of antibodies to AFM tips was characterized by a standardized test system, in which biotinylated IgG was bound to the tip and a dense monolayer of avidin on mica served as test sample. All three methods of aminofunctionalization were found fully satisfactory for attachment of single antibodies to AFM tips, only in a parallel macroscopic assay on silicon nitride chips a minor difference was found in that APTES appeared to yield a slightly lower surface density of amino groups.

  20. High-Q whispering-gallery mode sensor in liquids

    Science.gov (United States)

    Nadeau, Jay L.; Ilchenko, Vladimir S.; Kossakovski, Dmitri; Bearman, Gregory H.; Maleki, Lute

    2002-06-01

    Optical sensing of biomolecules on microfabricated glass surfaces requires surface coatings that minimize nonspecific binding while preserving the optical properties of the sensor. Microspheres with whispering-gallery (WG) modes can achieve quality factor (Q) levels many orders of magnitude greater than those of other WG-based microsensors: greater than 1010 in air, and greater than 109 in a variety of solvents, including methanol, H2O and phosphate buffered saline (PBS). The presence of dyes that absorb in the wavelength of the WG excitation in the evanescent zone can cause this Q value to drop by almost 3 orders of magnitude. Silanization of the surface with mercapto-terminal silanes is compatible with high Q (>109), but chemical cross-linking of streptavidin reduces the Q to 105-106 due to build-up of a thick, irregular layer of protein. However, linkage of biotin to the silane terminus preserves the Q at a ~2x107 and yields a reactive surface sensitive to avidin-containing ligands in a concentration-dependent manner. Improvements in the reliability of the surface chemistry show promise for construction of an ultrasensitive biosensor.

  1. 家鸽消化道生长抑素免疫活性内分泌细胞的研究%The Research of the Somatostatin Immunoreactive Endocrine Cells in Digestive Tract of Columbae Livia Domesticus

    Institute of Scientific and Technical Information of China (English)

    訾芙玮; 李淑兰

    2010-01-01

    应用卵白素-生物素-过氧化物酶复合物(avidin-biotin-peroxidase complex,ABC)免疫组织化学方法对家鸽(columbae livia domesticus)消化道生长抑素细胞的分布密度和形态学特点进行了观察.结果表明:生长抑素(somatostatin,简称SS)细胞主要分布于胃体及其以下部位,其中胃体部的分布密度最高,小肠各段的分布密度较低,且小肠各部位间差异不显著.而在食管和嗉囊中均未发现SS细胞.SS细胞的形态多样,主要有圆形、椭圆形和锥体形等.主要分布在上皮细胞基部、上皮细胞之间和腺泡上皮细胞之间.通过形态学观察认为:家鸽消化道生长抑素细胞的分布与其消化道各部位的功能有关.

  2. 家鸽消化道5-羟色胺细胞的形态与分布%The Shape and Distribution of 5-HT Cells in Digestive Tract of Columba livia domesticus

    Institute of Scientific and Technical Information of China (English)

    曹雷; 李淑兰

    2009-01-01

    采用ABC(avidin-biotin-peroxidase complex)免疫组织化学方法,观察家鸽(Columba livia domes-ticus)消化道内5-羟色胺(5-HT)免疫阳性内分泌细胞的分布及形态.结果显示,除食管外,5-羟色胺细胞从胃到直肠各段均有分布.细胞分布密度呈波浪式,其中直肠部分布密度最高(5.20±1.94),回肠部次之(3.75±1.29),胃部最低(1.7±0.80).5-HT阳性细胞广泛分布于消化道上皮细胞之间、上皮基部、腺泡上皮细胞之间.形态多样,呈圆形、锥体形、梭形等.结论:5-HT细胞分布型的形成与消化道各部位消化功能有关,5-HT细胞形态与其内、外分泌功能是相适应的.

  3. Effect of Buzhong Yiqi Decoction (补中益气汤) on Murine Liver Damage Induced by Food Allergy

    Institute of Scientific and Technical Information of China (English)

    陈虹; 董阳深; 陈奋华; 纪经智; 陈岩峰; 上野幸三; 饭仓洋治

    2004-01-01

    To investigate the effect of Buzhong Yiqi decoction ( 补中益气汤, BZYQD) on liver damage induced by food allergy in mice. Methods: Nc/Jic strain mice with high levels of serum IgE were sensitized by ovalbumin (OVA), and then divided into two groups and respectively treated with BZYQD (treated group) or normal saline (model group). Samples Of serum, liver tissues and small intestine were collected two weeks later, and another group of non-sensitized mice was set as the normal group. The levels of serum alanine aminotransferase (ALT) were measured with spectrophotometry. The liver tissue and small intestine were stained with hematoxylin and eosin (HE) for pathologic analysis. The liver samples were also subjected to analysis of CD4-T helper cell and cytokine (interleukin-4, IL-4, interleukin-6, IL-6) expression with immunohistochemical (avidin-biotin complex, ABC) method. Results: Serum ALT levels decreased and obvious pathologic improvements were seen in the mice treated with BZYQD. And compared with the model mice, the number of positive cells of IL-4, IL-6 and CD4 cell decreased significantly in those treated with BZYQD. Conclusion: BZYQD can effectively decrease the production of cytokines associated with allergic reaction in the liver of mice thus effective in treating liver damage caused by food allergy.

  4. Expression of epidermal growth factor and its receptor in gestational trophoblastic diseases

    Institute of Scientific and Technical Information of China (English)

    LI Ning; YUAN Yu-kang; LIU Hui-xi

    2005-01-01

    Objective: To determine whether epidermal growth factor (EGF) and its receptor have any possible correlation with etiology of gestational trophoblastic diseases. Methods: Avidin-biotin immunoperoxidase techniques with polyclonal antibodies against EGF, EGFR were used to examine 53 cases of GTD, including complete hydatidiform mole(16),invasive mole(20),gestational choriocarcinoma(17).Results:EGF was mainly localized on syncytiotrophoblasts (ST), and was found less on cytotrophoblasts. Cytologic localization of EGFR showed the similar results. The positive rate of EGF and EGFR were 0.625, 0.813 in hydatidiform mole, 0.405, 0.450 in invasive mole and 0.118, 0.235 in gestational choriocarcinoma. There was significant difference of EGF or EGFR among hydatidiform mole group and other groups, respectively (P<0.05). Conclusion:The cellular levels of EGF and EGFR decreased gradually in the development of GTD. It implied that the autocrine and paracrine mechanism may play an important role on the proliferation and differentiation of trophoblast cells and the disorder of the system may lead to GTD malignant transformation.

  5. Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria.

    Science.gov (United States)

    Bodegas, M E; Villaro, A C; Montuenga, L M; Moncada, S; Riveros-Moreno, V; Sesma, P

    1995-10-01

    Physiological and histochemical studies have recently supported the notion that nitric oxide (NO) is the transduction signal responsible for the non-adrenergic, non-cholinergic relaxation of the vasculature as well as the airways of the mammalian lung. We report the presence of immunoreactivity to NO synthase (NOS) in nerve cell bodies and nerve fibres in the neural plexus of the buccal cavity and lungs of the frog, Rana temporaria, using the indirect immunocytochemical technique of avidin-biotin and the NADPH-diaphorase technique. The neural ganglia located next to the muscle layer and within the connective tissue of the buccal cavity were partially immunoreactive for NOS. In the lungs, NOS immunoreactivity occurred in nerve cell bodies, as well as in both myelinated and unmyelinated nerve fibres. Fine nerve fibres immunoreactive to NOS were observed within the muscle fibre bundles and next to the respiratory epithelium. Both the presence of NOS immunoreactivity and the positive histochemical reaction for NADPH-diaphorase in the neural plexus of amphibian respiratory tract suggests a broad evolutionary role for NO as a peripheral neurotransmitter.

  6. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Supachai Sakkhachornphop

    2015-01-01

    Full Text Available The 3′-end processing (3′P of each viral long terminal repeat (LTR during human immunodeficiency virus type-1 (HIV-1 integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN, and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.

  7. Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Santos P.R.P.

    1997-01-01

    Full Text Available Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite

  8. Characterization of fully functional spray-on antibody thin films

    Energy Technology Data Exchange (ETDEWEB)

    Figueroa, Jhon [Department of Chemistry, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-5250 (United States); Magaña, Sonia; Lim, Daniel V. [Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-7115 (United States); Schlaf, Rudy, E-mail: schlaf@eng.usf.edu [Department of Electrical Engineering, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620-5101 (United States)

    2014-02-15

    The authors recently demonstrated that fully functional Escherichia coli O157:H7 antibody thin films can be prepared using a simple pneumatic nebulizer on glass surface [1]. This paper focuses on the investigation of the morphology and physical properties of these films with the aim to better understand their performance. A series of E. coli O157:H7 antibody spray-on thin films were investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), immunoassays, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), fluorescence microscopy, atomic force microscope (AFM) and contact angle analysis. These data were compared to measurements on films prepared with the biotin–avidin covalent bonding scheme. The investigation showed that films created by a 2 min pneumatic spray deposition time can capture antigens similar as the avidin–biotin wet-chemical method. The results also suggests that an influential factor for the comparable capture cell ability between sprayed and covalent films is an increased antibody surface coverage for the sprayed films (non-equilibrium technique), which compensates for the lack of its antibody orientation. There was no significant antibody denaturation detected on any of the sprayed films. Both techniques led to the formation of cluster-aggregates, a factor that seems unavoidable due to the natural tendency of protein to cluster. The avidin–biotin bridge films generally had a higher roughness, which manifested itself in a higher wettability compared to the sprayed films.

  9. C-cells in colloid goiter

    Directory of Open Access Journals (Sweden)

    Lima Marcus A.

    2003-01-01

    Full Text Available PURPOSE: The aim of this investigation was to quantitatively evaluate C-cells in colloid goiters, analyzing 36 thyroids that were obtained through thyroidectomy from 24 patients with goiter and 12 normal glands from adult patients without thyroid disease, which were used as the control group. MATERIAL AND METHODS: On average, 6 different thyroid areas were sampled and labeled by immunohistochemistry with a monoclonal anticalcitonin antibody, utilizing the avidin-biotin-peroxidase complex. C-cells were counted in fields measuring 1 square centimeter, and the mean number of cells per field was then calculated. Data were statistically analyzed using the Mann-Whitney test. RESULTS: In the colloid goiter group, the number of C-cells ranged from 0 to 23 per field, while in normal controls they ranged from 20 to 148 per field. CONCLUSIONS: These results demonstrate a significant decrease of C-cell number in the colloid goiter group compared with control group, indicating that the hyperplastic process is restricted to follicular cells, to the detriment of C-cells, which probably cease to receive trophic stimuli.

  10. Blood feeding patterns of Nyssomyia intermedia and Nyssomyia neivai (Diptera, Psychodidae in a cutaneous leishmaniasis endemic area of the Ribeira Valley, State of São Paulo, Brazil

    Directory of Open Access Journals (Sweden)

    Ana Maria Marassa

    2013-09-01

    Full Text Available Introduction The aim of this study was to identify the blood feeding sources of Nyssomyia intermedia (Ny. intermedia and Nyssomyia neivai (Ny. neivai, which are Leishmania vectors and the predominant sandfly species in the Ribeira Valley, State of São Paulo, Brazil, an endemic area for cutaneous leishmaniasis. Methods Specimens were captured monthly between February 2001 and December 2003 on a smallholding and a small farm situated in the Serra district in the Iporanga municipality. The blood meals of 988 engorged females were tested using the avidin-biotin immunoenzymatic enzyme-linked immunosorbent assay (ELISA. Seven blood meal sources were investigated: human, dog, chicken, bovine, pig, horse and rat. Results The results showed that among the females that fed on one or more blood sources, the respective percentages for Ny. intermedia and Ny. neivai, respectively, were as follows: human (23% and 36.8%, pig (47.4% and 26.4%, chicken (25.7% and 36.8% and dog (3.9% and 0%, and the differences in the blood sources between the two species were statistically significant (p = 0.043. Conclusions Both species had predominant reactivity for one or two blood sources, and few showed reactivity indicating three or four sources. Many different combinations were observed among the females that showed reactivity for more than one source, which indicated their opportunistic habits and eclecticism regarding anthropic environmental conditions.

  11. Determination of Biotin in Pharmaceutical Formulations by Potassium Permanganate-luminol-CdTe Nanoparticles Chemiluminescence System

    Institute of Scientific and Technical Information of China (English)

    TRAORE Zoumana Sékou; SU Xing-guang

    2012-01-01

    A sensitive flow-injection chemiluminescence method was developed for the determination of biotin in the pharmaceutical formulations.The affinity between avidin and biotin was used to adsorb biotin on the polystyrene,with subsequent quantification of biotin based on its ability to enhance the chemiluminescence(CL) signal generated by the redox reaction of potassium permanganate-luminol-CdTe nanoparticles CL system.The investigations prove that apart from 3-aminophthalate,the CdTe quantum dots(QDs) play both catalytic and emitter roles.Under optimum conditions,the linear range for the determination of biotin was 0.01-25 ng/mL with a detection limit of 7.3×10-3ng/mL(S/N=3).The relative standard deviation of 5 ng/L biotin was 2.06%(n=7).The proposed method was used to determine the biotin concentration in the pharmaceutical formulations and the recovery was between 96.4% and 104%.The proposed method is simple,convenient,rapid and sensitive.

  12. A Safe, Versatile and Translation-prone Strategy for Using Circulating Lipoproteins as Endogenous Drug Delivery Systems

    Directory of Open Access Journals (Sweden)

    Mahshid Foroozesh

    2010-10-01

    Full Text Available "nLipoproteins (LPs, the endogenous lipid-protein micro- and nanostructures involved in lipid metabolism, have attracted a high degree of interest in recent years for being used as novel drug delivery systems. Numerous diagnostic and therapeutic agents (in particular anti-cancer agents have been studied using native and (semisynthetic LPs as both prolonged and targeted drug delivery systems. Since all reported loading methods are basically in vitro or ex vivo procedures with related limitations, an idea has been raised for finding a completely new loading paradigm to overcome the limitations in using native particles as drug vehicles. The basis for this hypothesis is that we are able to load native and circulating LPs without extracting them from body via using specific monoclonal antibodies (MAb, already linked to desired drugs or to be linked to drug via proper linker system such as avidin-biotin bridges actively-targeted against specialized Apos available on the surface of all LPs. Obviously, by choosing the right anti-Apo antibody (preferably single chain variable fragment; scFv, we can select the right circulating LP subpopulation (i.e., LDL, HDL, VLDL, or CM. By considering all parameters and using the most appropriate strategy, this novel, safe, versatile, industrializable, and clinically translation-prone  paradigm could be used for both prolonged and (LP receptor- and non-LP receptor- targeted drug delivery purposes.

  13. Effects of histamine on growth and apoptosis of human melanoma cells A375

    Institute of Scientific and Technical Information of China (English)

    RAN Li-wei; TAN Sheng-shun; XU Xin-ling; ZHANG Jiang-an; WANG Wan-juan

    2005-01-01

    Objective: To investigate the effects of histamine on growth and apoptosis of human melanoma cells A375. Methods: The effect of histamine on growth of A375 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis by double staining with Annexin V-FITC and PI, and active caspase-3 analysis by staining FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. StreptAvidin-Biotin Complex (SABC)immunocytochemical assays were adopted to detect Bax/Bcl-2 protein expressions. Results: Histamine inhibited proliferation of A375 cells in a dose- and time-dependent manner, and altered cell cycle distribution of A375 cells revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. Histamine induced apoptosis of A375 cells (P<0. 05), elevated the cells population with detectable active caspase-3 (P<0. 05), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (P<0.05). Conclusion: That histamine inhibits cell cycle progress of A375 cells is one of the possible mechanisms of proliferation arrest of A375 cells elicited by histamine. Histamine mediates apoptosis in A375 cells that may be caspase-dependent through mitochondria routine. Histamine with high concentration inhibits growth of A375 cells in vitro by interfering proliferation and inducing apoptosis of cells.

  14. On the possible involvement of bovine serum albumin precursor in lipofection pathway

    Indian Academy of Sciences (India)

    Anubhab Mukherjee; Jayanta Bhattacharyya; Arabinda Chaudhuri

    2014-03-01

    Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ∼2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.

  15. Immunocytochemical Identification and Localization of Diffuse Neuroendocrine System (DNES) Cells in Gastrointestinal Tract of Channel Catfish (Ictalurus punctatus)

    Institute of Scientific and Technical Information of China (English)

    HE Min; WANG Kai-yu; ZHANG Yu

    2009-01-01

    To detect distribution and relative frequency of diffuse neuroendocrine system (DNES) cells in the gastrointestinal tract of channel catfish (Ictalurus punctatus), the intestinal tract of channel catfish was divided into seven portions from proximal to distal: the enlarged area after oesophagus, cardia, fundus, pylorus, and anterior, middle, and posterior intestine. Immunohistochemical method using the strept avidin-biotin-complex (SABC) was employed. All antisera between seven portions of the channel catfish were compared statistically using statistical package for the social science (SPSS). Five types of DNES cells were determined: neuropeptide Y-immunoreactive (NPY-IR) cells were demonstrated in both anterior and middle intestine; serotonin (5-HT) immunoreactive cells were detected throughout the whole gastrointestinal tract; vasoactive intestinal peptide (VIP) positive cells were at the highest frequency in pylorus; glucagon-immunoreactive (GLU-IR) cells were moderate in number in the fundus and anterior, middle intestine, and no immunoreactivity was determined in the other portions; somatostatin (SOM) positive cells were more abundant in the anterior and middle intestine. The regional distribution and relative frequency of immunoreactive cells in the channel catfish, Ictalurus punctatus, are essentially similar to those of other fish. However, some characteristics are observed in this species, which further proved that the diversity of the physiological function of DNES cells was based on their morphology.

  16. Expression of Androgen Receptor in Meningiomas

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to investigate the expression of androgen receptor (AR) in meningiomas and its relation to tumor proliferative potential, we examined the expression of AR and proliferating cell nuclear antigen (PCNA) by avidine-biotin complex immunohistochemistry in 39 cases of meningiomas. Of the 39 cases of meningiomas, 20(51 %) showed positive AR immunoreactivity. The AR expression positivity rates were 31 % (6/19) in benign meningiomas, 58 % (7/12) in atypical meningiomas, 87.5 % (7/8) in malignant meningiomas, respectively. In addition to the tumor cells, cells of microvascular endothelial proliferation were frequently AR positive. Malignant meningiomas had a significantly higher percentage of AR positive cells compared with atypical and benign meningiomas (P<0.05). The mean proliferating cell nuclear antigen labeling index (PCNA LI) was significantly higher in the malignant meningiomas when compared with atypical meningiomas (P<0.05) and benign meningiomas (P<0.05). AR positive meningiomas had higher PCNA LI than AR negative meningiomas (P<0.05). The expression of AR in tumor tissues was significantly related with PCNA LI. These data indicated that AR in the meningiomas was correlated with histological grade and AR might participate in the growth of these tumors and tumor angiogenesis. The measurement of AR in these tumors may indirectly represent tumor growth potential.

  17. Endothelin-1 and endothelin receptors in the basilar artery of the capybara.

    Science.gov (United States)

    Loesch, Andrzej; Gajkowska, Barbara; Dashwood, Michael R; Fioretto, Emerson T; Gagliardo, Karina M; Lima, Ana R De; Ribeiro, Antonio A C M

    2005-02-01

    Little is known about cerebral vasculature of capybara, which seems may serve as a natural model of studying changes in cerebral circulation due to internal carotid artery atrophy at animal sexual maturation. This is the first study of the light- and electron-immunocytochemical localisation of endothelin-1 (ET-1) and ETA and ETB endothelin receptors in the basilar artery of capybaras (6 to 12-month-old females and males) using an ExtrAvidin detection method. All animals examined showed similar patterns of immunoreactivity. Immunoreactivity for ET-1 was detected in the endothelium and adventitial fibroblasts, whilst immunoreactivity for ETA and ETB receptors was present in the endothelium, vascular smooth muscle, perivascular nerves and fibroblasts. In endothelial cells immunoreactivity to ET-1 was pronounced in the cytoplasm or on the granular endoplasmic reticulum. Similar patterns of immunolabelling were observed for ETA and ETB receptors, though cytoplasmic location of clusters of immunoprecipitate seems dominant. These results suggest that the endothelin system is present throughout the wall of the basilar artery of capybara.

  18. Magnetic Control of Rigid Achiral Microswimmers

    Science.gov (United States)

    Cheang, U.; Meshkati, Farshad; Fu, Henry; Kim, Minjun

    2013-11-01

    We report control of rigid achiral microswimmers in low Reynolds number environments. A rotating magnetic field was used to actuate the microswimmers wirelessly by rotating the microswimmers, which produces propulsion. Previous magnetically actuated microswimmers in bulk fluids have been designed with either flexibility or chiral geometry; we show that simpler geometries with neither flexibility nor chirality can produce propulsion. The microswimmer consists of three magnetic beads conjugated using avidin-biotin linkages into an arc formation. We designed a magnetic field generator consisting of electromagnetic coils arranged in an approximate Helmholtz configuration. A highspeed camera provided realtime imaging of the microswimmers' motion in a PDMS chamber. The rigidity of the microswimmer was characterized by tracking the position of the individual beads and calculating their relative distances. As a function of field strength and rotation frequency, we observed changes in the rotational axis of the microswimmers and the corresponding effects on their velocities. The achiral microswimmers exhibited active propulsion and were controllable in both speed and direction, which demonstrates the possibility for future biomedical applications such as drug delivery.

  19. Chromatographic Studies of Protein-Based Chiral Separations

    Science.gov (United States)

    Bi, Cong; Zheng, Xiwei; Azaria, Shiden; Beeram, Sandya; Li, Zhao; Hage, David S.

    2016-01-01

    The development of separation methods for the analysis and resolution of chiral drugs and solutes has been an area of ongoing interest in pharmaceutical research. The use of proteins as chiral binding agents in high-performance liquid chromatography (HPLC) has been an approach that has received particular attention in such work. This report provides an overview of proteins that have been used as binding agents to create chiral stationary phases (CSPs) and in the use of chromatographic methods to study these materials and protein-based chiral separations. The supports and methods that have been employed to prepare protein-based CSPs will also be discussed and compared. Specific types of CSPs that are considered include those that employ serum transport proteins (e.g., human serum albumin, bovine serum albumin, and alpha1-acid glycoprotein), enzymes (e.g., penicillin G acylase, cellobiohydrolases, and α-chymotrypsin) or other types of proteins (e.g., ovomucoid, antibodies, and avidin or streptavidin). The properties and applications for each type of protein and CSP will also be discussed in terms of their use in chromatography and chiral separations.

  20. Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

    Science.gov (United States)

    Hager, Roland; Burns, Jonathan R; Grydlik, Martyna J; Halilovic, Alma; Haselgrübler, Thomas; Schäffler, Friedrich; Howorka, Stefan

    2016-06-01

    The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays.

  1. Expression of ATP7B in human gastric cardiac carcinomas in comparison with distal gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Da-Long Wu; Hui-Xing Yi; Feng-Ying Sui; Xiao-Hong Jiang; Xiao-Ming Jiang; Ying-Ying Zhao

    2006-01-01

    AIM: To analyze expression of ATP7B in gastric cardiac adenocarcinomas, its clinicopathologic significance, in comparison with distal gastric adenocarcinomas.METHODS: Immunohistochemical avidin-biotin peroxidase complex method was applied to detect the expression of ATP7B in 49 cases of cardiac carcinomas,the corresponding adjacent non-neoplastic epithelium and 55 cases of distal gastric carcinomas.RESULTS: The proportion of ATP7B positive samples in gastric cardiac carcinomas (51.0%, 25 of 49) was significantly higher than that in the corresponding adjacent non-neoplastic epithelium (22.4%, 11 of 49)(P = 0.003). ATP7B expression in poorly differentiated gastric cardiac carcinomas was significantly higher than that in well/moderately differentiated gastric cardiac carcinomas (P = 0.030). ATP7B expression in gastric cardiac carcinomas was independent of age, tumor size, nodal stage and metastasis status. ATP7B protein was detected in 30.9% (17/55 cases) of distal gastric carcinomas, markedly lower than that in gastric cardiac carcinomas (P = 0.037).CONCLUSION: ATP7B protein is frequently overexpressed in gastric cardiac carcinomas, and correlated with the differentiation of cardiac carcinoma. ATP7B expression in gastric cardiac carcinomas is significantly higher than that in distal gastric carcinomas, which might partially explain the difference of chemotherapy response and prognosis between these two gastric carcinomas.

  2. The Distribution of MAP-2 Phosphorylation in Cerebral Cortex of Long-Tailed Monkey Fetuses (Macaca fascicularis in the Last Trimester of Gestation

    Directory of Open Access Journals (Sweden)

    Tri Wahyu Pangestiningsih

    2015-11-01

    Full Text Available Memories are storage in cholinoceptive cells, the cells which are enriched with microtubule-associated protein 2 (MAP-2 that localized in the neuronal dendrite and the cell bodies. Phosphorylation of MAP-2 may increase memory with reduce stability of dendrite by altered dendrite length and lead new side-branches of neuronal as a neuronal plasticity processes in cerebral cortex. The aim of this research is to study the distribution of MAP-2 phosphorylation neurons in cerebral cortex of long-tailed macaques in the third semester of gestationalimmunohistochemically using avidin biotin conjugated complex method. Neurons MAP-2 phosphorylation immunoreactive were located in dendrites and cell bodies, mostly in pyramidal neurons of cerebral cortex. Intensity of MAP-2 phosphorylation immunoreactivity in layer V were stronger than another layer and the neurons that very intensely stained were the pyramidal cells in frontal and parietal lobes, that was suggested that neurons in this areas more responsive to neuroplasticity. From the results we concluded that MAP-2 phosphorylation already distributed in the cerebral cortex of long-tailed macaque fetuses at the last trimester of gestation, mostly in the pyramidal cells of layer V that is suggested plays a role for preparation of memoryformation.Keywords: fetus, long-tailed monkey, cerebral cortex, memory, MAP-2 phosphorylation

  3. In vivo imaging of chromogranin A-positive endocrine tumours by three-step monoclonal antibody targeting

    Energy Technology Data Exchange (ETDEWEB)

    Siccardi, A.G. [Dipartimento di Ricerca Biologica e Tecnologica, Istituto Scientifico San Raffaele, Milano (Italy); Paganelli, G. [Dipartimento di Medicina Nucleare, Istituto Scientifico San Raffaele, Milano (Italy); Pontiroli, A.E. [Dipartimento di Medicina Interna, Istituto Scientifico San Raffaele, Milano (Italy); Pelagi, M. [Dipartimento di Ricerca Biologica e Tecnologica, Istituto Scientifico San Raffaele, Milano (Italy); Magnani, P. [Dipartimento di Medicina Nucleare, Istituto Scientifico San Raffaele, Milano (Italy); Viale, G. [Dipartimento di Ricerca Biologica e Tecnologica, Istituto Scientifico San Raffaele, Milano (Italy); Faglia, G. [Istituto di Scienze Endocrine, Universita di Milano (Italy); Fazio, F. [Dipartimento di Medicina Nucleare, Istituto Scientifico San Raffaele, Milano (Italy)

    1996-11-01

    The detection of chromogranins (Cg) by immunohistochemistry and serology represents a new in vitro diagnostic tool for endocrine tumours. We have recently reported on the feasibility of targeting chromogranin A (CgA) for in vivo detection of pituitary adenomas by immunoscintigraphy (ISG). The scintigraphic procedure, based on an anti-CgA monoclonal antibody and on the avidin-biotin three-step method (Cg-3S-ISG), was evaluated on a group of 29 consecutive patients with known or suspected endocrine tumours other than pituitary adenomas, i.e. medullary thyroid carcinoma, carcinoid, insulinoma and parathormone- or ACTH-producing tumours. Primary tumours (10) and recurrences (16) were visualised in 26 patients, whereas conventional imaging techniques (planar radiography, computerised tomography, magnetic resonance imaging and ultrasonography) failed to detect the tumour sites in ten of the same (Cg-3S-ISG-positive) patients. Therefore, these preliminary results indicate that Cg-3S-ISG, the first immunological method able to detect endocrine tumours in vivo, has a higher diagnostic accuracy than conventional imaging techniques (93.1% compared with 65.5%). (orig.). With 3 figs., 1 tab.

  4. THE EFFECT OF MEBT/MEBO ON EPIDERMAL REGENERATIVE STEM CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To investigate the mechanism of the effect of Moist Exposed Burn Therapy (MEBT) and Moist Exposed Burn Ointment (MEBO) on the spontaneous repair and healing of superficial third degree burn wound involving fatty layer. Method: A series of skin tissue samples were taken from deep burn wounds of 2 cases. Immunocytochemistry method, biotin avidin DCS system, and indirect immunofluorescence technique were applied. Mouse anti human keratin type 19 monoclonal antibody was used to detect regenerative epidermal stem cells in wound tissues. Results: Epidermal regenerative stem cells emerged at 24 hours post burn, and the number of epidermal regenerative stem cell increased on day 4 post burn. On days 7 and 14 post burn, the number of epidermal stem cells increased to the peak level. On days 21 and 28 post burn, it decreased and disappeared gradually as burn wound progressed to healing. Conclusion: MEBT/MEBO has the effect of promoting the activation and proliferation of epidermal regenerative stem cells in the residual viable tissue of superficial full thickness burn wound, and these stem cells play an unique role in spontaneous wound healing of deep burn.

  5. DERIVATIZACIÓN Y CARACTERIZACIÓN ESPECTROSCÓPICA DE UN BIOPOLÍMERO A BASE DE L -LISINA CON ANÁLOGOS DE LA D-BIOTINA: CO -POLI(L-LISINA-GRAFT -(ε-N-[X-D -BIOTINIL]-L-LISINA

    Directory of Open Access Journals (Sweden)

    Flavio Dolores Martínez-Mancera

    2016-01-01

    Full Text Available We report the single-step derivatization reaction of a biopolymer based onL -lysine with D -biotin analogs:Co -poly(L -lysine-graft-(ε-N -[X-D-biotinyl]-L -lysine (PLL-X-Biotin. The valeric acid carboxylate of D -biotin is activated to an NHS ester for direct modification of amine groups in proteins and other macromolecules. NHS esters react by nucleophilic attack of an amine in the carbonyl group, releasing the NHS group, and forming a stable amide linkage. NHS-X-Biotin is the simplest biotinylation reagent commercially available. In contrast withD -biotin, it has a longer spacer arm off the valeric acid side chain allowing better binding potential for avidin or streptavidin probes. Derivatization of poly(L -lysine (PLL with NHS-X-Biotin led to a copolymer PLL-X-Biotin. UV-Visible, IR-FT and 1H NMR characteristics derived from synthesis are briefly discussed.

  6. Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies.

    Science.gov (United States)

    Sasaki, Takanori; Terano, Yoshitake; Shibata, Tadayoshi; Kawamoto, Hiroyoshi; Kuzuguchi, Tsuyoshi; Kohyama, Erina; Watanabe, Toshihiro; Ohyama, Tohru; Gemba, Munekazu

    2005-01-01

    Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.

  7. THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    刘学光; 张志刚; 张秀荣; 朱虹光; 陈琦; 郭慕依

    2002-01-01

    Objective. To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). Methods. A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. Results. A specific monoclonal antibody against AM was successfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. Conclusion. AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

  8. THE LOCALIZATION OF ADRENOMEDULLIN IN RAT KIDNEY TISSUE AND ITS INHIBITORY EFFECT ON THE GROWTH OF CULTURED RAT MESANGIAL CELLSA

    Institute of Scientific and Technical Information of China (English)

    刘学光; 张志刚; 等

    2002-01-01

    Objective:To observe the localization of adrenomedullin(AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC).Methods:A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry.The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC)and MsC were investigated by Northern blot assay,and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H] thymidine incorporation as an index.Results:A specific monoclonal antibody against AM was successfull developed.AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells),some cortical proximal tubules,medullary collecting duct cells,interstitial cells,vascular smooth muscle cells and endothelial cells.Northern blot assay showed the AM mRNA was expressed only on cultured GEC,but not on MsC,however,AM receptor CRLR mRNA was only expressed on MsC.GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect.Conclusion:AM produced by GEC inhibits the proliferation of MsC,which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.

  9. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity.

    Science.gov (United States)

    Turunen, S; Käpylä, E; Terzaki, K; Viitanen, J; Fotakis, C; Kellomäki, M; Farsari, M

    2011-12-01

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  10. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Turunen, S; Kaepylae, E; Kellomaeki, M [Tampere University of Technology, Department of Biomedical Engineering, PO Box 692, 33101 Tampere (Finland); Terzaki, K; Fotakis, C; Farsari, M [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), N. Plastira 100, 70013, Heraklion, Crete (Greece); Viitanen, J, E-mail: elli.kapyla@tut.fi [VTT Technical Research Centre of Finland, PO Box 1300, 33101 Tampere (Finland)

    2011-12-15

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  11. AN IMMUNOCYTOCHEMICAL STUDY OF BONEMORPHOGENETIC PROTEIN IN EXPERIMENTAL FRACTURE HEALING OF THE RABBIT MANDIBLE

    Institute of Scientific and Technical Information of China (English)

    金岩; 杨连甲; FrankH.White

    1994-01-01

    A monoclonal antibody raised against bone morphogenetic protein (BMP-McAb)has been used to demonstrate the presence of bone mrphogenetic protein(BMP) in experimental fracture healing.Rabbit mandibles were frac-tured using standrdized methods and left to heal for 3,7,14,21and 24 d,respectively.The avidin-biotin com- plex(ABC)method demonstrated an accumulation of positively stained primitive mesenchyman cells at the fracture site in the hematoma stage of bone repair.These cells appeared to undergo differentiation into positively-stained chondroblasts and osteoblasts during the phase of callus formation.Undifferentiated mesenchymal cells showed a high positive reactivity in the early post-fracture stages but a much lower reactivity during the remodelling phase.The results of our study suggest that bone inductive processes are accompanied by the presence of BMP in osteopro-genitor cells during fracture healing of the mandible and that BMP may plqy a significant role in osteogenesis dur-ing bone healing.

  12. Microfluidics and nanoparticles based amperometric biosensor for the detection of cyanobacteria (Planktothrix agardhii NIVA-CYA 116) DNA.

    Science.gov (United States)

    Ölcer, Zehra; Esen, Elif; Ersoy, Aylin; Budak, Sinan; Sever Kaya, Dilek; Yağmur Gök, Mehmet; Barut, Serkan; Üstek, Duran; Uludag, Yildiz

    2015-08-15

    Some of the cyanobacteria produce protease inhibitor oligopeptides such as cyanopeptolins and cause drinking water contamination; hence, their detection has great importance to monitor the well-being of water sources that is used for human consumption. In the current study, a fast and sensitive nucleic acid biosensor assay has been described where cyanopeptolin coding region of one of the cyanobacteria (Planktothrix agardhii NIVA-CYA 116) genome has been used as target for monitoring of the fresh water resources. A biochip that has two sets of Au electrode arrays, each consist of shared reference/counter electrodes and 3 working electrodes has been used for the assay. The biochip has been integrated to a microfluidics system and all steps of the assay have been performed during the reagent flow to achieve fast and sensitive DNA detection. On-line hybridization of the target on to the capture probe immobilized surface resulted in a very short assay duration with respect to the conventional static assays. The binding of the avidin and enzyme modified Au nanoparticles to the biotinylated detection probe and the subsequent injection of the substrate enabled a real-time amperometric measurement with a detection limit of 6×10(-12) M target DNA (calibration curve r(2)=0.98). The developed assay enables fast and sensitive detection of cyanopeptolin producing cyanobacteria from freshwater samples and hence shows a promising technology for toxic microorganism detection from environmental samples.

  13. Simple and suitable immunosensor for β-lactam antibiotics analysis in real matrixes: milk, serum, urine.

    Science.gov (United States)

    Merola, Giovanni; Martini, Elisabetta; Tomassetti, Mauro; Campanella, Luigi

    2015-03-15

    The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics.

  14. Fabrication of molecular nanopatterns at aluminium oxide surfaces by nanoshaving of self-assembled monolayers of alkylphosphonates.

    Science.gov (United States)

    El Zubir, Osama; Barlow, Iain; Leggett, Graham J; Williams, Nicholas H

    2013-11-21

    Nanoshaving, by tracing an atomic force microscope probe across a surface at elevated load, has been used to fabricate nanostructures in self-assembled monolayers of alkylphosphonates adsorbed at aluminium oxide surfaces. The simple process is implemented under ambient conditions. Because of the strong bond between the alkylphosphonates and the oxide surface, loads in excess of 400 nN are required to pattern the monolayer. Following patterning of octadecylphosphonate SAMs, adsorption of aminobutyl phosphonate yielded features as small as 39 nm. Shaving of monolayers of aryl azide-terminated alkylphosphonates, followed by attachment of polyethylene glycol to unmodified regions in a photochemical coupling reaction, yielded 102 nm trenches into which NeutrAvidin coated, dye-labelled, polymer nanospheres could be deposited, yielding bright fluorescence with little evidence of non-specific adsorption to other regions of the surface. Structures formed in alkylphosphonate films by nanoshaving were used to etch structures into the underlying metal. Because of the isotropic nature of the etch process, and the large grain size, some broadening was observed, but features 25-35 nm deep and 180 nm wide were fabricated.

  15. Cytokeratin 5/6 expression in benign and malignant breast lesions

    Directory of Open Access Journals (Sweden)

    Bhalla Amarpreet

    2010-10-01

    Full Text Available Background: Cytokeratin s (CK are used for the fingerprinting of carcinomas in general. In breast tissue, the luminal epithelial cells express CK 8/18, CK 7 and CK 19, while basal/myoepithelial cells express CK 5/6, CK 14 and CK 17. Material and Methods: Immunohistochemical staining for cytokeratin 5/6 was applied on cell block sections of 23 cases of benign and 25 cases of malignant breast lesions using avidin biotin peroxidase technique. The distribution and intensity of staining was recorded and graded semiquantitatively. Result: All benign lesions showed positive immunoreaction, with the staining index varying from 6-9, except lactating adenoma. The malignant lesions comprised three cases of ductal carcinoma in situ (DCIS and 22 cases of infiltrating ductal carcinoma, not otherwise specified, IDC (NOS. None of the DCIS cases showed a positive immunoreaction. Among the IDC (NOS lesions, six cases of grade III breast carcinoma exhibited a positive immunohistochemical reaction, the staining index of which varied from 2-6. The staining reaction in the malignant lesions was only cytoplasmic and the intensity was significantly less than that of benign lesions. Conclusion: CK 5/6 expression breast carcinoma implies a ′basal like′ molecular phenotype and is associated with poor prognosis. This antibody is also used as a component of panels to differentiate benign and malignant breast lesions.

  16. Study of serum Helicobacter pylori soluble antigen

    Institute of Scientific and Technical Information of China (English)

    吴勤动; 朱永良

    2002-01-01

    Objective:to explore a new serological method for detecting Helicobacter pylori(H.pylori) infection.Methods:Serum soluble antigen of H.pylori was detected by using avidin-biotin ELISA technique to evaluate the status of H.pylori infection and for comparison with rapid urease test(RUT).histologic examination and serology,Results:The sensitivity,specificity,positive predictive value and negative predictive value were 77.46% ,91.07%,91.67% and 76.12%,respectively.The prevalence rate of werum H. pylori soluble antigen in 138 patients undergong endoscopy was similar to the rate obtained by 14 C-UBT methods(P>0.05).Conclusions:The detection of serum H.pylori soluble antigen(HpSAg) could be used as a new serological method which is accurate,and convenient,not affected by the memorizing raction of serum antibody;is more sensitive,more specific and suitable for dinical diagriosis,and evaluation of eradication and for follow-up of H.pylori as well as for detection in children and pregnant women.

  17. Selective tumor imaging by a novel tumor specific aralin-infrared-to-visible phosphor conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Kawasaki, Y; Gotoh, Y; Komeno, T; Todoroki, R; Tashiro, F [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, 278-8510 Chiba (Japan); Tokuzen, K; Nagasaki, Y; Soga, K [Polyscale Technology Research Center, 2641 Yamazaki, Noda, 278-8510 Chiba (Japan); Kamimura, M [Graduate School of Pure and Applied Science, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8573, Ibaraki (Japan); Tomatsu, M, E-mail: ftashir@rs.noda.tus.ac.j [Akita Research Institute for Food and Brewing (ARIF), 4-26 Azasanuki Arayamachi, Akita, 010-1623 Akita (Japan)

    2009-11-15

    Aralin is a novel cytotoxic protein from Aralia elata and selectively induces apoptosis in transformed cells as compared to normal cells (1). Aralin is a lectin specific for sugar chain such as galactose and possesses RNA N-glycosidase activity. In this study, antitumor potency of aralin was analyzed using the poly(ethyleneglycol) (PEG)/streptavidin co-immobilized infrared-to-visible upconversion phosphors, Y{sub 2}O{sub 3} nanoparticles (2). Cy3-conjugated aralin could clearly detect the surface of SV40-transformed VA13 and human cervical carcinoma HeLa cells, but to a lesser extent on the normal human fibroblast WI-38 cells. Conjugation of aralin with PEGylated Y{sub 2}O{sub 3} nanophosphor was carried out via biotin-avidin binding. The Y{sub 2}O{sub 3}-conjugated aralin also clearly visualize by a fluorescence microscope measurements equipped with near-infrared excitation source scanning in HeLa cells. It is also important to note that no remarkable damage to the cells was observed during these observations. Thus, these data imply that the Y{sub 2}O{sub 3}-conjugated aralin would potentially be useful material for tumor detection in vivo.

  18. Binding-regulated click ligation for selective detection of proteins.

    Science.gov (United States)

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins.

  19. Biological Applications of Extraordinary Electroconductance (EEC)

    Science.gov (United States)

    Tran, L. C.; Werner, F. M.; Solin, S. A.

    2014-03-01

    Rapid detection of biomolecular concentration is a fundamental goal for lab on a chip diagnostic systems. The Extraordinary Electroconductance (EEC) sensor, a stacked, AuTi-GaAs metal semiconductor hybrid structure (MSH), has been previously demonstrated to have an electric field sensitivity of 3.05V/cm in a mesoscopic-scale structure fabricated at the center of a parallel plate capacitor. In this work, we demonstrate the first successful application of EEC sensors as electrochemical detectors of molecular binding to the sensor surface. The negatively charged avidin derivative, captavidin, was applied with varying captavidin concentrations in phosphate buffered saline (PBS). The four-point measured resistance of bare EEC sensors was shown to increase by a factor of four due to captavidin binding at the sensor surface, as compared to a baseline binding assay in which the captavidin binding sites were blocked. Calculations for approximate electric field strengths introduced by a bound captavidin molecule will also presented. EEC sensors' four point measurements showed robustness and stability in spite of variations in the functional, linking layer. S.A.S. is a co-founder of and has a financial interest in PixelEXX, a start-up company whose mission is to market imaging arrays.

  20. Selective tumor imaging by a novel tumor specific aralin-infrared-to-visible phosphor conjugate

    Science.gov (United States)

    Kawasaki, Y.; Gotoh, Y.; Tokuzen, K.; Kamimura, M.; Komeno, T.; Tomatsu, M.; Todoroki, R.; Nagasaki, Y.; Soga, K.; Tashiro, F.

    2009-11-01

    Aralin is a novel cytotoxic protein from Aralia elata and selectively induces apoptosis in transformed cells as compared to normal cells (1). Aralin is a lectin specific for sugar chain such as galactose and possesses RNA N-glycosidase activity. In this study, antitumor potency of aralin was analyzed using the poly(ethyleneglycol) (PEG)/streptavidin co-immobilized infrared-to-visible upconversion phosphors, Y2O3 nanoparticles (2). Cy3-conjugated aralin could clearly detect the surface of SV40-transformed VA13 and human cervical carcinoma HeLa cells, but to a lesser extent on the normal human fibroblast WI-38 cells. Conjugation of aralin with PEGylated Y2O3 nanophosphor was carried out via biotin-avidin binding. The Y2O3-conjugated aralin also clearly visualize by a fluorescence microscope measurements equipped with near-infrared excitation source scanning in HeLa cells. It is also important to note that no remarkable damage to the cells was observed during these observations. Thus, these data imply that the Y2O3-conjugated aralin would potentially be useful material for tumor detection in vivo.

  1. Reversal of multidrug resistance phenotype in human breast cancer cells using doxorubicin-liposome-microbubble complexes assisted by ultrasound.

    Science.gov (United States)

    Deng, Zhiting; Yan, Fei; Jin, Qiaofeng; Li, Fei; Wu, Junru; Liu, Xin; Zheng, Hairong

    2014-01-28

    The circumvention of multidrug resistance (MDR) plays a critically important role in the success of chemotherapy. The aim of this work is to investigate the effectiveness and possible mechanisms of the reversal of MDR phenotype in human breast cancer cells by using doxorubicin-liposome-microbubble complexes (DLMC) assisted by ultrasound (US). DLMC is fabricated through conjugating doxorubicin (DOX)-liposome (DL) to the surface of microbubbles (MBs) via the biotin-avidin linkage. The resulting drug-loaded complexes are then characterized and incubated with MCF-7/ADR human breast cancer cells and followed by US exposure. Our results show the more rapid cellular uptake, evident enhancement of nuclear accumulation and less drug efflux in the resistant cells treated by DLMC+US than those treated by DL, DL+verapamil under the same US treatment or DLMC without US. The enhanced drug delivery and cellular uptake also associated with the increase of cytotoxicity against MCF-7/ADR cells, lower MCF-7/ADR cell viability and higher apoptotic cells. Mechanism investigations further disclose a significant increase of reactive oxygen species (ROS) level, enhanced DNA damage and obvious reduction of P-glycoprotein expression in the resistant cells treated with DLMC+US compared with the control cases of cells treated by DLMC, DL+US or DL+verapamil+US. In conclusion, our study demonstrates that DLMC in combination with US may provide an effective delivery of drug to sensitize cells to circumvent MDR and to enhance the therapeutic index of the chemotherapy.

  2. Paclitaxel-liposome-microbubble complexes as ultrasound-triggered therapeutic drug delivery carriers.

    Science.gov (United States)

    Yan, Fei; Li, Lu; Deng, Zhiting; Jin, Qiaofeng; Chen, Juanjuan; Yang, Wei; Yeh, Chih-Kuang; Wu, Junru; Shandas, Robin; Liu, Xin; Zheng, Hairong

    2013-03-28

    Liposome-microbubble complexes (LMC) have become a promising therapeutic carrier for ultrasound-triggered drug delivery to treat malignant tumors. However, the efficacy for ultrasound-assisted chemotherapy in vivo and the underlying mechanisms remain to be elucidated. Here, we investigated the feasibility of using paclitaxel-liposome-microbubble complexes (PLMC) as possible ultrasound (US)-triggered targeted chemotherapy against breast cancer. PTX-liposomes (PL) were conjugated to the microbubble (MB) surface through biotin-avidin linkage, increasing the drug-loading efficiency of MBs. The significant increased release of payloads from liposome-microbubble complexes was achieved upon US exposure. We used fluorescent quantum dots (QDs) as a model drug to show that released QDs were taken up by 4T1 breast cancer cells treated with QD-liposome-microbubble complexes (QLMC) and US, and uptake depended on the exposure time and intensity of insonication. We found that PLMC plus US inhibited tumor growth more effectively than PL plus US or PLMC without US, not only in vitro, but also in vivo. Histologically, the inhibition of tumor growth appeared to result from increased apoptosis and reduced angiogenesis in tumor xenografts. In addition, a significant increase of drug concentration in tumors was observed in comparison to treatment with non-conjugated PL or PLMC without US. The significant increase in an antitumor efficacy of PLMC plus US suggests their potential use as a new targeted US chemotherapeutic approach to inhibit breast cancer growth.

  3. Rapid plasmid library screening using RecA-coated biotinylated probes.

    Science.gov (United States)

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M

    1986-12-01

    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  4. Role of vasoactive intestinal peptide and nitric oxide in the modulation of electroacupucture on gastric motility in stressed rats

    Institute of Scientific and Technical Information of China (English)

    Guo-Ming Shen; Mei-Qi Zhou; Guan-Sun Xu; Ying Xu; Gang Yin

    2006-01-01

    AIM: To investigate the effects and mechanisms of vasoactive intestinal peptide (VIP) and nitric oxide (NO)in the modulation of electroacupucture (EA) on gastric motility in restrained-cold stressed rats.METHODS: An animal model of gastric motility disorder was established by restrained-cold stress. Gastric myoelectric activities were recorded by electrogastroent erography (EGG). VIP and NO concentrations in plasma and gastric mucosal and bulb tissues were detected by radioimmunoassay (RIA). VIP expression in the gastric wails was assayed using avidin-biotin-peroxidase complex (ABC) and image analysis.RESULTS: In cold restrained stressed rats, EGG was disordered and irregular. The frequency and amplitude of gastric motility were higher than that in control group (P< 0.01). VIP and NO contents of plasma, gastric mucosal and bulb tissues were obviously decreased (P < 0.01).Following EA at "Zusanli" (ST36), the frequency and amplitude of gastric motility were obviously lowered (P <0.01), while the levels of VIP and NO in plasma, gastric mucosal and bulb tissues increased strikingly (P < 0.01,P < 0.05) and expression of VIP in antral smooth muscle was elevated significantly (P < 0.01) in comparison with those of model group.CONCLUSION: VIP and NO participate in the modulatory effect of EA on gastric motility. EA at "Zusanli"acupoint (ST36) can improve gastric motility of the stressed rats by increasing the levels of VIP and NO.

  5. Polythiophene derivative on quartz resonators for miRNA capture and assay.

    Science.gov (United States)

    Palaniappan, Al; Cheema, Jamal Ahmed; Rajwar, Deepa; Ammanath, Gopal; Xiaohu, Liu; Koon, Lim Seng; Yi, Wang; Yildiz, Umit Hakan; Liedberg, Bo

    2015-12-07

    A novel approach for miRNA assay using a cationic polythiophene derivative, poly[3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrobromide] (PT), immobilized on a quartz resonator is proposed. The cationic PT enables capturing of all RNA sequences in the sample matrix via electrostatic interactions, resulting in the formation of PT-RNA duplex structures on quartz resonators. Biotinylated peptide nucleic acid (b-PNA) sequences are subsequently utilized for the RNA assay, upon monitoring the PT-RNA-b-PNA triplex formation. Signal amplification is achieved by anchoring avidin coated nanoparticles to b-PNA in order to yield responses at clinically relevant concentration regimes. Unlike conventional nucleic acid assay methodologies that usually quantify a specific sequence of RNA, the proposed approach enables the assay of any RNA sequence in the sample matrix upon hybridization with a PNA sequence complementary to the RNA of interest. As an illustration, successful detection of mir21, (a miRNA sequence associated with lung cancer) is demonstrated with a limit of detection of 400 pM. Furthermore, precise quantification of mir21 in plasma samples is demonstrated without requiring PCR and sophisticated instrumentation.

  6. Probing molecular interactions with methylene blue derivatized self-assembled monolayers

    Directory of Open Access Journals (Sweden)

    Eleni Koutsoumpeli

    2015-12-01

    Full Text Available The emergence of stratified and personalised medicine and the associated need for highly multiplexed detection strategies are driving the development of innovative sensor technology. Electronic immunosensor arrays capable of label-free and highly parallel monitoring of ligand binding have emerged as a particularly promising technology capable of meeting these new diagnostic challenges. In this study, we present an approach for interrogating molecular interactions electronically using redox active molecular monolayers. Specifically, we have synthesised self-assembled molecular monolayers assembled from long-chain alkanethiols (LCAT incorporating oligoethyleneglycol (OEG linkers that can be derivatized with a range of functional groups, including the redox active molecule methylene blue. Critically, we show that the electron transport properties of this redox-active monolayer are highly sensitive to the electrochemical environment, including the local concentration of protons and the electrostatic potential at the plane of electron transfer. Using a combination of cyclic voltammetry and QCM-D to study in detail the behaviour of the monolayer during functionalisation and analyte binding, we demonstrate that these redox properties can be exploited for the electrochemical sensing of molecular interactions (biotin–avidin in our case on SAMs. Given the versatility of LCAT-OEG monolayers, in terms of linker lengths, choice of functional group, and ability to create mixed component layers and the straight-forward assembly of mixed SAMs of high quality, our electrochemical sensing approach forms an excellent and generic label-free platform for probing a wide range of molecular interactions.

  7. Expression of intercellular adhesion molecule-1 in UVA-irradiated human skin cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Treina, G.; Scaletta, C.; Frenk, E.; Applegate, L.A. [University Hospital-CHUV, Lausanne (Switzerland). Laboratory of Photobiology; Fourtanier, A.; Seite, S. [L`Oreal-Centre de Recherche Charles Zviak (France). Recherche Avancee Biologie

    1996-08-01

    Ultraviolet A (UVA) radiation represents an important oxidative stress to human skin and certain forms of oxidative stress have been shown to modulate intercellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 has been shown to play an important part in many immune reactions and the perturbations of this molecule by ultraviolet radiation could have implications in many inflammatory responses. An enhancement immunohistochemical method with avidin/biotin was used for analysing the early effects of UVA radiation on human cell cultures and human skin (340-400 nm). Both in vitro and in vivo data show that ICAM-1 staining in epidermal keratinocytes, which was expressed constitutively, decreased in a UVA dose-dependent manner. The decrease was most noted at 3-6 h following UVA radiation with some ICAM-1 staining returning by 48 h post-UVA. ICAM-1 positive staining in the dermis was specific for vascular structures and was increased 24 h after UVA radiation. Cultured dermal fibroblasts exhibited ICAM-1 staining which increased slightly within 6-48 h post-UVA radiation. As epidermal ICAM-1 expression is depleted following UVA radiation and dermal expression increases due to an increase in the vascular structures, ICAM-1 provides a valuable marker following UVA radiation in human skin that can be readily measured in situ. (author).

  8. Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip.

    Science.gov (United States)

    Kim, Yeon Seok; Jung, Ho Sup; Matsuura, Toshihiko; Lee, Hea Yeon; Kawai, Tomoji; Gu, Man Bock

    2007-05-15

    An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.

  9. Nanopeptamers for the development of small-analyte lateral flow tests with a positive readout.

    Science.gov (United States)

    Vanrell, Lucía; Gonzalez-Techera, Andrés; Hammock, Bruce D; Gonzalez-Sapienza, Gualberto

    2013-01-15

    There is a great demand for rapid tests that can be used on-site for the detection of small analytes, such as pesticides, persistent organic pollutants, explosives, toxins, medicinal and abused drugs, hormones, etc. Dipsticks and lateral flow devices, which are simple and provide a visual readout, may be the answer, but the available technology for these compounds requires a competitive format that loses sensitivity and produces readings inversely proportional to the analyte concentration, which is counterintuitive and may lead to potential misinterpretation of the result. In this work, protein-multipeptide constructs composed of anti-immunocomplex peptides selected from phage libraries and streptavidin/avidin as core protein were used for direct detection of small compounds in a noncompetitive two-site immunoassay format that performs with increased sensitivity and positive readout. These constructs that we termed "nanopeptamers" allow the development of rapid point-of-use tests with a positive visual end point of easy interpretation. As proof of concept, lateral flow assays for the herbicides molinate and clomazone were developed and their performance was characterized with field samples.

  10. Conformal nano-thin modified polyelectrolyte coatings for encapsulation of cells.

    Science.gov (United States)

    Granicka, L H; Antosiak-Iwańska, M; Godlewska, E; Strawski, M; Szklarczyk, M; Maranowski, B; Kowalewski, C; Wiśniewsk, J

    2011-10-01

    Encapsulation of cells in polymeric shells allows for separation of biological material from produced factors, which may find biotechnological and biomedical applications. Human T-lymphocyte cell line Jurkat as well as rat pancreatic islets were encapsulated using LbL technique within shells of polyelectrolyte modified by incorporation of biotin complexed with avidin to improve cell coating and to create the potential ability to elicit specific biochemical responses. The coating with nano-thin modified shells allowed for maintenance of the evaluated cells' integrity and viability during the 8-day culture. The different PE impact may be observed on different biological materials. The islets exhibited lower mitochondrial activity than the Jurkat cells. Nevertheless, coating of cells with polyelectrolyte modified membrane allowed for functioning of both model cell types: 10 μm leukemia cells or 150 μm islets during the culture. Applied membranes maintained the molecular structure during the culture period. The conclusion is that applied modified membrane conformation may be recommended for coating shells for biomedical purposes.

  11. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

    Science.gov (United States)

    de Boer, Ernie; Rodriguez, Patrick; Bonte, Edgar; Krijgsveld, Jeroen; Katsantoni, Eleni; Heck, Albert; Grosveld, Frank; Strouboulis, John

    2003-06-01

    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

  12. Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip.

    Science.gov (United States)

    Wang, Shi-Hua; Wen, Ji-Kai; Zhou, Ya-Feng; Zhang, Zhi-Ping; Yang, Rui-Fu; Zhang, Ji-Bin; Chen, Jia; Zhang, Xian-En

    2004-11-01

    Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.

  13. Evanescent Wave Fiber Optic Biosensor for Salmonella Detection in Food

    Directory of Open Access Journals (Sweden)

    Arun K. Bhunia

    2009-07-01

    Full Text Available Salmonella enterica is a major food-borne pathogen of world-wide concern. Sensitive and rapid detection methods to assess product safety before retail distribution are highly desirable. Since Salmonella is most commonly associated with poultry products, an evanescent wave fiber-optic assay was developed to detect Salmonella in shell egg and chicken breast and data were compared with a time-resolved fluorescence (TRF assay. Anti-Salmonella polyclonal antibody was immobilized onto the surface of an optical fiber using biotin-avidin interactions to capture Salmonella. Alexa Fluor 647-conjugated antibody (MAb 2F-11 was used as the reporter. Detection occurred when an evanescent wave from a laser (635 nm excited the Alexa Fluor and the fluorescence was measured by a laser-spectrofluorometer at 710 nm. The biosensor was specific for Salmonella and the limit of detection was established to be 103 cfu/mL in pure culture and 104 cfu/mL with egg and chicken breast samples when spiked with 102 cfu/mL after 2–6 h of enrichment. The results indicate that the performance of the fiber-optic sensor is comparable to TRF, and can be completed in less than 8 h, providing an alternative to the current detection methods.

  14. Interdigitated microelectrode based impedance biosensor for detection of salmonella enteritidis in food samples

    Energy Technology Data Exchange (ETDEWEB)

    Kim, G [National Institute of Agricultural Engineering, 249 Seodun-dong, Suwon, Republic of Korea, 441-100 (Korea, Republic of); Morgan, M; Hahm, B K; Bhunia, A [Department of Food Science, Purdue University, West Lafayette, IN 47907 (United States); Mun, J H; Om, A S [Department of Food and Nutrient, Hanyang University, 17 Haengdang-dong, Seoul, Republic of Korea, 133-791 (Korea, Republic of)], E-mail: giyoungkim@rda.go.kr

    2008-03-15

    Salmonella enteritidis outbreaks continue to occur, and S. enteritidis-related outbreaks from various food sources have increased public awareness of this pathogen. Conventional methods for pathogens detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Recently developed biosensors have shown great potential for the rapid detection of foodborne pathogens. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on avidin-biotin binding on the surface of the IME to form an active sensing layer. To increase the sensitivity of the sensor, three types of sensors that have different electrode gap sizes (2 {mu}m, 5 {mu}m, 10 {mu}m) were fabricated and tested. The impedimetric biosensor could detect 10{sup 3} CFU/mL of Salmonella in pork meat extract with an incubation time of 5 minutes. This method may provide a simple, rapid and sensitive method to detect foodborne pathogens.

  15. Interdigitated microelectrode based impedance biosensor for detection of salmonella enteritidis in food samples

    Science.gov (United States)

    Kim, G.; Morgan, M.; Hahm, B. K.; Bhunia, A.; Mun, J. H.; Om, A. S.

    2008-03-01

    Salmonella enteritidis outbreaks continue to occur, and S. enteritidis-related outbreaks from various food sources have increased public awareness of this pathogen. Conventional methods for pathogens detection and identification are labor-intensive and take days to complete. Some immunological rapid assays are developed, but these assays still require prolonged enrichment steps. Recently developed biosensors have shown great potential for the rapid detection of foodborne pathogens. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on avidin-biotin binding on the surface of the IME to form an active sensing layer. To increase the sensitivity of the sensor, three types of sensors that have different electrode gap sizes (2 μm, 5 μm, 10 μm) were fabricated and tested. The impedimetric biosensor could detect 103 CFU/mL of Salmonella in pork meat extract with an incubation time of 5 minutes. This method may provide a simple, rapid and sensitive method to detect foodborne pathogens.

  16. Effect of Buzhong Yiqi Decoction(补中益气汤)on Murine Liver Damage Induced by Food Allergy

    Institute of Scientific and Technical Information of China (English)

    陈虹; 董阳深; 陈奋华; 纪经智; 陈岩峰; 上野幸三; 饭仓洋治

    2004-01-01

    Objective: To investigate the effect of Buzhong Yiqi decoction (, BZYQD) on liver damage induced by food allergy in mice. Methods: Nc/Jic strain mice with high levels of serum IgE were sensitized by ovalbumin (OVA), and then divided into two groups and respectively treated with BZYQD (treated group) or normal saline (model group). Samples of serum, liver tissues and small intestine were collected two weeks later, and another group of non-sensitized mice was set as the normal group. The levels of serum alanine aminotransferase (ALT) were measured with spectrophotometry. The liver tissue and small intestine were stained with hematoxylin and eosin (HE) for pathologic analysis. The liver samples were also subjected to analysis of CD4-T helper cell and cytokine (interleukin-4, IL-4, interleukin-6, IL-6) expression with immunohistochemical (avidin-biotin complex, ABC) method. Results; Serum ALT levels decreased and obvious pathologic improvements were seen in the mice treated with BZYQD. And compared with the model mice, the number of positive cells of IL-4, IL-6 and CD4 cell decreased significantly in those treated with BZYQD. Conclusion: BZYQD can effectively decrease the production of cytokines associated with allergic reaction in the liver of mice thus effective in treating liver damage caused by food allergy.

  17. Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage.

    Science.gov (United States)

    Brasino, Michael; Lee, Ju Hun; Cha, Jennifer N

    2015-02-01

    For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented-by either chemical functionalization or genetic engineering-with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

  18. Detection of Target Biomolecules by Magnetic Reporting Using Rod-Like Nanosensors

    Science.gov (United States)

    Guertin, R. P.; Goldberg, E.; Harrah, T. P.; Sonkusale, S.; Park, K.; Sun, S.; Oh, J. I.; Naughton, M.

    2008-03-01

    We describe the ongoing development of a device to assay a variety of cellular, viral and molecular targets by measuring the increase of the Brownian relaxation time, τ, in solution of magnetically-tagged nanoscale detectors. The shift shows as a frequency reduction of the peak of the complex magnetic susceptibility, χ(φ)''. Measurements of χ(φ)'' with 12 nm monodisperse nanoparticles of CoFe2O4 coated with polyethelyne glycol reveal spectra with the narrowest lines yet reported. Thin avidin coating of these particles reveals small shifts in χ(φ)''. Bacteriophage T4 tail fibers, engineered to specific lengths (30-150 nm), were employed as the platform for magnetic nanoparticle attachment and at the other end for an inserted target peptide epitope. Attachment of the nanoparticles to bacteriophage T4 tail fibers was successful, though no detectable shifts in χ(φ)'' were detected due to weak attachment. The advantages associated with non-spherical geometry detectors will be discussed, as will preliminary measurements with rare earth oxide magnetic nanoparticles. Progress on miniaturization and low power requirements of the electronic detection system will be reported. Supported by NERCE/BEID (NIAID).

  19. Hybridization chain reaction-based fluorescence immunoassay using DNA intercalating dye for signal readout.

    Science.gov (United States)

    Deng, Yan; Nie, Ji; Zhang, Xiao-hui; Zhao, Ming-Zhe; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2014-07-07

    A novel format of fluorescence immunosorbent assay based on the hybridization chain reaction (HCR) using a DNA intercalating dye for signal readout was constructed for the sensitive detection of targets, both in competitive and sandwich modes. In this platform, the capture and recognition processes are based on immunoreactions and the signal amplification depends on the enzyme-free, isothermal HCR-induced labelling event. After a competitive or a sandwich immunoreaction, a biotinylated capture DNA was bound to a biotinylated signal antibody through avidin, and triggered the HCR by two specific hairpins into a nicked double helix. Gene Finder (GF), a fluorescent probe for double-strand DNA, was intercalated in situ into the amplified chain to produce the fluorescence signal. The limit of detection (LOD) for rabbit IgG in competitive mode by HCR/GF immunoassay was improved at least 100-fold compared with the traditional fluorescence immunoassay using the fluorescein isothiocyanate-labelled-streptavidin or fluorescein isothiocyanate-labelled second antibody as the signal readout. The proposed fluorescence immunoassay was also demonstrated by using α-fetoprotein as the model target in sandwich mode, and showed a wide linear range from 28 ng mL(-1) to 20 μg mL(-1) with a LOD of 6.0 ng mL(-1). This method also showed satisfactory analysis in spiked human serum, which suggested that it might have great potential for versatile applications in life science and point-of-care diagnostics.

  20. Piezoresistive microcantilever aptasensor for ricin detection and kinetic analysis

    Science.gov (United States)

    Liu, Zhi-Wei; Tong, Zhao-Yang; Liu, Bing; Hao, Lan-Qun; Mu, Xi-Hui; Zhang, Jin-Ping; Gao, Chuan

    2015-04-01

    Up to now, there has been no report on target molecules detection by a piezoresistive microcantilever aptasensor. In order to evaluate the test performance and investigate the response dynamic characteristics of a piezoresistive microcantilever aptasensor, a novel method for ricin detection and kinetic analysis based on a piezoresistive microcantilever aptasensor was proposed, where ricin aptamer was immobilised on the microcantilever surface by biotin-avidin binding system. Results showed that the detection limit of ricin was 0.04μg L-1 (S/N ≥ 3). A linear relationship between the response voltage and the concentration of ricin in the range of 0.2μg L-1-40μg L-1 was obtained, with the linear regression equation of ΔUe = 0.904C + 5.852 (n = 5, R = 0.991, p soil, and flour sample) determination met the analysis requirements, which was 90.5˜95.5% and 7.85%˜9.39%, respectively. On this basis, a reaction kinetic model based on ligand-receptor binding and the relationship with response voltage was established. The model could well reflect the dynamic response of the sensor. The correlation coefficient (R) was greater than or equal to 0.9456 (p < 0.001). Response voltage (ΔUe) and response time (t0) obtained from the fitting equation on different concentrations of ricin fitted well with the measured values.

  1. Aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) for electrochemical analysis of tumor biomarkers.

    Science.gov (United States)

    Wang, Pengjuan; Wan, Ying; Deng, Shengyuan; Yang, Shulin; Su, Yan; Fan, Chunhai; Aldalbahi, Ali; Zuo, Xiaolei

    2016-12-15

    Herein, an aptamer-initiated on-particle template-independent enzymatic polymerization (aptamer-OTEP) strategy for electrochemical aptasensor (E-aptasensor) is developed for analysis of cancer biomarker carcino-embryonic antigen (CEA). A pair of DNA aptamers is employed which can be specifically bond with CEA simultaneously. One of the aptamer is thiolated at 3'-terminal and immobilized onto the gold electrode as a capture probe, while the other one has a thiol group at its 5'-terminal and is modified onto the gold nanoparticles surface to form a nanoprobe. In the present of target, the two aptamers can "sandwich" the target, thus the nanoprobe is attached to the electrode. Then terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the incorporation of biotin labeled dNTPs into the 3'-OH terminals of the DNA aptamer on the nanoprobe. The as-generated long DNA oligo tentacles allow specific binding of numerous avidin modified horseradish peroxidase (Av-HRP), resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. Taking advantage of the enzyme based nucleic acid amplification and nanoprobe, this strategy is demonstrated to possess the outstanding amplification efficiency.

  2. Relationship of abnormal Tamm-Horsfall glycoprotein localization to renal morphology and function.

    Science.gov (United States)

    Chambers, R; Groufsky, A; Hunt, J S; Lynn, K L; McGiven, A R

    1986-07-01

    Tamm-Horsfall glycoprotein (TH) distribution was studied using a biotin-avidin immunoperoxidase technique in renal biopsies from 166 consecutive patients and 8 normal kidneys. Tubulointerstitial damage was independently assessed and graded. In 109 patients TH antibodies were measured by ELISA and in 30 of these urinary TH and beta 2-microglobulin excretions were measured by radioimmunoassay. In 124 biopsies only distal tubular epithelium and casts were stained. Glomerular space (8) or interstitial (34) deposits were seen in 42 biopsies; 16/68 with glomerulonephritis, 4/14 with systemic vasculitis, 12/33 with chronic interstitial nephritis, 1/8 with acute interstitial nephritis, 9/43 with other nephropathies. There was no correlation between TH distribution and the degree of tubulointerstitial damage (p greater than 0.5), urinary TH excretion (p greater than 0.05), urinary beta 2-microglobulin excretion (p greater than 0.05), glomerular filtration rate, urinary concentrating ability, or the incidence of pyuria. TH antibodies did not correlate with TH distribution (p greater than 0.5) or the degree of tubulointerstitial damage. Abnormal TH distribution showed no statistical relationship to the degree of tubulointerstitial damage, changes in renal function or levels of TH antibodies.

  3. Biomolecule-recognition gating membrane using biomolecular cross-linking and polymer phase transition.

    Science.gov (United States)

    Kuroki, Hidenori; Ito, Taichi; Ohashi, Hidenori; Tamaki, Takanori; Yamaguchi, Takeo

    2011-12-15

    We present for the first time a biomolecule-recognition gating system that responds to small signals of biomolecules by the cooperation of biorecognition cross-linking and polymer phase transition in nanosized pores. The biomolecule-recognition gating membrane immobilizes the stimuli-responsive polymer, including the biomolecule-recognition receptor, onto the pore surface of a porous membrane. The pore state (open/closed) of this gating membrane depends on the formation of specific biorecognition cross-linking in the pores: a specific biomolecule having multibinding sites can be recognized by several receptors and acts as the cross-linker of the grafted polymer, whereas a nonspecific molecule cannot. The pore state can be distinguished by a volume phase transition of the grafted polymer. In the present study, the principle of the proposed system is demonstrated using poly(N-isopropylacrylamide) as the stimuli-responsive polymer and avidin-biotin as a multibindable biomolecule-specific receptor. As a result of the selective response to the specific biomolecule, a clear permeability change of an order of magnitude was achieved. The principle is versatile and can be applied to many combinations of multibindable analyte-specific receptors, including antibody-antigen and lectin-sugar analogues. The new gating system can find wide application in the bioanalytical field and aid the design of novel biodevices.

  4. Localization of metallothionein in the genital organs of the male rat.

    Science.gov (United States)

    Nishimura, H; Nishimura, N; Tohyama, C

    1990-07-01

    We studied the immunohistological localization of metallothionein (MT), a low molecular weight metal binding protein, in male rat genital organs (testis, epididymis, ejaculatory duct, seminal vesicle, coagulating gland, and prostate) by use of the avidin-biotin-peroxidase complex method. MT concentrations in testis, seminal vesicle, and prostate ranged from 15-30 micrograms/g tissue. In testis, seminiferous tubules with mature spermatozoa exhibited weak MT staining, whereas the tubules containing differentiating spermatogenic cells but not containing spermatozoa showed strong MT staining. No MT immunostaining was observed in Leydig cells. In growing rat testes, the pattern of MT immunostaining was found to change with development: MT was found in supporting cells only on Day 7, spermatogonia adjacent to basement membrane on Day 14, and spermatocytes localized in the central part of the tubules on Day 21. Strong MT immunostaining in the basal cells was a common feature in other genital tissues, except the ductus efferentes. In prostate, the strongest MT staining was found in the lateral lobe, and MT was localized in apocrine secretions in the dorsal lobe. The present results suggest a close association of MT with cell proliferation and differentiation, as well as possible involvement of MT in supply or storage of zinc ions.

  5. Surface characterization of binary grafting of AAc/NIPAAm onto poly(tetrafluoroethylene) (PTFE)

    Energy Technology Data Exchange (ETDEWEB)

    Adem, E. [Instituto de Fisica-UNAM, A. Postal 20-364, Mexico, DF 01000 (Mexico); Avalos-Borja, M. [Centro de Ciencias de la Materia Condensada-UNAM, A. Postal 2681, Ensenada BC 22800 (Mexico); Bucio, E. [Instituto de Ciencias Nucleares-UNAM, A. Postal 70-543, Mexico, DF 04510 (Mexico); Burillo, G. [Instituto de Ciencias Nucleares-UNAM, A. Postal 70-543, Mexico, DF 04510 (Mexico)]. E-mail: burillo@nuclecu.unam.mx; Castillon, F.F. [Centro de Ciencias de la Materia Condensada-UNAM, A. Postal 2681, Ensenada BC 22800 (Mexico); Cota, L. [Centro de Ciencias de la Materia Condensada-UNAM, A. Postal 2681, Ensenada BC 22800 (Mexico)

    2005-07-01

    The present study shows results on radiation grafting of acrylic acid (AAc) and N-isopropylacrylamide (NIPAAm) mixtures onto polytetrafluoroethylene (PTFE) films. The main objective is to use the modified polymer for immobilization of the avidin-streptavidin systems or other bio-compounds. Grafting onto PTFE was carried out by the pre-irradiation oxidative method in air at different dose rates and irradiation doses. The irradiation was produced with the electron beam of a 2 MV van de Graaff accelerator, and gamma-rays, from a Gamma Beam 651 PT. The samples were placed in glass ampoules with the AAc and NIPAAm mixtures, in an aqueous solution. Graft polymerization was performed by heating of the polymer-monomer composition in an argon atmosphere at temperature of 50 deg. C. Surface funcionalization was studied by X-ray photoelectron spectroscopy (XPS); morphology of the surface was studied by scanning electron microscopy (SEM) and hydrophylization of the surface, by contact angle measurements (static method)

  6. Cytokine expression in paraffin wax-embedded tissues from conventional calves.

    Science.gov (United States)

    Pedrera, M; Sánchez-Cordón, P J; Romero-Trevejo, J L; Raya, A I; Núñez, A; Gómez-Villamandos, J C

    2007-05-01

    The cross-reactivity of antibodies against human tumour necrosis factor (TNF)alpha, interleukin (IL)-1alpha, IL-1beta and porcine IL-6, and the distribution of immunolabelled cells were evaluated on paraffin wax-embedded tissues from five healthy calves. The tissues were fixed in 10% buffered formalin or Bouin's solution and processed for structural studies and immunohistochemical studies by the avidin-biotin-peroxidase technique. Bouin's solution proved to be the more suitable fixative and Tween 20 the most effective antigen unmasking technique for increasing detectable antigenicity. Constitutive expression of TNFalpha, IL-1alpha, IL-1beta and IL-6 by different cell populations, mainly macrophage-like cells, was detected. Lymphoid organs displayed a higher presence of immunolabelled cells than did lung, liver or kidney. TNFalpha and IL-1alpha appeared as the predominant cytokines, especially in the gut-associated lymphoid tissue of the ileum and in the regional mesenteric lymph nodes. The results will facilitate investigation of the role of these cytokine-producing cells in inflammatory disease processes in calves.

  7. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.

    Science.gov (United States)

    Sainsbury, Frank; Rhéaume, Ann-Julie; Goulet, Marie-Claire; Vorster, Juan; Michaud, Dominique

    2012-12-01

    Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

  8. Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin.

    Science.gov (United States)

    Yahiro, Kinnosuke; Morinaga, Naoko; Satoh, Mamoru; Matsuura, Gen; Tomonaga, Takeshi; Nomura, Fumio; Moss, Joel; Noda, Masatoshi

    2006-10-01

    Some shiga toxin-producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N-glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin-agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to beta1 integrin, and its identification was confirmed with anti-integrin beta1 antibody. The p155 protein was identified as alpha2 integrin using anti-integrin alpha2 antibody. In addition, treatment of Vero cells with beta1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes alpha2beta1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB-induced morphological changes in Vero cells.

  9. A multiplex method for the detection of serum antibodies against in silico-predicted tumor antigens.

    Science.gov (United States)

    Reuschenbach, Miriam; Dörre, Jonathan; Waterboer, Tim; Kopitz, Jürgen; Schneider, Martin; Hoogerbrugge, Nicoline; Jäger, Elke; Kloor, Matthias; von Knebel Doeberitz, Magnus

    2014-12-01

    Humoral immune responses against tumor antigens are studied as indirect markers of antigen exposure and in cancer vaccine studies. An increasing number of tumor antigens potentially translated from mutant genes is identified by advances in genomic sequencing. They represent an interesting source for yet unknown immunogenic epitopes. We here describe a multiplex method using the Luminex technology allowing for the detection of antibodies against multiple in silico-predicted linear neo-antigens in large sets of sera. The approach included 32 synthetic biotinylated peptides comprising a predicted set of frameshift mutation-induced neo-antigens. The antigens were fused to a FLAG epitope to ensure monitoring antigen binding to avidin-linked microspheres in the absence of monoclonal antibodies. Analytical specificity of measured serum antibody reactivity was proven by the detection of immune responses in immunized rabbits and a colorectal cancer patient vaccinated with peptides included in the assay. The measured antibody responses were comparable to peptide ELISA, and inter-assay reproducibility of the multiplex approach was excellent (R (2) > 0.98) for 20 sera tested against all antigens. Our methodic approach represents a valuable platform to monitor antibody responses against predicted antigens. It may be used in individualized cancer vaccine studies, thereby extending the relevance beyond the model system in the presented approach.

  10. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang [Asan Medical Center, University of Ulsan, Seoul (Korea, Republic of)

    1994-07-15

    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  11. Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material

    Directory of Open Access Journals (Sweden)

    Ülo Langel

    2010-03-01

    Full Text Available Interest in cell-penetrating peptides (CPPs as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin--galactosidase (ABG, which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases.

  12. Study of serum Helicobacter pylori soluble antigen

    Institute of Scientific and Technical Information of China (English)

    吴勤动; 朱永良

    2002-01-01

    Objective: to explore a new serological method for detecting Helicobac ter pylori ( H. pylori ) infection. Methods: Serum soluble antigen of H. p ylor i was detected by using avidin-biotin ELISA technique to evaluate the status of H. pylori infection and for comparison with rapid urease test ( RUT ), histo logi c examination and serology. Results: The sensitivity, specificity, positive pred ictive value and negative predictive value were 77.46%, 91.07%, 91.67% a nd 76.12 %, respectively. The prevalence rate of serum H. pylori soluble antigen in 138 patients undergoing endoscopy was similar to the rate obtained by 14 C-UBT met hods ( P>0.05 ). Conclusions: The detection of serum H. pylori solub le antigen( HpSAg) could be used as a new serological method which is accurate, and convenie nt, not affected by the memorizing reaction of serum antibody; is more sensitive , m ore specific and suitable for clinical diagnosis, and evaluation of eradication and for follow-up of H. pylori as well as for detection in children and pre gnant women.

  13. Screening for hereditary fructose intolerance mutations by reverse dot-blot.

    Science.gov (United States)

    Lau, J; Tolan, D R

    1999-02-01

    An assay is described which is useful for genetic screening of the two most prevalent mutations that cause hereditary fructose intolerance (HFI). Both mutations lie within exon 5 of the aldolase B gene. Amplification of exon 5 from genomic DNA isolated from peripheral lymphocytes using biotinylated aldolase B-specific primers yields a biotin-tagged probe. This probe is hybridized to complementary poly(dT)-tailed allele specific oligonucleotides (ASOs) that are bound to a nylon membrane. The length of the ASOs, the amount bound to the membrane and the time of hybridization are optimized for discrimination of all four alleles under the same hybridization conditions. Detection of biotinylated amplified DNA is performed by creating an avidin-alkaline phosphatase complex and visualization by chemiluminescence. This assay can rapidly detect the two mutations, A149P and A174D, which cause >70% of HFI worldwide, and offers a rapid and sensitive assay that is much less invasive for the diagnosis of this often difficult to diagnose disorder.

  14. Screening study on new tumor marker periplakin for lung cancer

    Institute of Scientific and Technical Information of China (English)

    Shuqin Dai; Wei Li; Mian Kong; Yuzhen Zheng; Shuying Chen; Junye Wang; Linquan Zang

    2013-01-01

    Objective: The aim of this study was to use lung cancer targeting binding polypeptide ZS-9 to screen cDNA library of human lung cancer and obtain ZS-9 specific ligand to confirm tumor marker of non small-cell lung cancer. Methods: Artificially synthesize biotin labeled peptide ZS-9, anchored ZS-9 in the enzyme label plate coupled by avidin, used ZS-9 as probe to screen cDNA library of human lung cancer, after screening, obtained bacteriophage clone specifically binding with anchored polypeptide ZS-9. Extracted plasmid of bacteriophage and performed sequencing after amplified by PCR. Results: It was demonstrated by bioinformatic analysis on the sequence of ligand binded by lung cancer specific peptide ZS-9 that the ligand was the cytoskeletal protein periplakin on the surface of lung cancer cells, suggesting that periplakin might be a new marker for non-small-cell lung cancer in lung cancer. Conclusion: Use specific lung cancer binding peptide to screen new tumor marker periplakin in lung cancer and further studies on its biologic functions in genesis and development of lung cancer are still needed.

  15. The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

    Institute of Scientific and Technical Information of China (English)

    WANG LiJiang; WEI QingShan; WU OhunSheng; HU ZhaoYing; JI Jian; WANG Ping

    2008-01-01

    This paper presents development of a quartz crystal microbalance (QCM) biosensor for real-time de-tection of E. coli O157:H7 DNA based on nanogold particles amplification. Many inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated sin-gle-stranded DNA (ssDNA) probes could be fixed through Au-SH bonding. The hybridization was in-duced by exposing the ssDNA probe to the complementary target DNA of E. coli O157:H7 gene eaeA, then resulted in a mass change and corresponding frequency shifts (△f) of the QCM. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass. The electro-chemical techniques, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were adopted to manifest and character each step. The target DNA corresponding to 2.0×103 colony forming unit (CFU)/mL E. coli O157:H7 cells can be detected by this biosensor, so it is practical to develop a sensitive and effective QCM biosensor for pathogenic bacteria detection based on specific DNA analy-sis. The piezoelectric biosensing system has potential for further applications, such as food safety and environment monitoring, and this approach lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.

  16. Rapid mastitis detection assay on porous nitrocellulose membrane slides.

    Science.gov (United States)

    Mujawar, Liyakat Hamid; Moers, Antoine; Norde, Willem; van Amerongen, Aart

    2013-09-01

    We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.

  17. EXPRESSION OF p16 AND p53 IN GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective:To investigate the clinical significance of p53 and p16 expression in gastric carcinoma with special reference to the prognosis.Methods:One hundred and fifty-two patients with gastric carcinoma undergoing operation in our hospital between 1991 and 1998 were evaluated for expression of p53 and p16 in formalin-fixed and paraffin-embedded tumor tissue utilizing Avidin-Biotin immunohistochemistry techniques. Statistical correlations with stage, histological type, differentiation degree, location, size, and overall survival were done. The Cox proportional hazard model was also performed to evaluate which factors had an independent prognostic value.Results:In 152 cases of resected gastric cancer, 110 (72.4%) were p16 positive and 49 (32.2%) showed p53 overexpression. Differences were observed in the frequency of p16 positivity with respect to age, gender and tumor size. The frequency of p53 positivity cells in well-differentiated tumors was significantly higher than that in poorly differentiated tumors (41.9% vs. 25.6%; P= 0.034). In a multivariate analysis, tumor TNM stage, perioperation chemotherapy and the expression of p16 were independent prognostic factors in gastric cancer.Conclusions:The results of the current study suggest that expression of p16 may be a useful prognostic factor for patients with gastric carcinoma, but the expression of p53 as detected by immunohistochemistry were of no value in predicting the prognosis of patients with gastric carcinoma independently.

  18. Immunohistochemical Localization of 5-HT Cells in Gastrointestinal Tract of Physignathus cocincinus%长鬣蜥胃肠道5-羟色胺细胞的免疫组织化学定位

    Institute of Scientific and Technical Information of China (English)

    陈霞; 李淑兰; 赵文阁

    2007-01-01

    研究长鬣蜥消化道内含有的5-羟色胺(5-HT)内分泌细胞的位置和形态,采用ABC(avidin-biotin-peroxidase complex)免疫组织化学法,应用5-HT特异性抗血清,对其消化道内的5-HT细胞进行免疫化学定位.结果: 5-HT细胞从胃部到直肠的胃肠道各段均有分布,细胞密度分布以胃部最高,十二指肠次之,回肠最低.5-HT细胞的形态呈圆形、椭圆形和锥体形等,其中胃体部和直肠部以圆形和椭圆形为主,小肠部以锥体形为主;5-HT细胞广泛分布于上皮基部、上皮细胞之间、腺泡上皮及腺泡之间,有时可见于固有膜内.结论:5-HT细胞的密度分布与其食性、食物组成和生活环境有关,5-HT细胞的形态与其内、外分泌是相适应的.

  19. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    Directory of Open Access Journals (Sweden)

    M. Anthony Moody

    2012-03-01

    Full Text Available The traditional enzyme-linked immunospot (ELISpot assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay. Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs. We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.

  20. Detection of bovine herpesvirus 1 sequences in yaks (Bos grunniens) with keratoconjunctivitis, using a highly sensitive nested polymerase chain reaction.

    Science.gov (United States)

    Bandyopadhyay, S; Das, S; Baruah, K K; Chakravarty, P; Chakrabarty, D; Sarkar, T; Pal, B; De, S; Pan, D; Bera, A K; Bandyopadhyay, S; Bhattacharya, D

    2010-12-01

    Thirty-seven yaks (Bos grunniens) with keratoconjunctivitis and 22 healthy yaks were used to investigate the role of bovine herpesvirus 1 (BoHV-1) in keratoconjunctivitis in yaks. Nucleic acid sequences of BoHV-1 glycoproteins B and E were detected in conjunctival swabs from all yaks with keratoconjunctivitis using a nested polymerase chain reaction (PCR). In 21 yaks, BoHV-1 sequences were detected along with Moraxella bovis (M. bovis) and Neisseria spp. The amplified BoHV-1 sequences were identical, and no nucleotide variation was observed when compared with a BoHV-1 reference strain using single-strand conformation polymorphism analysis of the amplified DNA sequences. Interestingly, BoHV-1 sequences could not be detected in samples from healthy yaks. However, conjunctival swabs from two healthy yaks (9.09%) yielded M. bovis and Neisseria spp. Samples from 35 yaks with keratoconjunctivitis showed positive reactions in an avidin biotin enzyme-linked immunosorbent assay for BoHV-1 antibodies; all the healthy yaks were seronegative. This is the first report of a possible association of BoHV-1 with keratoconjunctivitis in yaks.

  1. Distribution Patterns and Developmental Changes of GnRH and GnRHR-Immunopositive Cells in the Pituitary of Ji Ning Gray Goats

    Directory of Open Access Journals (Sweden)

    Liu Xiao, Wang Shu-Ying, Huang Li-Bo, Hou Yan-Meng and Shi Yun-Zhi

    2014-01-01

    Full Text Available Immunohistochemical Strept Avidin Biotin-Peroxidase Complex (SABC three-step method was used to investigate the distribution patterns and developmental changes of GnRH and GnRHR immunopositive (GnRH-ip and GnRHR-ip cells in the pituitary of Ji Ning Gray goats during 0-180 days of age. The results showed that GnRH-ip and GnRHR-ip cells were detected only in the pars distalis of adenohypophysis. There were no positive cells in the pars intermedia and neurohypophysis. GnRH-ip and GnRHR-ip cells were not observed in the anterior pituitary at birth day and 30 days of age. At 60 days, a number of GnRH-ip and GnRHR-ip cells were found in the anterior pituitary. GnRH-ip cells were pale brown which scattered or clustered around the sinusoid capillary; GnRHR-ip cells were brown and the cytomembrane was darker. The size and percentage of GnRH-ip and GnRHR-ip cells increased with the age growth. The numbers of GnRH-ip and GnRHR-ip cells after sexual maturity were significantly bigger than that before sexual maturity. The results above suggested that GnRH and GnRHR in the pituitary of Ji Ning Gray goats play a pivotal role in the sexual development and sexual maturity.

  2. Layer-by-layer-assembled microfiltration membranes for biomolecule immobilization and enzymatic catalysis.

    Science.gov (United States)

    Smuleac, V; Butterfield, D A; Bhattacharyya, D

    2006-11-21

    Multilayer assemblies of polyelectrolytes, for protein immobilization, have been created within the membrane pore domain. This approach was taken for two reasons: (1) the high internal membrane area can potentially increase the amount of immobilized protein, and (2) the use of convective flow allows uniform assembly of layers and eliminates diffusional limitations after immobilization. To build a stable assembly, the first polyelectrolyte layer was covalently attached to the membrane surface and inside the pore walls. Either poly(L-glutamic acid) (PLGA) or poly(L-lysine) (PLL) was used in this step. Subsequent deposition occurs by multiple electrostatic interactions between the adsorbing polyelectrolyte [poly(allylamine) hydrochloride (PAH) or poly(styrenesulfonate) (PSS)] and the oppositely charged layer. Three-layer membranes were created: PLL-PSS-PAH or PLGA-PAH-PSS, for an overall positive or negative charge, respectively. The overall charge on both the protein and membrane plays a substantial role in immobilization. When the protein and the membrane are oppositely charged, the amount immobilized and the stability within the polyelectrolyte assembly are significantly higher than for the case when both have similar charges. After protein incorporation in the multilayer assembly, the active site accessibility was comparable to that obtained in the homogeneous phase. This was tested by affinity interaction (avidin-biotin) and by carrying out two reactions (catalyzed by glucose oxidase and alkaline phosphatase). Besides simplicity and versatility, the ease of enzyme regeneration constitutes an additional benefit of this approach.

  3. Mechanically robust, rapidly actuating, and biologically functionalized macroporous poly(N-isopropylacrylamide)/silk hybrid hydrogels.

    Science.gov (United States)

    Gil, Eun Seok; Park, Sang-Hyug; Tien, Lee W; Trimmer, Barry; Hudson, Samuel M; Kaplan, David L

    2010-10-05

    A route toward mechanically robust, rapidly actuating, and biologically functionalized polymeric actuators using macroporous soft materials is described. The materials were prepared by combining silk protein and a synthetic polymer (poly(N-isopropylacrylamide) (PNIAPPm)) to form interpenetrating network materials and macroporous structures by freeze-drying, with hundreds of micrometer diameter pores and exploiting the features of both polymers related to dynamic materials and structures. The chemically cross-linked PNIPAAm networks provided stimuli-responsive features, while the silk interpenetrating network formed by inducing protein β-sheet crystallinity in situ for physical cross-links provided material robustness, improved expansion force, and enzymatic degradability. The macroporous hybrid hydrogels showed enhanced thermal-responsive properties in comparison to pure PNIPAAm hydrogels, nonporous silk/PNIPAAm hybrid hydrogels, and previously reported macroporous PNIPAAm hydrogels. These new systems reach near equilibrium sizes in shrunken/swollen states in less than 1 min, with the structural features providing improved actuation rates and stable oscillatory properties due to the macroporous transport and the mechanically robust silk network. Confocal images of the hydrated hydrogels around the lower critical solution temperature (LCST) revealed macropores that could be used to track changes in the real time morphology upon thermal stimulus. The material system transformed from a macroporous to a nonporous structure upon enzymatic degradation. To extend the utility of the system, an affinity platform for a switchable or tunable system was developed by immobilizing biotin and avidin on the macropore surfaces.

  4. Biological Applications of Extraordinary Electroconductance and Photovoltaic Effects in Inverse Extraordinary Optoconductance

    Science.gov (United States)

    Tran, Lauren Christine

    The Extraordinary Electroconductance (EEC) sensor has been previously demonstrated to have an electric field sensitivity of 3.05V/cm in a mesoscopic-scale structure fabricated at the center of a parallel plate capacitor. In this thesis, we demonstrate the first successful application of EEC sensors as electrochemical detectors of protein binding and biological molecule concentration. Using the avidin derivative, captavidin, in complex with the vitamin biotin, the change in four-point measured resistance with fluid protein concentration of bare EEC sensors was shown to increase by a factor of four in the presence of biomolecular binding as compared to baseline. Calculations for approximate field strengths introduced by a bound captavidin molecule are also presented. The development of Inverse-Extraordinary Optoconductance (I-EOC), an effect which occurs in nanoscale sensors, is also discussed. In the I-EOC effect, electron transport transitions from ballistic to diffusive with increasing light intensity. In these novel, room temperature optical detectors, the resistance is low at low light intensity and resistance increases by 9462% in a 250nm device mesa upon full illumination with a 5 mW HeNe laser. This is the inverse of bulk and mesoscopic device behavior, in which resistance decreases with increasing photon density.

  5. Ultrastructural and lectin-histochemical differences between the scolex/strobila and bladder teguments of the Taenia taeniaeformis strobilocercus.

    Science.gov (United States)

    Olson, E J; Oaks, J A; Osmundson, G D; Hildreth, M B

    2000-02-01

    The strobilocercus stage of the cat tapeworm Taenia taeniaeformis is surrounded by a single syncytial sheet of cytoplasm called the tegument. The outer membrane of the tegument covers both the scolex/strobila (S/S) and the bladder portions of the strobilocercus, but only the S/S region is resistant to intestinal digestion. It has been suggested that the glycocalyx, the surface-exposed glycoconjugates of the outer membrane, may serve to insulate underlying surface membrane components from digestion. In this study, we used lectin binding to test the hypothesis that the glycocalyx of the S/S is different from that of the bladder and that this may serve as the resistance mechanism of the S/S to digestion. Biotin-labeled lectins and an avidin-glucose oxidase detection system were applied to whole strobilocerci and to 1-microm epon-araldite plastic-embedded sections. Lectins bound to either both regions of the strobilocerci, to the S/S regions only, or did not bind at all. The restriction of some glycoconjugates to the glycocalyx of the S/S region only is consistent with our hypothesis.

  6. Quantitative analysis of plasma interleiukin-6 by immunoassay on microchip

    Science.gov (United States)

    Abe, K.; Hashimoto, Y.; Yatsushiro, S.; Yamamura, S.; Tanaka, M.; Ooie, T.; Baba, Y.; Kataoka, M.

    2012-03-01

    Sandwich enzyme-linked immunoassay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. We employed the piezoelectric inkjet printing for deposition and fixation of 1st antibody on the microchannnel surface (300 μm width and 100 μm depth). Model analyte was interleukin-6 (IL-6) which was one of the inflammatory cytokine. After blocking the microchannel, antigen, biotin-labeled 2nd antibody, and avidin-labeled peroxidase were infused into the microchannel and incubated for 20 min, 10 min, and 5 min, respectively. This assay could detect 2 pg/ml and quantitatively measure the range of 0-32 pg/ml. Liner regression analysis of plasma IL-6 concentration obtained by microchip and conventional methods exhibited a significant relationship (R2 = 0.9964). This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. This assay enables us to determine plasma IL-6 with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

  7. Development and evaluation of a novel VEGFR2-targeted nanoscale ultrasound contrast agents

    Science.gov (United States)

    Yu, Houqiang; Li, Chunfang; He, Xiaoling; Zhou, Qibing; Ding, Mingyue

    2016-04-01

    Recent literatures have reported that the targeted nanoscale ultrasound contrast agents are becoming more and more important in medical application, like ultrasound imaging, detection of perfusion, drug delivery and molecular imaging and so on. In this study, we fabricated an uniform nanoscale bubbles (257 nm with the polydispersity index of 0.458) by incorporation of antibody targeted to vascular endothelial growth factor receptor 2 (VEGFR2) into the nanobubbles membrane by using avidin-biotin interaction. Some fundamental characterizations such as nanobubble suspension, surface morphology, particle size distribution and zeta potential were investigated. The concentration and time-intensity curves (TICs) were obtained with a self-made ultrasound experimental setup in vitro evaluation. In addition, in order to evaluate the contrast enhancement ability and the potential tumor-targeted ability in vivo, normal Wistar rats and nude female BALB/c mice were intravascular administration of the nanobubbles via tail vein injection, respectively. Significant contrast enhancement of ultrasound imaging within liver and tumor were visualized. These experiments demonstrated that the targeted nanobubbles is efficient in ultrasound molecular imaging by enhancement of the contrast effect and have potential capacity for targeted tumor diagnosis and therapy in the future.

  8. Radiation-Force Assisted Targeting Facilitates Ultrasonic Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Shukui Zhao

    2004-07-01

    Full Text Available Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to αvβ3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.

  9. Micro-emulsion synthesis, surface modification, and photophysical properties of Zn(1-x) Mn(x)S nanocrystals for biomolecular recognition.

    Science.gov (United States)

    Mohagheghpour, Elham; Moztarzadeh, Fathollah; Rabiee, Mohammad; Tahriri, Mohammadreza; Ashuri, Maziar; Sameie, Hassan; Salimi, Reza; Moghadas, Shahab

    2012-12-01

    In this research, we mainly focused on the micro-emulsion synthesis of biotinylated ZnS (zinc sulfide) nanocrystals for avidin recognition. Various samples of Zn(1-x)Mn(x) S, with x = 0.0001, 0.007, 0.02, 0.03, 0.055, 0.09 and 0.13, prepared by quaternary W/O (water-in-oil) microemulsion system. Cyclohexane was used as oil, Triton X-100 as surfactant, n-hexanol as a co-surfactant and mercaptoethanol and thioglycolic acid as linking agents. The obtained products were evaluated by commonly techniques such as: scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta meter for measurement ZP (zeta potential) and fluorescence spectroscopy analyses. The above-experimental results indicated that the optimum doping concentration of Mn was ~ 5.5% . The fluorescence spectra of the doped crystals consist of orange-red emissions. Eventually, this research showed with increasing more than 18 μl biotin to nanocrystals, no changes were observed in the emission intensity spectra.

  10. Direct Biomolecules Binding on Nonfouling Surface via Newly Discovered Supramolecular Self-assembly of Lysozyme under Physiological Condition

    Science.gov (United States)

    Yang, Peng

    2013-01-01

    A major challenge in the development of low cost and practical strategies for biomolecules immobilization on solid supports is that the multi-step chemical/physical activating and following deactivating procedures on nonfouling substrates often increase the cost and complexity of surface functional group types as well as deteriorate the surface integrity. Herein, we show a novel phase transition of lysozyme could be used to constitute a major step to address the above problem. It is found that when lysozyme is dissolved in a neutral buffer solution of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 7.4) with 1–50 mM tris(2-carboxyethyl)phosphine (TCEP) added, a fast phase transition process occurs and the resulting novel fibra-like hierarchical supramolecular assemblies made by primary spherical particles aggregation would function as a “superglue” that strongly and quickly bind onto non-fouling coatings. This binding is highly selective towards lysozyme, and excludes completely tedious synthetical, chemical/physical activation/deactivation (blocking) steps. When biotin is conjugated with lysozyme, such phase transition quickly constructs a perfect biotinylated surface on nonfouling surface for avidin binding, showing great potential for the development of low-cost and practical biochips. PMID:22707360

  11. Evaluation of Three Mycobacterium leprae Monoclonal Antibodies in Mucus and Lymph Samples from Ziehl-Neelsen Stain Negative Leprosy Patients and their Household Contacts in an Indian Community

    Directory of Open Access Journals (Sweden)

    Nora Cardona-Castro

    1998-07-01

    Full Text Available Mucus and lymph smears collected from leprosy patients (9 and their household contacts (44 in the Caño Mochuelo Indian Reservation, Casanare, Colombia, were examined with monoclonal antibodies (MoAb against Mycobacterium leprae. The individuals studied were: 5 borderline leprosy (BB patients, 4 with a lepromatous leprosy (LL, all of whom were undergoing epidemiological surveillance after treatment and 44 household contacts: 21 of the LL and 23 contacts of the BB patients. The MoAb were reactive with the following M. leprae antigens: 65 kd heat shock protein, A6; soluble antigen G7 and complete antigen, E11. All the samples were tested with each of the MoAb using the avidin-biotin-peroxidase technique and 3,3 diaminobenzidine as chromogen. The patients and household contacts studied were all recorded as Ziehl-Neelsen stain negative. The MoAb which showed optimal reaction was G7, this MoAb permited good visualization of the bacilli. Five patients with BB diagnosis and one with LL were positive for G7; of the BB patients' household contacts, 9 were positive for G7; 7 of the LL patients' household contacts were positive for the same MoAb. MoAb G7 allowed the detection of bacillar Mycobacterium spp. compatible structures in both patients and household contacts. G7 permited the visualization of the complete bacillus and could be used for early diagnosis and follow-up of the disease in patients.

  12. Ultrathin coatings from isocyanate-terminated star PEG prepolymers: layer formation and characterization.

    Science.gov (United States)

    Groll, Juergen; Ameringer, Thomas; Spatz, Joachim P; Moeller, Martin

    2005-03-01

    In this study we present the preparation of thin and ultrathin coatings from six-arm star-shaped isocyanate-terminated prepolymers on amino-functionalized silicon wafers. The backbone of the stars is a statistical copolymer of ethylene oxide and propylene oxide in the ratio 80:20 (Star PEG). Film preparation by spin coating from aqueous THF resulted in a variety of film morphologies that are determined by the water content of the solvent. Water is indispensable for activation of the isocyanate-terminated stars in solution and for proper cross-linking of the coatings on the substrate. This cross-linking results in a dense network of PEG chains on the substrate linked via urea groups with a mesh size of the network that corresponds to the arm length of the stars. Layer thickness variations between 3 and 500 nm revealed a strong dependence of the contact angle with water on the layer thickness which is explained by the chemical composition of the coatings. Due to the high functionality of the star-shaped prepolymers, free amino groups remain in the films that were detected by fluorescence microscopy after reaction with 4-chloro-7-nitrobenzofurazan (NBF). To test the system for the ability to prevent unspecific interaction with proteins, adsorption of fluorescence-labeled avidin was examined with fluorescence microscopy. For layer thicknesses between 3 and 50 nm, no protein adsorption could be detected.

  13. Comb-type prepolymers consisting of a polyacrylamide backbone and poly(L-lysine) graft chains for multivalent ligands.

    Science.gov (United States)

    Asayama, S; Maruyama, A; Akaike, T

    1999-01-01

    The comb-type copolymers consisting of a polyacrylamide (PAAm) backbone and poly(L-lysine) (PLL) graft chains have been prepared as the "prepolymer" for designing multivalent ligands. To regulate the length and density of the clusters of primary amino groups, the Nalpha-carboxyanhydride of Nepsilon-carbobenzoxy (CBZ)-L-lysine was first polymerized using p-vinylbenzylamine as an initiator. The resulting poly(CBZ-L-lysine) macromonomer was then radically copolymerized with AAm, followed by the deprotection of amino groups. For the model study, the reactive clusters of primary amino groups were completely converted into anion clusters by the reaction with succinic anhydride. The model multivalent ligands having the biotin label on the PAAm backbone were prepared by the terpolymerization of the macromonomer, AAm, and the biotin derivative having a vinyl group. The enzyme-linked immunosorbent assay showed that the biotin with no spacer on the PAAm backbone was recognized by the avidin-peroxidase conjugate specifically. Therefore, the highly sensitive detection of the interaction between cells and various model multivalent ligands was possible. The selective labeling onto the PAAm backbone revealed that the converted anion clusters of graft chains interacted exclusively with the cell and that the backbone was inert to the interaction with the cell. These results indicate that the various PAAm-graft-PLL comb-type copolymers with the defined length and density of the PLL-grafts are the potential prepolymers to investigate and to optimize the affinity of the multivalent ligands for receptors.

  14. IMMUNOHISTOCHEMICAL OBSERVATION OF MACROPHAGE COLONY STIMULATING FACTOR AND ITS RECEPTOR IN BREAST CANCER AND HEPATOMA TISSUES

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To study the potential role of cellular macrophageolony-stimulating factor (cM-CSF) and cellular macrophage colony-stimulating factor receptor (cM-CSF-R) with breast cancer and hepatoma and search the way for clinical application. Methods: Frozen surgical specimens from 48 breast cancer patients, including 29 cases of histological grade II and 19 eases of grade III, and 16 hepatoma patients were investigated by Avidin Biotin Complex (ABC) immunohistochemical assay with anti-M-CSF monoclonal antibody (Mab) and anti-M-CSF-R Mab. Pathohistological examination was performed as well. Results: cM-CSF and cM-CSF-R were detected in tested specimens. The expression levels of cM-CSF and cM-CSF-R in grade III group were higher than in grade II group and more higher than control group hyperplasia of breast. Hepatoma tissues also showed higher expression level of cM-CSF and cM-CSF-R than normal adult and fetal liver. Conclusion: Breast cancer and hepatoma tissues presented higher expression levels of cM-CSF and cM-CSF-R than control and expression level might be related with tumor's process.

  15. Signal Amplification by Enzymatic Reaction in an Immunosensor Based on Localized Surface Plasmon Resonance (LSPR

    Directory of Open Access Journals (Sweden)

    Yong-Beom Shin

    2010-03-01

    Full Text Available An enzymatic reaction was employed as a means to enhance the sensitivity of an immunosensor based on localized surface plasmon resonance (LSPR. The reaction occurs after intermolecular binding between an antigen and an antibody on gold nano-island (NI surfaces. For LSPR sensing, the gold NI surface was fabricated on glass substrates using vacuum evaporation and heat treatment. The interferon-g (IFN-g capture antibody was immobilized on the gold NIs, followed by binding of IFN-g to the antibody. Subsequently, a biotinylated antibody and a horseradish peroxidase (HRP conjugated with avidin were simultaneously introduced. A solution of 4-chloro-1-naphthol (4-CN was then used for precipitation; precipitation was the result of the enzymatic reaction catalyzed the HRP on gold NIs. The LSPR spectra were obtained after each binding process. Using this method, the enzyme-catalyzed precipitation reaction on the gold NI surface was found to effectively amplify the change in the signal of the LSPR immunosensor after intermolecular binding.

  16. Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device.

    Science.gov (United States)

    Battle, Katrina N; Jackson, Joshua M; Witek, Małgorzata A; Hupert, Mateusz L; Hunsucker, Sally A; Armistead, Paul M; Soper, Steven A

    2014-03-21

    We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

  17. Evaluation of Liver Ischemia-Reperfusion Injury in Rabbits Using a Nanoscale Ultrasound Contrast Agent Targeting ICAM-1.

    Directory of Open Access Journals (Sweden)

    Fang Xie

    Full Text Available To assess the feasibility of ultrasound molecular imaging in the early diagnosis of liver ischemia-reperfusion injury (IRI using a nanoscale contrast agent targeting anti-intracellular adhesion molecule-1 (anti-ICAM-1.The targeted nanobubbles containing anti-ICAM-1 antibody were prepared using the avidin-biotin binding method. Human hepatic sinusoidal endothelial cells (HHSECs were cultured at the circumstances of hypoxia/reoxygenation (H/R and low temperature. The rabbit liver IRI model (I/R group was established using the Pringle's maneuver. The time-intensity curve of the liver contrast ultrasonographic images was plotted and the peak intensity, time to peak, and time of duration were calculated.The size of the targeted nanobubbles were 148.15 ± 39.75 nm and the concentration was 3.6-7.4 × 109/ml, and bound well with the H/R HHSECs. Animal contrast enhanced ultrasound images showed that the peak intensity and time of duration of the targeted nanobubbles were significantly higher than that of common nanobubbles in the I/R group, and the peak intensity and time of duration of the targeted nanobubbles in the I/R group were also significantly higher than that in the SO group.The targeted nanobubbles have small particle size, stable characteristic, and good targeting ability, which can assess hepatic ischemia-reperfusion injury specifically, noninvasively, and quantitatively at the molecular level.

  18. Dispersion and shape engineered plasmonic nanosensors

    Science.gov (United States)

    Jeong, Hyeon-Ho; Mark, Andrew G.; Alarcón-Correa, Mariana; Kim, Insook; Oswald, Peter; Lee, Tung-Chun; Fischer, Peer

    2016-04-01

    Biosensors based on the localized surface plasmon resonance (LSPR) of individual metallic nanoparticles promise to deliver modular, low-cost sensing with high-detection thresholds. However, they continue to suffer from relatively low sensitivity and figures of merit (FOMs). Herein we introduce the idea of sensitivity enhancement of LSPR sensors through engineering of the material dispersion function. Employing dispersion and shape engineering of chiral nanoparticles leads to remarkable refractive index sensitivities (1,091 nm RIU-1 at λ=921 nm) and FOMs (>2,800 RIU-1). A key feature is that the polarization-dependent extinction of the nanoparticles is now characterized by rich spectral features, including bipolar peaks and nulls, suitable for tracking refractive index changes. This sensing modality offers strong optical contrast even in the presence of highly absorbing media, an important consideration for use in complex biological media with limited transmission. The technique is sensitive to surface-specific binding events which we demonstrate through biotin-avidin surface coupling.

  19. Ultrasensitive detection and rapid identification of multiple foodborne pathogens with the naked eyes.

    Science.gov (United States)

    Zhang, Heng; Zhang, Yali; Lin, Yankui; Liang, Tongwen; Chen, Zhihua; Li, Jinfeng; Yue, Zhenfeng; Lv, Jingzhang; Jiang, Qing; Yi, Changqing

    2015-09-15

    In this study, a novel approach for ultrasensitive detection and rapid high-throughput identification of a panel of common foodborne pathogens with the naked eyes is presented. As a proof-of-concept application, a multiple pathogen analysis array is fabricated through immobilizing three specific polyT-capture probes which can respectively recognize rfbE gene (Escherichia coli O157:H7), invA gene (Salmonella enterica), inlA gene (Listeria monocytogenes) on the plastic substrates. PCR has been developed for amplification and labeling target genes of rfbE, invA, inlA with biotin. The biotinated target DNA is then captured onto the surface of plastic strips through specific DNA hybridization. The succeeding staining of biotinated DNA duplexes with avidin-horseradish peroxidise (AV-HRP) and biotinated anti-HRP antibody greatly amplifies the detectable signal through the multiple cycle signal amplification strategy, and thus realizing ultrasensitive and specific detection of the above three pathogens in food samples with the naked eyes. Results showed approximately 5 copies target pathogenic DNA could be detected with the naked eyes. This simple but very efficient colorimetric assay also show excellent anti-interference capability and good stability, and can be readily applied to point-of-care diagnosis.

  20. Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers

    Energy Technology Data Exchange (ETDEWEB)

    Kandpal, R.P.; Kandpal, G.; Weissman, S.M. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-01-04

    The authors describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotde. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)[sub n] microsatellites with this approach contained clones with inserts containing CA repeats. They have also used this protocol for enrichment of (CAG)[sub n] and (AGAT)[sub n] sequence repeats and for Not I jumping clones. They have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

  1. Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

    Science.gov (United States)

    Wright, A. D.; Chapes, S. K.

    1999-01-01

    The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

  2. NEPHROPATHIES IN THE EUROPEAN CAPTIVE CHEETAH (ACINONYX JUBATUS) POPULATION.

    Science.gov (United States)

    Url, Angelika; Krutak, Verena; Kübber-Heiss, Anna; Chvala-Mannsberger, Sonja; Robert, Nadia; Dinhopl, Nora; Schmidt, Peter; Walzer, Chris

    2016-09-01

    According to previous studies in captive cheetah ( Acinonyx jubatus ) populations, one of the most threatening diseases besides amyloidosis, myelopathy, veno occlusive disease, and gastritis, is renal failure. Contrary to captive cheetahs in North America and South Africa, morphological data concerning renal lesions in the cheetah European Endangered Species Program (EEP) are lacking. This study details the histological characterization as well as immunohistochemical and morphometrical analysis of nephropathies in 35 captive cheetahs from the EEP, which were necropsied between 1985 and 2003. Examination of paraffin- and glycolmethacrylate-methylmethacrylate (GMA-MMA) embedded kidney samples by light microscopy revealed glomerulonephritis in 91%, with a high prevalence for glomerulosclerosis and glomerulonephritis with the histologic pattern of membranous glomerulonephritis (77%). Besides these predominating glomerulopathies, a wide range of other renal lesions, like acute tubular necrosis, interstitial nephritis, calcinosis, and amyloidosis, were present. Pathological expression of collagen type IV, complement C3, fibronectin, and IgG was demonstrated in the glomeruli of the cheetah kidneys with the use of the avidin-biotin complex method. Morphometrical analysis was performed on GMA-MMA embedded kidney samples to obtain glomerulosclerosis index and glomerulosclerosis incidence.

  3. Single and multiple molecular beacon probes for DNA hybridization studies on a silica glass surface

    Science.gov (United States)

    Fang, Xiaohong; Liu, Xiaojing; Tan, Weihong

    1999-05-01

    Surface immobilizable molecular beacons have been developed for DNA hybridization studies on a silica glass plate. Molecular beacons are a new class of oligonucleotide probes that have a loop-and-stem structure with a fluorophore and a quencher attached to the two ends of the stem. They only emit intense fluorescence when hybridize to their target molecules. This provides an excellent selectivity for the detection of DNA molecules. We have designed biotinylated molecular beacons which can be immobilized onto a solid surface. The molecular beacon is synthesized using DABCYL as the quencher and an optical stable dye, tetramethylrhodamine, as the fluorophore. Mass spectrometry is used to confirm the synthesized molecular beacon. The molecular beacons have been immobilized onto a silica surface through biotin-avidin binding. The surface immobilized molecular beacons have been used for the detection of target DNA with subnanomolar analytical sensitivity. have also immobilized two different molecular beacons on a silica surface in spatially resolved microscopic regions. The hybridization study of these two different molecular beacon probes has shown excellent selectivity for their target sequences. The newly designed molecular beacons are intended for DNA molecular interaction studies at an interface and for the development of ultrasensitive DNA sensors for a variety of applications including disease diagnosis, disease mechanism studies, new drug development, and in the investigation of molecular interactions between DNA molecules and other interesting biomolecules.

  4. Immunoradiometric assay for quantification of prostate-specific antigen (PSA)

    Energy Technology Data Exchange (ETDEWEB)

    Ruiz, Miriam [Institute of Nuclear Sciences and Technology, Havana (Cuba). Dept.of Radiochemistry]. E-mail: mirian@fctn.isctn.edu.cu

    2002-07-01

    To develop an immunoradiometric assay (IRMA) for quantification of total PSA in serum, we carried out the evaluation of a panel of 12 monoclonal antibodies (MAbs). The mAbs (CB-PSA 1 to CB-PSA 10) were developed in the Center of Genetic Engineering and Biotechnology (CIGB) Havana, Cuba and mAbs (CMC-H9, CMC-A5) in the Center of Immunology and Biologics (CENIB), Camaguey, Cuba. The inhibition assays were carried out using as reference a commercial equimolar assay for total PSA. For the evaluation, the mAbs were labeled with {sup 125} I by the method of Chloramine T and the capture mAbs were used conjugated with biotin. For separation, it was used the avidin coupled to magnetic particles. To validate the assay we evaluated the sensibility, inter and intraassay precision and the correlation with reference assay in 65 samples of patient under suspicion of prostate cancer. The partner CB-PSA 4 / CB-PSA 9 (capture / tracer) showed the best results in the IRMA. The developed assay presented a detection limit of 0.025 mg/L, a good intra and interassay precision and a high correlation with the reference assay. (author)

  5. New Tools for the site-specific attachment of proteins to surface

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kwon, Y; Coleman, M A

    2005-06-17

    Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. Although a variety of methods are available for attaching proteins on solid surfaces. Most of them rely on non-specific adsorption methods or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with complementary reactive groups. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue, however in most of the cases the interaction of the tags are reversible (e.g., glutathione S-transferase, maltose binding protein and poly-His) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-avidin and antigen antibody). The key for the covalent attachment of a protein to a solid support with a total control over the orientation is to introduce two unique and mutually reactive groups on both the protein and the surface. The reaction between these two groups should be highly selective thus behaving like a molecular 'Velcro'.

  6. New Tools for the site-specific attachment of proteins to surface

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Y; Coleman, M A; Camarero, J A

    2006-06-27

    Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays (''protein chips'') can be used very efficiently to analyze all kind of protein interactions en masse. Although a variety of methods are available for attaching proteins on solid surfaces. Most of them rely on non-specific adsorption methods or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with complementary reactive groups. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue, however in most of the cases the interaction of the tags are reversible (e.g., glutathione S-transferase, maltose binding protein and poly-His) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-avidin and antigen antibody). The key for the covalent attachment of a protein to a solid support with a total control over the orientation is to introduce two unique and mutually reactive groups on both the protein and the surface. The reaction between these two groups should be highly selective thus behaving like a molecular ''velcro''.

  7. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    Science.gov (United States)

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen

    2002-01-01

    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

  8. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

    Directory of Open Access Journals (Sweden)

    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

  9. On the possibility of the unification of drug targeting systems. Studies with liposome transport to the mixtures of target antigens.

    Science.gov (United States)

    Trubetskoy, V S; Berdichevsky, V R; Efremov, E E; Torchilin, V P

    1987-03-15

    In order to make the drug targeting system more effective, simple and technological, we suggest creation of drug-bearing conjugates capable of simultaneous binding with different antigenic components of the target via specific antibodies. It is supposed that the targeted therapy should include sequential administration of the mixture of modified antibodies (or other specific vectors) against different components of affected tissue and, upon antibody accumulation in the desired region, administration of modified drugs or drug carrying systems which can recognize and bind with the target via accumulated antibodies due to the interaction between vector modifier and carrier modifier. Using as a model system monolayers consisting of the mixture of extracellular antigens and appropriated antibodies, it was shown that the treatment of the target with the mixture of biotinylated antibodies against all target components and subsequent binding with the target of biotinylated liposomes via avidin permits high liposome accumulation on the monolayer. The binding achieved is always higher than in the case of the utilization of single antibody-bearing liposomes. Besides, the system suggested is very simple and its components can be easily obtained on technological scale in standardized conditions.

  10. Detection of avian influenza virus H5N1 subtype in organs of chicken affected by higly pathogenic avian infuenza in East and West Java by using immunohistochemical technique

    Directory of Open Access Journals (Sweden)

    R Damayanti

    2004-10-01

    Full Text Available The study was conducted to detect antigen H5N1 of highly pathogenic Avian Influenza (HPAI virus in various farms in East and West Java. The immunohistochemical technique was applied due to Hematoxilin-eosin (H&E staining was impossible to visualize the antigen in tissue. Immunohistochemical staining was applied for some visceral organs collected from the areas where the outbreaks occurred in September-October 2003. The specimens were processed as histopathological paraffin blocks using standard method. The blocks that were suspected to have antigen H5N1 were cut and rabbit antisera to H5N1 produced from the local isolate was applied as the primary antibody. Biotinylated secondary antibody and avidin biotin peroxidase from a commercial kit were administered. The antigen present in the tissues were visualized by adding a substrate called Amino Ethyl Carbazole (AEC resulting in reddish brown colour. This immunostaining proved to be accurate and reliably quick method to detect H5N1 antigen present in the avian tissues. In conclusion, the outbreak of bird flu was caused by H5N1 strain and the antigen could be found in wattles, combs, brain, trachea, lungs, heart, proventriculus, liver, spleen, kidney and ovary.

  11. Proteomic profiling of fetal esophageal epithelium, esophageal cancer, and tumor-adjacent esophageal epithelium and immunohistochemical characterization of a representative differential protein, PRX6

    Science.gov (United States)

    Guo, Jun-Hui; Xing, Guo-Lan; Fang, Xin-Hui; Wu, Hui-Fang; Zhang, Bo; Yu, Jin-Zhong; Fan, Zong-Min; Wang, Li-Dong

    2017-01-01

    AIM To understand the molecular mechanism of esophageal cancer development and provide molecular markers for screening high-risk populations and early diagnosis. METHODS Two-dimensional electrophoresis combined with mass spectrometry were adopted to screen differentially expressed proteins in nine cases of fetal esophageal epithelium, eight cases of esophageal cancer, and eight cases of tumor-adjacent normal esophageal epithelium collected from fetuses of different gestational age, or esophageal cancer patients from a high-risk area of esophageal cancer in China. Immunohistochemistry (avidin-biotin-horseradish peroxidase complex method) was used to detect the expression of peroxiredoxin (PRX)6 in 91 cases of esophageal cancer, tumor-adjacent normal esophageal tissue, basal cell hyperplasia, dysplasia, and carcinoma in situ, as well as 65 cases of esophageal epithelium from fetuses at a gestational age of 3-9 mo. RESULTS After peptide mass fingerprint analysis and search of protein databases, 21 differential proteins were identified; some of which represent a protein isoform. Varying degrees of expression of PRX6 protein, which was localized mainly in the cytoplasm, were detected in adult and fetal normal esophageal tissues, precancerous lesions, and esophageal cancer. With the progression of esophageal lesions, PRX6 protein expression showed a declining trend (P PRX6 protein expression showed a declining trend with age (P PRX6 protein expression was significantly higher in well-differentiated esophageal cancer tissues than in poorly differentiated esophageal cancer tissues (P PRX6 protein is associated with fetal esophageal development and cancer differentiation. PMID:28293090

  12. Characterization of receptor proteins using affinity cross-linking with biotinylated ligands.

    Science.gov (United States)

    Shinya, Tomonori; Osada, Tomohiko; Desaki, Yoshitake; Hatamoto, Masahiro; Yamanaka, Yuko; Hirano, Hisashi; Takai, Ryota; Che, Fang-Sik; Kaku, Hanae; Shibuya, Naoto

    2010-02-01

    The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

  13. p53 expression is of independent predictive value in lymph node-negative breast carcinoma.

    Science.gov (United States)

    Fresno, M; Molina, R; Pérez del Río, M J; Alvarez, S; Díaz-Iglesias, J M; García, I; Herrero, A

    1997-07-01

    The aim of this study was to evaluate p53 expression, determined by immunohistochemistry, in 151 infiltrating ductal breast carcinomas with negative axillary lymph nodes, and to determine whether p53 can be considered as an independent prognostic value for overall and disease-free survival. A monoclonal antibody (DO-7) that reacts with an epitope on the N terminal portion of the human protein p53 was used to detect p53 in paraffin-embedded sections, utilising a standard avidin-biotin-peroxidase complex (ABC) technique with a microwave oven antigen retrieval. Overexpression of p53 (more than 50% of stained cells) was found in 45 cases (30%). Forty-five cases were negative and occasionally or moderately stained cells were present in 61 cases. p53 protein overexpression was significantly associated with high histological grade and tumour necrosis, high MIB-1 value (MIB-1 > 30%) and negative oestrogen receptor status. Univariate analysis (log-rank) showed a shorter overall survival (P = 0.003) in patients with high tumour p53 positivity. This statistical significance was also seen on multivariate analysis (Cox's logistic regression, P = 0.004). p53 protein overexpression is an independent prognostic marker in node-negative breast carcinoma for overall survival and should be used with other prognostic factors.

  14. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

    Science.gov (United States)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  15. A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Feng Zhou

    2015-09-01

    Full Text Available Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs as donors in luminescence resonance energy transfer (LRET. UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR excitation.

  16. Changes of IL-6 and relevant cytokines in patients with hepatocellularcarcinoma and their clinical significance

    Institute of Scientific and Technical Information of China (English)

    Cai Yah Zhao; Yue Lin Li; Su Xia Liu; Zhong Jun Feng

    2000-01-01

    AIM To study the changes of IL-6,IL-2, sIL-2R and TNF ir patients with hepatocellular carcinoma(HCC)and their clinical significance.METHODS IL-6, IL-2, sIL-2R and TNF were detected by avidin-biotin-system ELISA, double-sandwichELISA respectively in 60 patients with HCC and 36 patients with liver cirrhosis (LC) and 66 healthy persons.RESULTS The levels of IL-6, sIL-2R and TNF increased, but IL-2 level was lower in patients with HCCthan that in normal controls (NC) (t test, t=8.21, 4.71, 3.87, 2.13, P<0.01 or 0.05). IL-6 level in HCCwas 10 fold higher than NC, and also much higher than LC. IL-6 level was higher in later stage than that inearlier stage. There was a positive correlation between IL-6 and sIL-2R, TNF, while no positive correlationwas found between IL-2 and IL-6, sIL-2R in HCC.CONCLUSION The remarkably higher level of IL-6 is helpful for the early diagnosis of HCC.

  17. Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

    Institute of Scientific and Technical Information of China (English)

    You-Hong Yuan; En-Hua Xiao; Jian-Bin Liu; Zhong He; Ke Jin; Cong Ma; Jun Xiang; Jian-Hua Xiao; Wei-Jian Chen

    2008-01-01

    AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC)on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and StTept Avidin-Biotin Complex (SABC)immunohistochemical staining. RESULTS" The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P<0. 001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P<0. 001). PCNA and nm23 expression in the VX-2 tumor periphery first increased and then decreased (P<0. 001 and P=0. 03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P<0. 001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

  18. Design of Biotin-Functionalized Luminescent Quantum Dots

    Directory of Open Access Journals (Sweden)

    Kimihiro Susumu

    2007-01-01

    Full Text Available We report the design and synthesis of a tetraethylene glycol- (TEG- based bidentate ligand functionalized with dihydrolipoic acid (DHLA and biotin (DHLA—TEG—biotin to promote biocompatibility of luminescent quantum dots (QD's. This new ligand readily binds to CdSe—ZnS core-shell QDs via surface ligand exchange. QDs capped with a mixture of DHLA and DHLA—TEG—biotin or polyethylene glycol- (PEG- (molecular weight average ∼600 modified DHLA (DHLA—PEG600 and DHLA—TEG—biotin are easily dispersed in aqueous buffer solutions. In particular, homogeneous buffer solutions of QDs capped with a mixture of DHLA—PEG600 and DHLA—TEG—biotin that are stable over broad pH range have been prepared. QDs coated with mixtures of DHLA/DHLA—TEG—biotin and with DHLA—PEG600/DHLA—TEG—biotin were tested in surface binding assays and the results indicate that biotin groups on the QD surface interact specifically with NeutrAvidin-functionalized microtiter well plates.

  19. Lanthanide-doped NaScF4 nanoprobes: crystal structure, optical spectroscopy and biodetection

    Science.gov (United States)

    Ai, Yu; Tu, Datao; Zheng, Wei; Liu, Yongsheng; Kong, Jintao; Hu, Ping; Chen, Zhuo; Huang, Mingdong; Chen, Xueyuan

    2013-06-01

    Trivalent lanthanide ions (Ln3+)-doped inorganic nanoparticles (NPs) as potential luminescent bioprobes have been attracting tremendous interest because of their unique upconversion (UC) and downconversion (DC) luminescence properties. NaScF4, as an important host material, has been rarely reported and its crystal structure remains unclear. Herein, based on the single crystal X-ray diffraction, the space group of NaScF4 crystals was determined to be P31 containing multiple sites of Sc3+ with crystallographic site symmetry of C1, which was verified by high-resolution photoluminescence spectroscopy of Eu3+ at low temperature (10 K). Furthermore, monodisperse and size-controllable NaScF4:Ln3+ NPs were synthesized via a facile thermal decomposition method. The biotinylated NaScF4:Er3+/Yb3+ NPs were demonstrated for their applications as a heterogeneous UC luminescence bioprobe to detect avidin with a detection limit of 180 pM. After bioconjugation with amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), NaScF4:Ln3+ NPs also exhibited specific recognition of cancer cells overexpressed with uPA receptor (uPAR, an important marker of tumor biology and metastasis), showing great potentials in tumor-targeted bioimaging.Trivalent lanthanide ions (Ln3+)-doped inorganic nanoparticles (NPs) as potential luminescent bioprobes have been attracting tremendous interest because of their unique upconversion (UC) and downconversion (DC) luminescence properties. NaScF4, as an important host material, has been rarely reported and its crystal structure remains unclear. Herein, based on the single crystal X-ray diffraction, the space group of NaScF4 crystals was determined to be P31 containing multiple sites of Sc3+ with crystallographic site symmetry of C1, which was verified by high-resolution photoluminescence spectroscopy of Eu3+ at low temperature (10 K). Furthermore, monodisperse and size-controllable NaScF4:Ln3+ NPs were synthesized via a facile thermal

  20. Study on Electrochemical Biosensor Based on Alumina Nanochannels%基于氧化铝纳米通道的电化学生物传感器研究

    Institute of Scientific and Technical Information of China (English)

    陈燕; 李雪梅; 李秋顺; 史建国

    2014-01-01

    The perfectly ordered alumina nanochannels prepared by binary anodic oxidation was modified with amino-groups on the surface by reacting with 3-aminopropylethoxysilane and then reacted for 12 h in a buffer solution (pH 5.5)containing biotin to immobilize biotin on the surface of the nanochannels with bore diameters of ca.50 nm.Circular slice of polymer membrane with 7 mm was glued with PVC/THF solution on the top of a length of PVC tubing,and the modified alumina nanochannels prepared was glued on the base of the membrane with organic silicone rubber,which was to be used as working electrode.Potentiometric study was made with biotin-avidin system as a mode,using the modified alumina nano-channels as discerning carrier.Linear relationship between values of potential change and mass concentration of avidin was kept in the range of 0.10 to 0.60 mg·L-1 , with detection limit (3σ)of 0.05 mg·L-1 .Feasibility of determination of bio-materials of macro-molecules using the modified alumina nanochannels as bio-sensor in potentiometry was proved.%将制备好的氧化铝纳米通道经与3-氨丙基三乙氧基硅烷反应使其表面修饰了氨基后,再在含生物素的缓冲溶液(pH 5.5)中反应12 h,制成表面固定了生物素的氧化铝纳米通道,通道孔径约50 nm。另取 PVC 管一段,在其顶端用 PVC/THF 混合液粘附制备好的聚合物膜,再将上述修饰好的氧化铝纳米通道用有机硅橡胶粘在敏感膜的底部,作为工作电极待用。以生物素-亲和素体系为模型,用经修饰的氧化铝纳米通道为识别载体进行电位法检测,实现了亲和素的检测。亲和素的质量浓度在0.10~0.60 mg·L-1范围内与相应的电位变化值之间呈线性关系,检出限(3σ)为0.05 mg·L-1。试验结果验证了氧化铝纳米通道电位生物传感器测定生物大分子的可行性。

  1. 液态氟碳脂质微球靶向损伤心肌细胞的体外实验%In Vitro Experimental Study of Rat Cardiomyocyte Injury with Targeting of Perfluorocarbon Lipid Particles

    Institute of Scientific and Technical Information of China (English)

    何佰永; 李赵欢; 唐红; 李国华; 陈松; 王炼; 宋海波; 方华; 曾俊

    2011-01-01

    The present study was to investigate in vitro the rat cardiomyocyte injury with targeting of home-made per-fluorocarbon lipid particles with avidin-biotin interaction. Neonatal rat cardiomyocytes were cultured in vitro and divided into two groups: TNF-a activated group and non-activated group. Those in the TNF-α activated group were exposed to 200ng/ml TNF-a solution for 6 hours and then cardiomyocytes in both groups were pretargeted with bioti-nylated ICAM-1 monoclonal antibodies, and were exposed to streptavidin, and then to home-made green fluorescent-ly-labeled biotinylated perfluorocarbon lipid particles. Cardiomyocytes nucleus stained with Hoechst. The results were detected with fluorescence microscope. As a result, in TNF-a activated group, around blue fluorescent cardiomyocytes nucleus, a great amount of green fluorescent particles were found, while there were few green fluorescent particles in non-TNF activated group. It has been shown that ICAM-1 is expressed in the surface of cardiomyocytes when they are stimulated by TNF-a. Perfluorocarbon lipid particles associated with ICAM-1 monoclonal antibodies can be targeted to injured cardiomyocytes by avidin-biotin interaction.%采用生物素-亲和素作用实现自制液态氟碳脂质微球与损伤后心肌细胞的靶向结合.先体外培养大鼠原代心肌细胞,将培养出的心肌细胞分为TNF-α处理组和非处理组,TNF-α处理组的心肌细胞给予200 ng/ml TNF-α溶液刺激.6h后两组均先后加入等量的生物素化的抗大鼠细胞间黏附分子-1(ICAM-1)单克隆抗体、链酶亲和素和自制生物素化液态氟碳脂质微球,然后用Hoechst液对两组心肌细胞核染色,最后在荧光显微镜下观察.结果显示TNF-α处理组染成蓝色的心肌细胞核周围可见大量绿色荧光微球,但非处理组蓝色的心肌细胞核周围有极少许绿色荧光微球.研究表明心肌细胞在TNF-α刺激下可产生ICAM-1炎症细胞因子,液

  2. Localization of vesicular glutamate transporters in the peripheral vestibular system of rat%囊泡膜谷氨酸转运体在前庭外周系统中的分布

    Institute of Scientific and Technical Information of China (English)

    王远; 庞有旺; 董玉琳; 张富兴; 李金莲; 李云庆

    2007-01-01

    Objective To examine the vesicular glutamate transporters (VGluTs: VGluT1-VGluT3) in the peripheral vestibular system. Methods The vestibular structures, including Scarpa's ganglion (vestibular ganglion, VG), maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs,by avidin-biotinylated peroxidase complex method, with 3-3'-diaminobenzidine (DAB) as chromogen. Results (1) VGluT1was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs. (2)Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3-positive afferent fibers was in the maculae and ampullary cristae. (3) No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. Conclusion These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.%目的 检查囊泡膜谷氨酸转运体(vesicular glutamate transporter,VGluT)在前庭外周系统中的分布特征.方法 采用ABC(avidin-biotinylated peroxidase complex)免疫组织化学方法,二氨基联苯作为染色剂,观察VGluT1-3在正常成年SD大鼠前庭外周系统,包括球囊、椭圆囊、壶腹嵴和前庭神经节(Scarpa神经节)的表达.结果 (1)VGluT1样免疫阳性产物位于前庭神经节和传入纤维支配的前庭外周终末感受器.(2)大部分感觉上皮细胞表达高密度的VGluT3样免疫阳性产物,但在球囊斑和椭圆囊斑,VGluT3样免疫阳性传入纤维表达较弱.(3)在上述部位,没有或仅有很弱的VGluT2样免疫反应.结论 VGluT1和VGluT3参与了初级前庭传入纤维和毛细胞中将谷氨酸转运入囊泡的过程,可能在调节前庭终末感受器通过前庭核向大脑传递信息的传导通路中具有重要作用.

  3. Morphometric characteristics of Neuropeptide Y immunoreactive neurons of human cortical amygdaloid nucleus

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    Mališ Miloš

    2008-01-01

    Full Text Available Introduction Cortical amygdaloid nucleus belongs to the corticomedial part of the amygdaloid complex. In this nucleus there are neurons that produce neuropetide Y. This peptide has important roles in sleeping, learning, memory, gastrointestinal regulation, anxiety, epilepsy, alcoholism and depression. Material and methods We investigated morphometric characteristics (numbers of primary dendrites, longer and shorter diameters of cell bodies and maximal radius of dendritic arborization of NPY immunoreactive neurons of human cortical amygdaloid nucleus on 6 male adult human brains, aged 46 to 77 years, by immunohistochemical avidin-biotin technique. Results Our investigation has shown that in this nucleus there is a moderate number of NPY immunoreactive neurons. 67% of found neurons were nonpyramidal, while 33% were pyramidal. Among the nonpyramidal neurons the dominant groups were multipolar neurons (41% - of which 25% were multipolar irregular, and 16% multipolar oval. Among the pyramidal neurons the dominant groups were the neurons with triangular shape of cell body (21%. All found NPY immunoreactive neurons (pyramidal and nonpyramidal altogether had intervals of values of numbers of primary dendrites 2 to 6, longer diameters of cell bodies 13 to 38 µm, shorter diameters of cell bodies 9 to 20 µm and maximal radius of dendritic arborization 50 to 340 µm. More than a half of investigated neurons (57% had 3 primary dendrites. Discussion and conclusion The other researchers did not find such percentage of pyramidal immunoreactive neurons in this amygdaloid nucleus. If we compare our results with the results of the ather researchers we can conclude that all pyramidal NPY immunoreactive neurons found in this human amygdaloid nucleus belong to the class I of neurons, and that all nonpyramidal NPY immunoreactive neurons belong to the class II of neurons described by other researchers. We suppose that all found pyramidal neurons were projectional.

  4. Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    XING Yifei; XIAO Yajun; ZENG FuQing; ZHAO Jun; XIAO Chuanguo; XIONG Ping; FENG Wei

    2007-01-01

    Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

  5. Level of proinsulin in association with cardiovascular risk factors and sleep snoring

    Institute of Scientific and Technical Information of China (English)

    En-Zhi Jia; Hai-Yan Wang; Wen-Zhu Ma; Zhi-Jian Yang; Shi-Wei Chen; Guang-Yao Qi; Chun-Fa You; Jian-Feng Ma; Jing-Xin Zhang; Zhen-Zhen Wang; Wei-Chong Qian

    2005-01-01

    AIM: To explore the relationship between the level of proinsulin with cardiovascular risk factors and sleep snoring.METHODS: Based on the random stratified sampling principle, 1 193 Chinese residents in Pizhou City, Jiangsu Province (530 males and 663 females, aged 35-59 years with an average age of 46.69 years) were recruited. Their sleep snoring habits were investigated. Biotin-avidin based double mAbs ELISA was used to detect specific insulin and proinsulin, and a risk factor score was established to evaluate the individuals according to the number of their risk factors.RESULTS: The results of Spearman correlation analysis and covariate ANOVA analysis after age and sex were controlled, indicated that not only the level of proinsulin (r = 0.156, P = 0.000, F= 5.980 P = 0.000), but also cardiovascular risk factors score (r = 0.194, P = 0.000,F= 11.135, P = 0.000) significantly associated with the frequency of sleep snoring, and the significant relationship between true insulin and frequency of sleep snoring was only shown in the covariate ANOVA analysis (F = 2.868,P = 0.022). The result of multivariate stepwise logistic regression after age, sex, body mass index, waist circumference and true insulin were controlled showed that proinsulin (division by interval of quartile) was an independent risk factor for sleep snoring (OR = 1.220,95%CI: 1.085-1.373, P = 0.001).CONCLUSION: The interaction of cardiovascular risk factors clustering, high proinsulin level and sleep breathing disorder may be a syndrome, which has not been recognized in human beings so far.

  6. Increased electrocatalyzed performance through hairpin oligonucleotide aptamer-functionalized gold nanorods labels and graphene-streptavidin nanomatrix: Highly selective and sensitive electrochemical biosensor of carcinoembryonic antigen.

    Science.gov (United States)

    Wen, Wei; Huang, Jing-Yi; Bao, Ting; Zhou, Jun; Xia, Hong-Xing; Zhang, Xiu-Hua; Wang, Sheng-Fu; Zhao, Yuan-Di

    2016-09-15

    We report a triplex signal amplification strategy for sensitive biosensing of cancer biomarker by taking advantage of hairpin-shaped oligonucleotide-functionalized gold nanorods (HO-GNRs), graphene and the avidin-biotin reation. The strategy expands electrochemical detection of carcinoembryonic antigen (CEA) by using an aptamer as biosensor's recognition element and HO-GNRs as signal enhancer. To construct this biosensor, the GNR was used as a carrier of horseradish peroxidase (HRP) and HO aptamer with a biotin at the 3'-end and a thiol at the 5'-end, which amplified the electrochemical response because of a large molar ratio of HRP to HO. In the presence of target CEA, the binding reactions of CEA with the loop portions of the HOs caused HOs' loop-stem structure opened and exposed the biotins, and then HRP-GNRs-HO conjugates were captured on graphene and streptavidin modified electrodes via the reaction between the exposed biotins and preimmobilized streptavidins. The accumulation of HRP effectively catalyzed the hydrogen peroxide-mediated oxidation of o-phenylenediamine to generate an electrochemical reduction current for CEA detection. Under optimal conditions, the electrochemical biosensor exhibited a wide dynamic range of 5pgmL(-1) and 50ngmL(-1) toward CEA standards with a low detection limit of 1.5pgmL(-1) (signal-to-noise ratio of 3). The proposed biosensor accurately detected CEA concentration in 8 human serum samples from patients with lung diseases, showing excellent correlations with standard chemiluminescence immunoassay. Furthermore, these results of target DNA detection made it abundantly clear that the proposed strategy can also be extended for detection of other relative biomarkers using different functional DNA structures, which shows great prospects in single-nucleotide polymorphisms analysis, biomedical sensing and application for accurate clinical diseases diagnostic.

  7. Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

    Directory of Open Access Journals (Sweden)

    Monteavaro Cristina Esther

    2000-01-01

    Full Text Available The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed[tm] was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT in an experimental model.

  8. Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

    Science.gov (United States)

    Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

    2017-01-01

    Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

  9. Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

    Directory of Open Access Journals (Sweden)

    I. Valpotic

    2009-12-01

    Full Text Available Colidiarrhea and colienterotoxemia caused by F4+ and/or F18+ enterotoxigenic E. coli (ETEC strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac+ non-ETEC vaccine candidate strain against challenge infection with F4ac+ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs. We also evaluated levamisole as an immune response modifier (IRM and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0 or with levamisole and perorally given F18ac+ non-ETEC strain (at day 0, and challenged with F4ac+ ETEC strain 7 days later. At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3+, CD45RA+, CD45RC+, CD21+, IgA+ and myeloid SWC3+ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3+, CD45RC+ and SWC3+ cells (p<0.05 as compared to those recorded in nontreated control pigs. In the ileum of these pigs we have recorded that only CD21+ cells were significantly increased (p<0.01. The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac+ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.

  10. Dual-targeted and pH-sensitive Doxorubicin Prodrug-Microbubble Complex with Ultrasound for Tumor Treatment

    Science.gov (United States)

    Luo, Wanxian; Wen, Ge; Yang, Li; Tang, Jiao; Wang, Jianguo; Wang, Jihui; Zhang, Shiyu; Zhang, Li; Ma, Fei; Xiao, Liling; Wang, Ying; Li, Yingjia

    2017-01-01

    In this study, we investigated the potential of a dual-targeted pH-sensitive doxorubicin prodrug-microbubble complex (DPMC) in ultrasound (US)-assisted antitumor therapy. The doxorubicin prodrug (DP) consists of a succinylated-heparin carrier conjugated with doxorubicin (DOX) via hydrazone linkage and decorated with dual targeting ligands, folate and cRGD peptide. Combination of microbubble (MB) and DP, generated via avidin-biotin binding, promoted intracellular accumulation and improved therapeutic efficiency assisted by US cavitation and sonoporation. Aggregates of prepared DP were observed with an inhomogeneous size distribution (average diameters: 149.6±29.8 nm and 1036.2±38.8 nm, PDI: 1.0) while DPMC exhibited a uniform distribution (average diameter: 5.804±2.1 μm), facilitating its usage for drug delivery. Notably, upon US exposure, DPMC was disrupted and aggregated DP dispersed into homogeneous small-sized nanoparticles (average diameter: 128.6±42.3 nm, PDI: 0.21). DPMC could target to angiogenic endothelial cells in tumor region via αvβ3-mediated recognition and subsequently facilitate its specific binding to tumor cells mediated via recognition of folate receptor (FR) after US exposure. In vitro experiments showed higher tumor specificity and killing ability of DPMC with US than free DOX and DP for breast cancer MCF-7 cells. Furthermore, significant accumulation and specificity for tumor tissues of DPMC with US were detected using in vivo fluorescence and ultrasound molecular imaging, indicating its potential to integrate tumor imaging and therapy. In particular, through inducing apoptosis, inhibiting cell proliferation and antagonizing angiogenesis, DPMC with US produced higher tumor inhibition rates than DOX or DPMC without US in MCF-7 xenograft tumor-bearing mice while inducing no obvious body weight loss. Our strategy provides an effective platform for the delivery of large-sized or aggregated particles to tumor sites, thereby extending their

  11. Development of an aptamer beacon for detection of interferon-gamma.

    Science.gov (United States)

    Tuleuova, Nazgul; Jones, Caroline N; Yan, Jun; Ramanculov, Erlan; Yokobayashi, Yohei; Revzin, Alexander

    2010-03-01

    Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)-based detection of interferon-gamma (IFN-gamma)--an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-gamma (K(d) = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-gamma binding with an aptamer beacon occurred within 15 min of analyte introduction--suggesting dynamic replacement of the quencher-complementary strand by IFN-gamma molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-gamma. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-gamma. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFN-gamma. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells.

  12. High density array fabrication and readout method for a fiber optic biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, Daniel (Walnut Creek, CA); Gray, Joe (San Francisco, CA)

    1997-01-01

    The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

  13. High density array fabrication and readout method for a fiber optic biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, D.; Gray, J.

    1997-11-25

    The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its ``sensor end`` biological ``binding partners`` (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor. 9 figs.

  14. High density array fabrication and readout method for a fiber optic biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, Daniel (Walnut Creek, CA); Gray, Joe (San Francisco, CA); Albertson, Donna G. (Lafayette, CA)

    2002-01-01

    The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

  15. High density array fabrication and readout method for a fiber optic biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Pinkel, Daniel (Walnut Creek, CA); Gray, Joe (San Francisco, CA); Albertson, Donna G. (Lafayette, CA)

    2000-01-01

    The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

  16. Comparing the expression of myoepithelial cell markers CD10 and smooth muscle actin with the estrogen receptor status in the invasive carcinoma breast: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Gaurav Arora

    2013-01-01

    Full Text Available Background: Breast cancer is the most frequent cancer in females throughout the world. Around 20% of breast carcinomas are estrogen receptor alpha (ER-negative. Thus, theoretically this negativity could be either the result of down-regulation of ER expression in the tumor cells, or the result of the tumor being derived from cells which normally lack that expression. Normal basal, including myoepithelial cells of the breast is ER-negative. CD10 and smooth muscle actin (SMA are used as markers for the demonstration of these basal cells. Aims: To compare the expression of positive staining for CD10 and SMA in ER-negative and ER-positive invasive breast carcinomas. Materials and Methods: The study was performed on 40 paraffin-embedded tissues of already diagnosed cases of invasive breast carcinomas with known ER status, i.e., thirty ER-negative and ten ER-positive cases. Expression of CD10 and SMA was demonstrated using avidin-biotin-peroxidase complex (ABC technique. Tumor was considered to be positive for both markers only when more than 10% of tumor cells were stained positive. Results: Overall, CD10 tumor cell staining was seen in eight, 23.3% (7/30 ER-negative cases and in 10% (1/10 ER-positive cases. Also the staining intensity was considered to be strong. SMA tumor cell staining was seen in only 6.7% (2/30 ER-negative cases and the staining intensity was considered to be moderate. Percentages of positively stained tumor cells varied between 13% to 72% and 23% to 45% for CD10 and SMA, respectively. Conclusion: CD10 is a better marker when compared to SMA, as it is expressed in more number of cases and gives strong positivity in tumor cells. Higher expression of CD10 and SMA is correlated with higher tumor grade and ER negativity.

  17. Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β.

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    Annamari Paino

    Full Text Available Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

  18. Immunocytochemical localization of the surfactant apoprotein and Clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice.

    Science.gov (United States)

    Ward, J. M.; Singh, G.; Katyal, S. L.; Anderson, L. M.; Kovatch, R. M.

    1985-01-01

    The localization of surfactant apoprotein (SAP) and the Clara cell antigen(s) (CCA) was studied in naturally occurring and experimentally induced pulmonary hyperplasias and neoplasms by avidin-biotin peroxidase complex (ABC) immunocytochemistry. Lungs of B6C3F1 and A strain mice with naturally occurring lesions, B6C3F1 mice given injections of N-nitrosodiethylamine (DEN), BALB/c nu/nu or nu/+ mice exposed transplacentally on Day 16 of gestation to ethylnitrosourea (ENU), or BALB/c nu/+ mice exposed to ENU at 8-12 weeks of age were preserved in formalin or Bouin's fixative. After ABC immunocytochemistry, SAP was found in the cytoplasm of normal alveolar Type II cells; in the majority of cells in focal alveolar and solid hyperplasias originating in peribronchiolar or peripheral locations; and in solid, tubular, papillary, and mixed adenomas and carcinomas. The larger mixed-pattern neoplasms and small or large tubular neoplasms usually had the least number of cells with SAP. The majority of large papillary adenomas and carcinomas in BALB/c mice exposed to ENU and in untreated A strain mice contained SAP in the nuclei of many neoplastic cells but only in the cytoplasm of a few neoplastic cells. CCA was found in normal Clara cells of bronchi and bronchioles but not in any hyperplastic or neoplastic lesion of any mouse studied. This study provided immunocytochemical evidence that the vast majority of naturally occurring and experimentally induced pulmonary neoplasms of mice are alveolar Type II cell adenomas and carcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:3883798

  19. Immunohistochemical demonstration of Clara cell antigen in lung tumors of bronchiolar origin induced by N-nitrosodiethylamine in Syrian golden hamsters.

    Science.gov (United States)

    Rehm, S.; Takahashi, M.; Ward, J. M.; Singh, G.; Katyal, S. L.; Henneman, J. R.

    1989-01-01

    Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffusely that were immunoreactive for cytokeratin. Immunoreactivity for cytokeratin was also noted for hyperplastic bronchiolar neuroepithelial bodies. Cellular hyperplasias extending out into the alveolar parenchyma contained ciliated cells and frequently consisted of cells immunoreactive for CCA, showing them to be of bronchiolar Clara cell origin. Tumors developed from bronchiolar cell hyperplasias localized within bronchioles and from bronchiolar cells lining former alveolar walls. Neoplastic growth patterns were tubulo-papillary, forming loose networks or densely cellular areas. Immunoreactivity for cytoplasmic CCA was found in 50% of the tumors and was seen most frequently in small cuboidal cells and larger, vacuolated cells scattered throughout the neoplasms. In summary, evidence is presented that NDEA-induced pulmonary tumors of the Syrian golden hamster originated from cells lining bronchioles and from extrabronchiolar Clara cell hyperplasias of the terminal bronchioles. As the

  20. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    Science.gov (United States)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  1. SUPERSENSITIVE IN SITU HYBRIDIZATION BY TYRAMIDE SIGNAL AMPLIFICATION AND NANOGOLD SILVER STAINING: THE CONTRIBUTION OF AUTOMETALLOGRAPHY AND CATALYZED REPORTER DEPOSITION TO THE REJUVENATION OF IN SITU HYBRIDIZATION.

    Energy Technology Data Exchange (ETDEWEB)

    TUBBS,R.R.PETTAY,J.GROGAN,T.CHEUNG,A.L.M.POWELL,R.D.HAINFELD,J.HAUSER-KRONBERGER,C.HACKER,G.W.

    2002-04-17

    It is peculiar that in situ hybridization (ISH), a technique with many similarities to immunohistochemistry (IHC), has not enjoyed the phenomenal growth in both basic research and clinical applications as has its sister technique IHC. Since the late 1970s, when immunoperoxidase techniques began to be applied to routine diagnostic material and to numerous research applications, there has been a natural evolution of the IHC procedure. Namely, only a few primary antibodies were available commercially at the onset, and only one indirect and the peroxidase-antiperoxidase (PAP) technique detection systems were in place. With the advent of avidin-biotin detection systems and monoclonal antibodies, and a viable commercial market, extraordinary growth of the procedure's applications in clinical research and diagnostic pathology occurred during the subsequent two decades. Today, IHC is automated and widely used for research purposes and, to a large extent, has become a routine diagnostic ''special stain'' in most clinical laboratories. During the same period, ISH enjoyed very little growth in both research and diagnostic applications. What has accounted for this lack of maturation of the technique? The success of IHC is part of the reason measuring a gene's encoded protein routinely and inexpensively, particularly as automation evolved, rendered IHC a more viable choice in many instances. Inherent comparative sensitivity of the procedures has also clearly been a factor. Unfortunately, the chromogenic procedures in place are often insufficiently sensitive to detect the relatively low amounts of DNA and RNA levels at which the clinical utility is to be found.

  2. Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin–Darby bovine kidney cell line

    Science.gov (United States)

    Samrath, Devprabha; Shakya, Sanjay; Rawat, Nidhi; Gilhare, Varsha Rani; Singh, Fateh

    2016-01-01

    Aim: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test. Materials and Methods: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. Results: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. Conclusion: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test. PMID:27051213

  3. Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin–Darby bovine kidney cell line

    Directory of Open Access Journals (Sweden)

    Devprabha Samrath

    2016-02-01

    Full Text Available Aim: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1 from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK cell line. Further, the virus was identified by agar gel immunodiffusion (AGID test. Materials and Methods: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. Results: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. Conclusion: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.

  4. Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells.

    Science.gov (United States)

    Yamazaki, Daiki; Yoshida, Shigeo; Asami, Tadao; Kuchitsu, Kazuyuki

    2003-07-01

    Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

  5. Soybean isoflavones alter parvalbumin in hippocampus of mid-aged normal female, ovariectomized female, and normal male rats

    Institute of Scientific and Technical Information of China (English)

    In Koo HWANG; Moo Ho WON; Yoon-bok LEE; Ki-yeon YOO; Tae-cheon KANG; Soon Sung LIM; Sang Moo KIM; Heon-soo SOHN; Woo-jung KIM; Hyun Kyung SHIN

    2006-01-01

    Aim: To investigate the long-term effect of soybean isoflavones on changes in parvalbumin (PV) immunoreactivity in the hippocampus in normal female, ovariectomized (OVX) female and normal male rats. Methods: Ten-month-old rats were assigned to one of 9 groups (n=7 in each group) based on body weight using arandomized complete-block design. The groups were: control diet-treated females,OVX females, and males; 0.3 g/kg isoflavone-treated females, OVX females, and males; and 1.2 g/kg isoflavone-treated females, OVX females, and males. The PV immunostaining was conducted by using the standard avidin-biotin complex method. Results: PV immunoreactivity and the number of PV-immunoreactive neurons in all the groups after isoflavone treatment were significantly changed in the hippocampal CA1 region and in the dentate gyrus, but not in the hippocampal CA2/3 region. PV immunoreactivity and the number of PV-immunoreactive neurons in the control diet OVX females were similar to those in the control diet, and were greater than those in the control diet normal females. PV immunoreactivity and the number of PV-immunoreactive neurons in all the isoflavone-treated groups decreased dose-dependently after isoflavone treatment. Conclusion: Long-term administration of isoflavones may induce a reduction of PV in interneurons in the hippocampal CA1 region and in the dentate gyrus. The reduction of PV in these regions suggests that the long-term administration of isoflavones may cause a change in calcium homeostasis in the hippocampal CA1 region and in the dentate gyrus.

  6. Muscle fibre types of the lumbrical, interossei, flexor, and extensor muscles moving the index finger.

    Science.gov (United States)

    Hwang, Kun; Huan, Fan; Kim, Dae Joong

    2013-09-01

    The aim of this study was to determine the fibre types of the muscles moving the index fingers in humans. Fifteen forearms of eight adult cadavers were used. The sampled muscles were the first lumbrical (LM), first volar interosseous (VI), first dorsal interosseus (DI), second flexor digitorum profundus (FDP), second flexor digitorum superficialis (FDS), and extensor digitorum (ED). Six micrometer thick sections were stained for fast muscle fibres. The procedure was performed by applying mouse monoclonal anti-skeletal myosin antibody (fast) and avidin-biotin peroxidase complex staining. Rectangular areas (0.38 mm × 0.38 mm) were photographed and the boundaries of the muscle areas were marked on the translucent film. The numbers and sizes of the muscle fibres in each part were evaluated by the image analyser program and calculated per unit area (1 mm(2)). The proportion of the fast fibres was significantly (p = 0.012) greater in the intrinsic muscles (55.7 ± 17.1%) than in the extrinsic muscles (45.9 ± 17.1%). Among the six muscles, the VI had a significantly higher portion (59.3%) of fast fibres than the FDS (40.6%) (p = 0.005) or the FDP (45.1%) (p = 0.023). The density of the non-fast fibres was significantly (p = 0.015) greater in the extrinsic muscles (539.2 ± 336.8/mm(2)) than in the intrinsic muscles (383.4 ± 230.4/mm2). Since the non-fast fibres represent less fatigable fibres, it is thought that the extrinsic muscles have higher durability against fatigue, and the intrinsic muscles, including the LM, should move faster than the FDS or FDP because the MP joint should be flexed before the IP joint to grip an object.

  7. Assessment of S100 protein expression in the epididymis of juvenile and adult European bison.

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    Joanna Surdyk-Zasada

    2010-11-01

    Full Text Available In our study, we decided to compare S100 protein expression in the material obtained from the epididymes of 5- and 12-month-old calves, and adult European bison, and to detect any differences in S100 expression according to the animal age and size of the organ examined. We used the epididymes obtained from 6 adult European bison aged 6-12 years, from 6 at the age of 12 months and 6 calves aged 5 months. Immunocytochemical reactions were performed using the avidin-biotinylated-peroxidase (ABC technique according to HSU. Specific polyclonal rabbit antiserum against bovine S100 protein (Bio Genex Laboratories at a dilution at 1:400 was applied. We found the expression of S100 protein in endothelial cells of arteries, veins and lymphatic vessels in all the study animals. At the same time, we found no differences in the expression of S100 protein in vascular endothelial cells. Our observations seem to indicate that S100 expression in endothelial cells of European bison epididymis is not correlated with age or maturity of the organ tested. We found S100 protein in smooth muscle cells of arteries and veins in all European bison specimens examined. Interestingly in the current study, in young 5-month-old sexually immature European bison specimens we observed weaker expression of S100 protein in smooth muscle cells of small vessels as compared to the same cell type both in large vessels in these animals and in small vessels in adult specimens.

  8. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

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    A Barkhordari

    2014-09-01

    Full Text Available [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists.Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP compared to those found in normal tissue.Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system.Results: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (β1,4GlcNAc and Wisteria floribunda (GalNAc as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue.Conclusion: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

  9. Age-associated decrease in GDNF and its cognate receptor GFRα-1 protein expression in human skin.

    Science.gov (United States)

    Adly, Mohamed A; Assaf, Hanan A; Hussein, Mahmoud Rezk Abdelwahed

    2016-06-01

    Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are expressed in normal human skin. They are involved in murine hair follicle morphogenesis and cycling control. We hypothesize that 'GDNF and GFRα-1 protein expression in human skin undergoes age-associated alterations. To test our hypothesis, the expression of these proteins was examined in human skin specimens obtained from 30 healthy individuals representing three age groups: children (5-18 years), adults (19-60 years) and the elderly (61-81 years). Immunofluorescent and light microscopic immunohistologic analyses were performed using tyramide signal amplification and avidin-biotin complex staining methods respectively. GDNF mRNA expression was examined by RT-PCR analysis. GDNF mRNA and protein as well as GFRα-1 protein expressions were detected in normal human skin. We found significantly reduced epidermal expression of these proteins with ageing. In the epidermis, the expression was strong in the skin of children and declined gradually with ageing, being moderate in adults and weak in the elderly. In children and adults, the expression of both GDNF and GFRα-1 proteins was strongest in the stratum basale and decreased gradually towards the surface layers where it was completely absent in the stratum corneum. In the elderly, GDNF and GFRα-1 protein expression was confined to the stratum basale. In the dermis, both GDNF and GFRα-1 proteins had strong expressions in the fibroblasts, sweat glands, sebaceous glands, hair follicles and blood vessels regardless of the age. Thus there is a decrease in epidermal GDNF and GFRα-1 protein expression in normal human skin with ageing. Our findings suggest that the consequences of this is that GFRα-1-mediated signalling is altered during the ageing process. The clinical and therapeutic ramifications of these observations mandate further investigations.

  10. Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus.

    Science.gov (United States)

    Dong, Shibiao; Zhao, Rongtao; Zhu, Jiangong; Lu, Xiao; Li, Yang; Qiu, Shaofu; Jia, Leili; Jiao, Xiong; Song, Shiping; Fan, Chunhai; Hao, RongZhang; Song, HongBin

    2015-04-29

    A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3',5,5'-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens.

  11. Radiation-induced apoptosis in SCID Mousespleen after a low-dose irration

    Science.gov (United States)

    Ohnishi, T.; Takahashi, A.; Ohnishi, K.

    Purpose: To estimate the effects of space radiation on health of space crews, we aimed to clarify whether pre-irradiation at a low-dose interferes in a p53-centered signal transduction pathway induced by radiation. By using a severe combined immunodeficiency (Scid) mouse defective DNA-PK activity, we examined the role of DNA-PK activity in radioadaptation induced by low-dose irradiation. Methodology: Specific pathogen free 5-week-old fe male mice of Scid and the parental mice (CB-17 Icr+/+) were irradiated with X-rays at 3.0 Gy 1, 2, 3 or 4 weeks after conditioning irradiation at 0.15, 0.30, 0.45 or 0.60 Gy. The mice spleens were fixed for immunohistochemistry 12 h after irradiation. Bax on formalin-fixed paraffin-embedded sections were stained by the avidin-biotin peroxidase complex method using HISTOFINE SAB-PO(R) kit (Nichirei Co., Tokyo, Japan). Apoptosis incidence in the sections was measured by staining with HE staining. Results: The frequency of Bax- and apoptosis -positive cells increased up to 12 h after irradiation at 3.0 Gy in the spleen of CB-17 Icr+/+ and Scid mice. However, they were not observed by irradiation with low dose at 0.15-0.60 Gy. When pre-irradiation at 0.45 Gy 2 weeks before challenging acute irradiation at 3.0 Gy was performed, Bax accumulation and apoptosis induced by irradiation at 3.0 Gy was depressed in the spleen of CB-17 Icr+/+ mice, but not Scid mice. Conclusions: These data suggest that DNA-PKcs (expressed in CB-17 Icr+/+, not Scid mice) might play a major role on radioadaptation induced by pre-irradiation at low dose in mice spleen. We expect that the present findings will provide useful information for the care of space crews' health.

  12. Seroconversion and seroreactivity patterns of dairy goats naturally exposed to caprine arthritis-encephalitis virus in Brazil Soroconversão e sororeatividade de cabras leiteiras naturalmente expostas ao virus da artrite-encefalite caprina no Brasil

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    Roberto Soares Castro

    2002-08-01

    Full Text Available A labelled avidin-biotin ELISA (lab-ELISA using repeated serum samples of goats showed a progressive seroconversion with higher seroconversion rate at the period going from the beginning of the breeding up to the last half of lactation (35.0%, compared to that recorded at the beginning of breeding (17.8%(pForam realizados exames sorológicos em cabras leiteiras, utilizando-se ELISA marcado com avidina-biotina (LAB-ELISA. Esses exames mostraram soroconversão progressiva, com uma taxa maior entre os animais a partir do início da reprodução até a última metade da lactação (35% comparada à observada nos animais até o início da reprodução (17,8%(p<0,05. Além disso, o padrão de sororeatividade das amostras colhidas a cada 30-40 dias, durante 12 meses, avaliado pelo LAB-ELISA, foi caracterizado por alta variabilidade individual. Não foi observada sororeversão, e títulos mais altos foram obtidos mais no grupo constituído por animais que entraram em lactação (n=6, média de títulos=913,4 do que no grupo constituído por animais que cruzaram, mas não conceberam (n=4, média de títulos=261,2(p<0,01.

  13. Development and distribution of mast cells and neuropeptides in human fetus duodenum

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yu Chen; Xue-Mei Jia; You-Su Jia; Xiao-Rong Chen; Hui-Zhu Wang; Wei-Qin Qi

    2004-01-01

    AIM: To study the developmental regularities and heterogeneity of mast cells (MC) in human fetus duodenum and the distribution and developmental regularities of substance P(SP), calcitonin gene-related peptide (CGRP)-immunoreactive (IR) peptidergic nerves in fetus duodenum,as well as the relationship between MC, SP and CGRP- IR peptidergic nerves.METHODS: Duodena from 21 cases of human fetus and one term infant were stained by hematoxylin-eosin (HE),toluidine blue (TB) and immunohistochemical avidin-biotinylated peroxidase complex (ABC) method.RESULTS: Lobe-shape intestinal villi in duodenum were already developed at the twelfth week. At the 21st wk,muscular mucosa appeared gradually, and four layers were observed in the wall of duodenum. TB staining showed that the granules in the immature MC were pale violet,while the mature MC were strong violet in color by TB staining. Connective tissue MC (CTMC) appeared occasionally in submucosa and muscular layer of duodenum at the16th wk. While the mucosa MC (MMC) appeared at the18th wk. At the 22nd wk, both CTMC and MMC were activated, and distributed in the surrounding blood vessels and ganglions. The verge of some MC were unclear, and showed degranular phenomena. At the 14th wk, SP and CGRP-IR nerve fibers and cells appeared in the myenteric and submucous plexuses in small intestine, and the responses were turn strongly. Neurons were light to deep brown, and nerve fibers were present as varicose and liner profiles. On the corresponding site of serial sections, SP and CGRP immunohistochemical reactions were coexisted in one nerve fiber or cell. Some of MC showed SP and CGRP-IR positive staining.CONCLUSION: There are two heterogeneous kinds of MC in duodenum, MMC and CTMC. MC might play an important role in regulating blood circulation and sensation.

  14. Expression of TGF-β in Region of Bone Defect Repaired by Collagen/Nano-beta-Tricalcium Phosphate Composite Artificial Bone

    Institute of Scientific and Technical Information of China (English)

    凌翔; 陈卫民; 刘胜洪; 王罡

    2003-01-01

    The distribution and function of transforming growth factor-beta (TGF-β) in the regionof bone defect repaired by collagen/nano-beta-tricalcium phosphate composite artificial bone (Co/N-TCP) and the ability of Co/N-TCP recruiting osteoblasts to precipitate the repair of bone defectwere investigated. Twenty-four domestic rabbits were operated on bilateral cranial bone to create anexperimental bone defect of 8.0 mm in diameter through the whole bone. On the left, Co/N-TCPwas implanted as experimental group, but on the right, Co/TCP was implanted as control group.At 2nd, 4th, 8th, 12th week after operation, all animals were sacrificed and the implanted materi-als with surrounding bone were taken out. Immunohistochemical staining was performed for TGF-βassay by avidin-biotin complex method (SABC). Simultaneously, TGF-β was quantitatively ana-lyzed by HPIAS-1000 imaging analysis system. The inmmunohistochemical staining for TGFβ re-vealed that osteoblasts and immature osteocytes highly expressed TGF-β. Diffused TGF-β positivestaining particles appeared in the mesenchymal and fibrous-tissue. There was no significant differ-ence in the TGF-β positive staining between two groups in the medial region to original osseous bedsat different time points (P>0. 05). However, in distal original osseous bed of the defected region,the positive expression of TGF-β in the Co/N-TCP group was significantly stronger than in the con-trol group (P<0.05 or 0.01). The Co/N-TCP has good bioactivities and ability of stimulating andconducting TGF-β to aggregate and precipitate the healing of bone defect.

  15. Improvement of radioimmunotherapy using pretargeting

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    Eric eFrampas

    2013-06-01

    Full Text Available During the past two decades, considerable research has been devoted to radionuclide therapy using radiolabelled monoclonal antibodies and receptor binding agents. Conventional Radioimmunotherapy (RIT is now an established and important tool in the treatment of hematologic malignancies such as Non-Hodgkin lymphoma. For solid malignancies, the efficacy of RIT has not been as successful due to lower radiosensitivity, difficult penetration of the antibody into the tumor and potential excessive radiation to normal tissues. Innovative approaches have been developed in order to enhance tumor absorbed dose while limiting toxicity to overcome the different limitations due to the tumor and host characteristics.Pretargeting techniques (pRIT are a promising approach that consists of decoupling the delivery of a tumor monoclonal antibody (mAb from the delivery of the radionuclide. This results in a much higher tumor-to-normal tissue ratio and is favorable for therapy as well and imaging. This includes various strategies based on avidin/streptavidin-biotin, DNA-complementary DNA and bispecific antibody-hapten bindings. PRIT continuously evolves with the investigation of new molecular constructs and the development of radiochemistry. Pharmacokinetics improve dosimetry depending on the radionuclides used (alpha, beta and Auger emitters with prediction of tumor response and host toxicities. New constructs such as the Dock and Lock technology allow production of a variety of mABs directed against tumor-associated antigens. Survival benefit has already been shown in medullary thyroid carcinoma. Improvement in delivery of radioactivity to tumors with these pretargeting procedures associated with reduced hematologic toxicity will become the next generation of RIT. The following review addresses actual technical and clinical considerations and future development of pRIT.

  16. Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas.

    Science.gov (United States)

    Linnoila, R I; Mulshine, J L; Steinberg, S M; Funa, K; Matthews, M J; Cotelingam, J D; Gazdar, A F

    1988-12-01

    Bronchial carcinoids and small cell lung cancer (SCLC) are currently recognized as neuroendocrine (NE) neoplasms. However, non-SCLC (NSCLC) may also express NE properties. Paraffin-embedded sections of a comprehensive panel of 113 lung carcinomas were analyzed for the expression of three general markers common to all NE cells, namely, chromogranin A, Leu-7 and neuron-specific enolase (NSE), five specific NE secretory products, and four other tumor markers by immunohistochemistry using the sensitive avidin-biotinylated peroxidase technique. The authors were able to demonstrate the following: (1) most, but not all carcinoids and SCLCs expressed multiple NE markers in a high percentage of tumor cells; (2) up to a half of NSCLC cases contained small subpopulations of cells expressing NE in a high percentage of tumor cells; (2) up to half of NSCLC cases contained small subpopulations of cells expressing NE markers; and (3) occasional NSCLCs showed staining patterns indistinguishable from SCLC. Specifically, 7 of 77 NSCLCs expressed four or more NE markers. NE markers in NSCLCs were more commonly expressed in adenocarcinomas and large cell carcinomas and rarely in squamous cell carcinomas. For comparison, the mean number of NE markers expressed by all cases of NSCLC was 1.5, carcinoids 6.0, and SCLCs 3.8. Individual "marker counts" were not useful in categorizing lung tumors as carcinoids and SCLC versus NSCLC. Instead, 95% of the tumors were correctly classified, applying a statistical model created from staining indices of the three general NE markers (chromogranin A, Leu-7, NSE) and three other tumor markers (carcinoembryonic antigen, keratin, vimentin). Because NSCLCs with NE features might have different clinical characteristics than other NSCLCs, immunohistochemistry provides an effective manner to identify this biologically interesting subset of NSCLCs in routine paraffin sections.

  17. Piezoresistive microcantilever aptasensor for ricin detection and kinetic analysis

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    Zhi-Wei Liu

    2015-04-01

    Full Text Available Up to now, there has been no report on target molecules detection by a piezoresistive microcantilever aptasensor. In order to evaluate the test performance and investigate the response dynamic characteristics of a piezoresistive microcantilever aptasensor, a novel method for ricin detection and kinetic analysis based on a piezoresistive microcantilever aptasensor was proposed, where ricin aptamer was immobilised on the microcantilever surface by biotin-avidin binding system. Results showed that the detection limit of ricin was 0.04μg L−1 (S/N ≥ 3. A linear relationship between the response voltage and the concentration of ricin in the range of 0.2μg L−1-40μg L−1 was obtained, with the linear regression equation of ΔUe = 0.904C + 5.852 (n = 5, R = 0.991, p < 0.001. The sensor showed no response for abrin, BSA, and could overcome the influence of complex environmental disruptors, indicating high specificity and good selectivity. Recovery and reproducibility in the result of simulated samples (simulated water, soil, and flour sample determination met the analysis requirements, which was 90.5∼95.5% and 7.85%∼9.39%, respectively. On this basis, a reaction kinetic model based on ligand-receptor binding and the relationship with response voltage was established. The model could well reflect the dynamic response of the sensor. The correlation coefficient (R was greater than or equal to 0.9456 (p < 0.001. Response voltage (ΔUe and response time (t0 obtained from the fitting equation on different concentrations of ricin fitted well with the measured values.

  18. Oncogene amplification detected by in situ hybridization in radiation induced rat skin tumors. [C-myc:a3

    Energy Technology Data Exchange (ETDEWEB)

    Yi Jin.

    1991-02-01

    Oncogene activation may play an important role in radiation induced carcinogenesis. C-myc oncogene amplification was detected by in situ hybridization in radiation-induced rat skin tumors, including squamous and basal cell carcinomas. In situ hybridization was performed with a biotinylated human c-myc third exon probe, visualized with an avidin-biotinylated alkaline phosphate detection system. No c-myc oncogene amplification was detected in normal rat skin at very early times after exposure to ionizing radiation, which is consistent with the view that c-myc amplification is more likely to be related to carcinogenesis than to normal cell proliferation. The incorporation of tritiated thymidine into the DNA of rat skin cells showed that the proliferation of epidermal cells reached a peak on the seventh day after exposure to ionizing radiation and then decreased. No connection between the proliferation of epidermal cell and c-myc oncogene amplification in normal or irradiated rat skin was found. The results indicated that c-myc amplification as measured by in situ hybridization was correlated with the Southern bolt results, but only some of the cancer cells were amplified. The c-myc positive cells were distributed randomly within regions of the tumor and exhibited a more uniform nuclear structure in comparison to the more vacuolated c-myc negative cells. No c-myc signal was detected in unirradiated normal skin or in irradiated skin cells near the tumors. C-myc amplification appears to be cell or cell cycle specific within radiation-induced carcinomas. 28 refs., 3 figs., 3 tabs.

  19. A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

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    Tsai Chueh-Jen

    2010-01-01

    Full Text Available Abstract There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α and nuclear factor-kappa B (NF-κB were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

  20. Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration.

    Science.gov (United States)

    McCormick, Aleesha M; Jarmusik, Natalie A; Endrizzi, Elizabeth J; Leipzig, Nic D

    2014-01-22

    Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

  1. Influence of superior cervical ganglionectomy on hippocampal neurogenesis and learning and memory in adult rats

    Institute of Scientific and Technical Information of China (English)

    Yanping Ding; Baoping Shao; Shiyuan Yu; Shanting Zhao; Jianlin Wang

    2009-01-01

    BACKGROUND: Studies have shown that neurogenesis in the dentate gyrus plays an important role in learning and memory. However, studies have not determined whether the superior cervical ganglion or the sympathetic nerve system influences hippocampal neurogenesis or learning and memory in adult rats. OBJECTIVE: To observe differences in dentate gyrus neurogenesis, as well as learning and memory, in adult rats following superior cervical ganglionectomy. DESIGN, TIME AND SETTING: A randomized, controlled, animal study was performed at the Immunohistochemistry Laboratory of the School of Life Sciences in Lanzhou University from July 2006 to July 2007.MATERIALS: Doublecortin polyclonal antibody was provided by Santa Cruz Biotechnology, USA;avidin-biotin-peroxidase complex was purchased from Zhongshan Goldenbride Biotechnology, China;Morris water maze was bought from Taimeng Technology, China. METHODS: A total of 20 adult, male, Wistar rats were randomly divided into surgery and control groups, with 10 rats in each group. In the surgery group, the bilateral superior cervical ganglions were transected. In the control group, the superior cervical ganglions were only exposed, but no ganglionectomy was performed. MAIN OUTCOME MEASURES: To examine distribution, morphology, and number of newborn neurons in the dentate gyrus using doublecortin immunohistochemistry at 36 days following surgical procedures. To examine ability of learning and memory in adult rats using the Morris water maze at 30 days following surgical procedures. RESULTS: Doublecortin immunohistochemical results showed that a reduction in the number of doublecortin-positive neurons in the surgery group compared to the control group (P<0.05), while the distribution of doublecortin-positive neurons was identical in the two groups. The surgery group exhibited significantly worse performance in learning and spatial memory tasks compared to the control group (P<0.05). CONCLUSION: Superior cervical ganglionectomy

  2. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    Science.gov (United States)

    Park, Seonhwa; Shin, Yu Mi; Seo, Jeongwook; Song, Ji-Joon; Yang, Haesik

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

  3. Brainstem gliomas - A clinicopathological study of 45 cases with p53 immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Badhe Prerna

    2004-01-01

    Full Text Available BACKGROUND: Brainstem tumors represent 10% of central nervous system tumors, accounting for 30% of pediatric posterior fossa tumors. AIMS: The aim of this study was to clinicopathologically correlate 45 cases of brain stem gliomas and determine the occurrence and prognostic significance of p53 expression. MATERIALS AND METHOD: 45 cases of brain stem gliomas encountered during a 19-year period. 30 were diagnosed by surgical biopsy and 15 at autopsy. In 25 cases p53 immunohistochemistry (Avidin Biotinylated technique was performed. The WHO brain tumor classification and Stroink's CT classification were applied. STATISTICAL ANALYSIS USED: Chi square test. RESULTS AND CONCLUSIONS: 51 % of gliomas were observed in the first decade of life. The female to male ratio was 1.04: 1. The commonest presenting features were cranial nerve palsies (33% and cerebellar signs (29.8%. 55.55% of cases were located in the pons, 31.01% in the medulla and 13.33% in the midbrain. Diffuse astrocytomas were seen in 40 cases (5% were Grade I, 47.5%Grade II, 32.5% Grade III and 15% Grade IV and pilocytic astrocytomas in 5 cases. Grade IV patients had 2- 3 mitoses /10 high power fields and had a poorer survival rate. Grade II astrocytomas were treated with excision and radiotherapy, while grade III and IV tumors were treated with radiotherapy and chemotherapy (CCNU. Improvement was noted in 20% of patients postoperatively. The outcome was better in patients who were treated surgically. p53 is a frequently mutated gene in brain stem astrocytomas. It was found in 50 % of glioblastoma multiforme, 28.57% of grade III astrocytoma and 12.5% of grade II astrocytoma, while grade 1 astrocytomas failed to express p53 protein. p53 positivity was more in high grade lesions, decreasing significantly in lower grade lesions.

  4. Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobicity of the mycelium.

    Science.gov (United States)

    Peñalver, M C; Casanova, M; Martínez, J P; Gil, M L

    1996-07-01

    Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface. It was found that mature mycelium was hydrophobic. Treatment of intact mycelium with beta-mercaptoethanol (beta ME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with beta ME extracts from mycelium inhibited their attachment to intact mycelial cells. A. fumigatus mycelium was tagged in vivo with biotin and treated with beta ME. The beta ME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA). This procedure allowed identification of cell wall surface proteins and glycoproteins. Rabbit polyclonal antisera were raised against beta ME extracts obtained from cells grown in YPD and Vogel's N media. These antisera defined some major cell-wall-bound antigens. SDS-PAGE and Western blotting analysis of the cell wall material released by beta ME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa. Treatment of cells grown in YPD, but not those grown in Vogel's N medium, with beta ME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera. The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by beta ME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium.

  5. Detection of Human Papillomavirus 18 in Cervical Cancer Samples Using PCR-ELISA (DIAPOPS

    Directory of Open Access Journals (Sweden)

    KN Tafreshi

    2011-12-01

    Full Text Available Background and Objectives: Human Papillomavirus (HPV infection is a major risk factor for adenocarcinoma of the cervix. The high-risk types of the virus such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, are responsible for approximately 50% of all cervical cancers. A rapid, sensitive and specific test has been proposed for detection of HPV to improve cervical cancer screening programs.Objectives: The aim of this study was to develop a fast PCR-ELISA assay designated as DIAPOPS (Detection of Immobilized Amplified Products in a One Phase Systemfor detection of HPV16 and HPV18 types in SCC samples and Pap smears. The type specific primers and probes were designed for PCR and PCR-ELISA. The amplified products were hybridized with a specific biotin-labeled probe for HPV18 inner amplicons. The hybrids were detected with peroxidase conjugated avidin. The test was performed on the paraffin block and Pap smear samples from the cervical cancer patients, and the results of DIAPOPS were compared with conventional PCR assay.Results: The 70 samples (SCC and Pap smear samples were collected from Imam Khomeini and Mirzakoochak Khan Hospitals in Tehran. The PCR-based method detected six HPV16 positive, three HPV18 positive and Two HPV33 positive samples. DIAPOPS results were compared with the conventional PCR results and they showed an increase in sensitivity of the DIAPOPS test. Not only all of them were confirmed by PCR-ELISA but also three samples that conventional PCR showed negative for HPV18, were demonstrated positive by the PCR-ELISA method.Conclusion: The results of the study show that modified PCR-ELISA assay is more sensitive to detect HPV types and can be used for diagnostic purposes.

  6. Immunohistochemical analysis of p53, cyclinD1, RB1, c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

    Institute of Scientific and Technical Information of China (English)

    SJ Moghaddam; EN Haghighi; S Samiee; N Shahid; AR Keramati; S Dadgar; MR Zali

    2007-01-01

    AIM: To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma.METHODS: Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method (avidin-biotin-peroxidase)for detection of p53, cyclinD1, RB1, c-fos and N-ras proteins.RESULTS: Six (24%), 5 (20%), 12 (48%) and 2 samples (8%) were positive for p53, cyclinD1, C-fos and N-ras expression, respectively. Twenty-two (88%) samples had alterations in the G1 cell-cycle checkpoint protein expression (RB1 or cyclinD1). P53 positive samples showed a higher (9 times) risk of being positive for RB1 protein than p53 negative samples. Loss of expression of RB1 in association with p53 over-expression was observed in 4 (66.7%) of 6 samples. Loss of expression of RB1 was seen in all cyclinD1 positive, 20 (90.9%) N-ras negative, and 11 (50%) C-fos positive samples,respectively. CyclinD1 positive samples showed a higher (2.85 and 4.75 times) risk of being positive for c-fos and N-ras expression than cyclinD1 negative samples.CONCLUSION: The expression of p53, RB1 and c-fos genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

  7. Chlamydiosis in mariculture-reared green sea turtles (Chelonia mydas).

    Science.gov (United States)

    Homer, B L; Jacobson, E R; Schumacher, J; Scherba, G

    1994-01-01

    From August 1990 to June 1991, a moderate die-off of 4- to 5-year-old green sea turtles (Chelonia mydas) occurred at Cayman Turtle Farm, Grand Cayman, British West Indies. Clinical signs included lethargy, anorexia, and inability to dive. Many of the ill turtles floated on the surface of their tanks. There was no apparent sex predilection. Complete necropsies, including histopathologic examination of tissues, were performed on eight turtles. Necropsies revealed multiple irregular discrete to patchy 1-10 mm pale gray foci throughout the hearts of four turtles. By light microscopic examination, the most severe and consistent lesions were necrotizing myocarditis, histiocytic to fibrinous splenitis, and hepatic lipidosis and necrosis. A mixed leukocytic infiltrate of acidophils, macrophages, and lymphocytes was present in affected areas of the heart. Other lesions included lymphocytic/plasmacytic interstitial nephritis, subacute interstitial pneumonia, subacute mesenteric vasculitis, chronic/active enteritis of the small intestine, and occasional granulomas associated with spirorchid trematode ova. Chlamydiae could be demonstrated in macrophages in sections of paraffin-embedded heart, liver, and spleen and in myocardial fibers and hepatocytes using a modified Macchiavello's stain. Chlamydial antigen was detected by light microscopic examination in the cytoplasm of myocardial fibers and in occasional hepatocytes using a commercially available genus-specific antichlamydial monoclonal antibody and the avidin biotin peroxidase complex staining method. Electron microscopic examination of the heart of the most severely affected turtle revealed developmental stages of chlamydial organisms. A suspension of heart from this turtle was inoculated into the yolk sacs of chicken embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase.

    Science.gov (United States)

    Truong, Van-Den; Clare, Debra A; Catignani, George L; Swaisgood, Harold E

    2004-03-10

    Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.

  9. Rapid high-throughput genotyping of HBV DNA using a modified hybridization-extension technique.

    Science.gov (United States)

    Bao, Han; Zhao, Wenliang; Ruan, Banjun; Wang, Qing; Zhao, Jinrong; Lei, Xiaoying; Wang, Weihua; Liu, Yonglan; Sun, Jianbing; Xiang, An; Guo, Yanhai; Yan, Zhen

    2013-11-07

    China has the highest incidence of hepatitis B virus (HBV) infection worldwide. HBV genotypes have variable impacts on disease pathogenesis and drug tolerance. We have developed a technically simple and accurate method for HBV genotyping that will be applicable to pre-treatment diagnosis and individualized treatment. Multiple sequence alignments of HBV genomes from GenBank were used to design primers and probes for genotyping of HBV A through H. The hybridization was carried out on nitrocellulose (NC) membranes with probes fixed in an array format, which was followed by hybrid amplification by an extension step with DNA polymerase to reinforce the double-stranded DNA hybrids on the NC membrane and subsequent visualization using an avidin-biotin system. Genotyping results were confirmed by DNA sequencing and bioinformatics analysis using the National Center for Biotechnology Information genotyping database, and compared with results from the line probe assay. The data show that multiple sequence alignment defined a 630 bp region in the HBV PreS and S regions that was suitable for genotyping. All genotyping significant single nucleotides in the region were defined. Two-hundred-and-ninety-one HBV-positive serum samples from Northwest Chinese patients were genotyped, and the genotyping rate from the new modified hybridization-extension method was 100% compared with direct sequencing. Compared with line probe assay, the newly developed method is superior, featuring reduced reaction time, lower risk of contamination, and increased accuracy for detecting single nucleotide mutation. In conclusion, a novel hybridization-extension method for HBV genotyping was established, which represents a new tool for accurate and rapid SNP detection that will benefit clinical testing.

  10. Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Leitner, Michael [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Stock, Lorenz G. [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Traxler, Lukas [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Leclercq, Laurent [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Bonazza, Klaus; Friedbacher, Gernot [Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna (Austria); Cottet, Hervé [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Stutz, Hanno [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Ebner, Andreas, E-mail: andreas.ebner@jku.at [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria)

    2016-08-03

    Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated. - Highlights: • SMIL coating allows generation of homogeneous ultra-flat surfaces. • Molecular electrostatic adhesion forces can be determined in the inner wall of CZE capillary with picoNewton accuracy. • Topographical images and simultaneously acquired adhesion maps yield morphological and chemical information at the nanoscale.

  11. Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis.

    Directory of Open Access Journals (Sweden)

    Marta Matulova

    Full Text Available In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection. To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis with or without previous oral vaccination was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of transcription were identified. The most inducible genes encoded avidin (AVD, extracellular fatty acid binding protein (EXFABP, immune responsive gene 1 (IRG1, chemokine ah221 (AH221, trappin-6-like protein (TRAP6 and serum amyloid A (SAA. Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and γδ T-lymphocytes, we found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221, TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42 of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization of the course of S. Enteritidis infection in chickens.

  12. Solid lipid nanoparticles as a vehicle for brain-targeted drug delivery: two new strategies of functionalization with apolipoprotein E

    Science.gov (United States)

    Rute Neves, Ana; Fontes Queiroz, Joana; Weksler, Babette; Romero, Ignacio A.; Couraud, Pierre-Olivier; Reis, Salette

    2015-12-01

    Nanotechnology can be an important tool to improve the permeability of some drugs for the blood-brain barrier. In this work we created a new system to enter the brain by functionalizing solid lipid nanoparticles with apolipoprotein E, aiming to enhance their binding to low-density lipoprotein receptors on the blood-brain barrier endothelial cells. Solid lipid nanoparticles were successfully functionalized with apolipoprotein E using two distinct strategies that took advantage of the strong interaction between biotin and avidin. Transmission electron microscopy images revealed spherical nanoparticles, and dynamic light scattering gave a Z-average under 200 nm, a polydispersity index below 0.2, and a zeta potential between -10 mV and -15 mV. The functionalization of solid lipid nanoparticles with apolipoprotein E was demonstrated by infrared spectroscopy and fluorimetric assays. In vitro cytotoxic effects were evaluated by MTT and LDH assays in the human cerebral microvascular endothelial cells (hCMEC/D3) cell line, a human blood-brain barrier model, and revealed no toxicity up to 1.5 mg ml-1 over 4 h of incubation. The brain permeability was evaluated in transwell devices with hCMEC/D3 monolayers, and a 1.5-fold increment in barrier transit was verified for functionalized nanoparticles when compared with non-functionalized ones. The results suggested that these novel apolipoprotein E-functionalized nanoparticles resulted in dynamic stable systems capable of being used for an improved and specialized brain delivery of drugs through the blood-brain barrier.

  13. XIAP as a prognostic marker of early recurrence of nonmuscular invasive bladder cancer

    Institute of Scientific and Technical Information of China (English)

    LI Ming; SONG Tao; YIN Zhen-fei; NA Yan-qun

    2007-01-01

    Background Dysregulation of apoptosis has been implicated not only in carcinogenesis and tumor progression but also in tumor recurrence. We investigated whether the expression of X-linked inhibitor of apoptosis (XIAP) might predict early recurrence in patients with non-muscular invasive bladder cancer.Methods The cohort comprised 176 consecutive patients with primary superficial bladder cancer treated with transurethral resection. Immunohistochemical staining using the standard avidin-biotin-peroxidase technique and RT-PCR were used to detect XIAP protein and mRNA expressions in cancer tissues. The relationship between XIAP expression and clinicopathological characteristics, cancer recurrence were analyzed.Results XIAP expression was observed in 108 cases (61.4%) and no expression in 68. There was no correlation between XIAP expression rate and the tumor pathological grade, but was an apparent trend toward the increased XIAP levels from well (G1) to poor (G3) differentiated cancer. Eighty-two (46.6%) patients experienced tumor recurrence at a mean of 28.6 months of the follow-up; 66 of them expressed XIAP (61.1%) and 16 were XIAP negative (23.5%). Twelve patients presented with invasive disease at the time of relapse and all of them expressed XIAP. Patients without XIAP expression or with low tumor grades had significantly higher recurrence-free survival than those with XIAP expression(log rank test P=0.0015) or high tumor grades (log rank test P<0.001). Multivariate analysis revealed that XIAP expression, tumor grade, and tumor number were independent predictors for the recurrence of non-muscular invasive bladder cancer (P=-0.004, 0.016, and 0.043, respectively).Conclusions XIAP may be considered as a new independent prognostic marker for early recurrence of non-muscular invasive bladder cancer.

  14. A Cell Lysis and Protein Purification - Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins

    Science.gov (United States)

    Nakyama, Tetsuya; Tabata, Kazuhito; Noji, Hiroyuki; Yokokawa, Ryuji

    We have developed two devices applicable to evaluate genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification - assay device. A motor protein, F1-ATPase expressed in E.coli, was focused in this report as a target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were demonstrated in order to detect F1-ATPase and to evaluate its activity. The specific bonding between his-tag in F1-ATPase and Ni-NTA coated on a glass surface was utilized for the purification process. After immobilization of F1-ATPase, avidin-coated microspheres and adenosine tri-phosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, at the optimum condition, extracts enough amount of protein for single molecule assay. Once cell lysate was injected to the purification - assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentratioin provides an optimal concentration for the assay i.e. the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected to the device. The optimum concentration was achieved by our cell lysis device not by the conventional method by ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification - assay device, and the results were compatible to that of conventional assay in which F1-ATPase was purified in bulk scale. In conclusion, we have demonstrated on-chip cell lysis and assay appropriate for the sequential analysis without any pretreatment. On-chip devices replacing conventional bioanalytical methods will be

  15. Loss of Ab-nerve endings associated with the Merkel cell-neurite complex in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis

    Institute of Scientific and Technical Information of China (English)

    Daniela Caldero n Carrio n; Yu ksel Korkmaz; Britta Cho; Marion Kopp; Wilhelm Bloch; Klaus Addicks; Wilhelm Niedermeier

    2016-01-01

    The Merkel cell-neurite complex initiates the perception of touch and mediates Ab slowly adapting type I responses. Lichen planus is a chronic inflammatory autoimmune disease with T-cell-mediated inflammation, whereas hyperkeratosis is characterized with or without epithelial dysplasia in the oral mucosa. To determine the effects of lichen planus and hyperkeratosis on the Merkel cell-neurite complex, healthy oral mucosal epithelium and lesional oral mucosal epithelium of lichen planus and hyperkeratosis patients were stained by immunohistochemistry (the avidin-biotin-peroxidase complex and double immunofluorescence methods) using pan cytokeratin, 20 (K20, a Merkel cell marker), and neurofilament 200 (NF200, a myelinated Ab- and Ad-nerve fibre marker) antibodies. NF200-immunoreactive (ir) nerve fibres in healthy tissues and in the lesional oralmucosa epitheliumof lichen planus and hyperkeratosis were counted and statistically analysed. In the healthy oral mucosa, K20-positive Merkel cells with and without close association to the intraepithelial NF200-ir nerve fibres were detected. In the lesional oral mucosa of lichen planus and hyperkeratosis patients, extremely rare NF200-ir nerve fibres were detected only in the lamina propria. Compared with healthy tissues, lichen planus and hyperkeratosis tissues had significantly decreased numbers of NF200-ir nerve fibres in the oral mucosal epithelium. Lichen planus and hyperkeratosis were associated with the absence of Ab-nerve endings in the oral mucosal epithelium. Thus, we conclude that mechanosensation mediated by the Merkel cell-neurite complex in the oral mucosal epithelium is impaired in lichen planus and hyperkeratosis.

  16. Detection of Neospora caninum in tissue sections using a murine monoclonal antibody.

    Science.gov (United States)

    Cole, R A; Lindsay, D S; Dubey, J P; Blagburn, B L

    1993-10-01

    A murine monoclonal antibody (MAb 6G7), isotype IgG2a, produced against tachyzoites of Neospora caninum (isolate NC-1) reacted specifically with tachyzoites of N. caninum in an indirect immunofluorescent antibody test. MAb 6G7 did not react with tachyzoites of Toxoplasma gondii, sporozoites of Isospora suis, Eimeria bovis, or E. tenella, or merozoites of E. bovis in the indirect immunofluorescent antibody test. MAb 6G7 reacted positively with both tachyzoites and bradyzoites of N. caninum in an avidin-biotin peroxidase complex immunohistochemical test on formalin-fixed paraffin-embedded tissues. No reaction was observed with the following: tachyzoites and bradyzoites of T. gondii, T. gondii-like parasites, or Hammondia hammondi; bradyzoites of Frenkelia microti; schizonts and merozoites of Sarcocystis-like organisms; schizonts, sarcocysts, and oocysts/sporocysts of S. cruzi; schizonts and merozoites of S. canis; schizonts of S. hirsuta, S. tenella, and S. capracanis; merozoites of S. neurona and S. neurona-like organisms, E. bovis, or Haemoproteus sp.; bradyzoites and merozoites of S. montanaensis; bradyzoites of S. odocoileocanis, S. cruzi, and S. tenella; meronts, sexual stages, and caryocysts of Caryospora sp. and C. bigenetica; micromerozoites, macromerozoites, and schizonts of Hepatozoon canis; sporozoites, sexual stages, and oocysts of Cryptosporidium parvum and C. baileyi; trophozoites of Monocystis lumbrici, Tritrichomonas foetus, and Balantidium coli; tissue cysts and bradyzoites of Besnoitia sp. and B. jellisoni; amastigotes of Leishmania sp.; and trophic theronts of Ichthyopthirius multifilis. MAb 6G7 reacted with tachyzoites and bradyzoites of N. caninum in natural and experimental infections in dogs, cattle, mice, rats, sheep, and goats, indicating that host origin of the tissues did not affect the performance of the test.

  17. Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice I. Identification of leukocyte response with monoclonal antibodies.

    Science.gov (United States)

    Letonja, T; Hammerberg, C

    1987-10-01

    Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained by an immunoperoxidase technique (avidin-biotin complex, ABC) for the differentiation antigens Lyt-1, Lyt-2, Mac-1, Mac-2, Mac-3, and B220. Binding of ABC to the cytoplasm of hepatocytes around the developing parasite was observed at 4 days postinfection (PI) in all 3 strains of mice, persisting in BALB/cJ and C3H/HeJ liver sections at 5 and 6 days PI, suggesting the presence of high concentrations of biotin, a fatty acid synthesis mediator. Two cell populations were labeled with B220 monoclonal antibodies: lymphocytes and polymorphonuclear (PMN) cells. At 4 days PI the number of labeled PMN cells peaked in infected C3H/HeJ and BALB/cJ mice; however a low number of PMN cells were labeled in infected C3H/HeDub mice. Few lymphocytes bound the B220 antibody in either BALB/cJ, C3H/HeJ, or C3H/HeDub infected mice. The number of Mac-1+ cells detected in infected C3H/HeJ and BALB/cJ liver sections were similar whereas fewer Mac-1+ cells were present in infected C3H/HeDub mice. Mac-2+ cells appeared in high numbers around the growing parasite at 5 and 6 days PI in the liver of C3H/HeDub mice, but not in the liver of BALB/cJ mice. Mac-3+ cells followed a similar pattern to that of the cell population defined by Mac-2. Few Lyt-1+ and Lyt-2+ cells were detected around the parasite site in the 3 strains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. NMR-DMF: a modular nuclear magnetic resonance-digital microfluidics system for biological assays.

    Science.gov (United States)

    Lei, Ka-Meng; Mak, Pui-In; Law, Man-Kay; Martins, Rui P

    2014-12-07

    We present a modular nuclear magnetic resonance-digital microfluidics (NMR-DMF) system as a portable diagnostic platform for miniaturized biological assays. With increasing number of combinations between designed probes and a specific target, NMR has become an accurate and rapid assay tool, which is capable of detecting particular kinds of proteins, DNAs, bacteria and cells with a customized probe quantitatively. Traditional sample operation (e.g., manipulation and mixing) relied heavily on human efforts. We herein propose a modular NMR-DMF system to allow the electronic automation of multi-step reaction-screening protocols. A figure-8 shaped coil is proposed to enlarge the usable inner space of a portable magnet by 4.16 times, generating a radio frequency (RF) excitation field in the planar direction. By electronically managing the electro-wetting-on-dielectric (EWOD) effects over an electrode array, preloaded droplets with the inclusion of biological constituents and targets can be programmed to mix and be guided to the detection site (3.5 × 3.5 mm(2)) for high-sensitivity NMR screening (static B field: 0.46 T, RF field: 1.43 mT per ampere), with the result (voltage signal) displayed in real-time. To show the system's utility, automated real-time identification of 100 pM of avidin in a 14 μL droplet was achieved. The system shows promise as a robust and portable diagnostic device for a wide variety of biological analyses and screening applications.

  19. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    Science.gov (United States)

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format.

  20. ELISA Progress in the Diagnosis of Foot and Mouth Disease%酶联免疫吸附试验在口蹄疫诊断中的研究进展

    Institute of Scientific and Technical Information of China (English)

    厍大亮; 李冬

    2011-01-01

    Timely and accurate diagnosis is the key to control foot and mouth disease. Both in the laboratory or field, ELISA is the most common diagnostic methods of FMD because it has many advantages. We introduce the principles, characteristics and research progress of conventional and novel ELISA. The conventional ELISA, including Liquid-phase Blocking sandwich ELISA, Indirect ELISA, Solid-phase Competition ELISA and Indirect Sandwich ELISA .While McAb ELISA, RT-PCR ELISA, Biotin-avidin ELISA and Dot-ELISA belong to the novel ELISA .The direction of the development of ELISA is sensitive, stable, simple and easy to automate. ELISA would be more widely used in clinical diagnosis and epidemiological investigation, with advances in technology.%防治口蹄疫的关键是做出及时、准确的诊断.酶联免疫吸附试验(ELISA)有诸多优点,因此不论在实验室还是在田间,都是常用的口蹄疫诊断方法.介绍了传统ELISA和新型ELISA方法的原理、特点及最新研究进展.传统ELISA包括液相阻断ELISA、间接ELISA、固相竞争ELISA、间接夹心ELISA.新型ELISA包括单克隆抗体ELISA、PCR-ELISA、生物素-亲和素ELISA及Dot-ELISA.ELISA的发展方向应当是既灵敏稳定,又操作方便、易于实现自动化.随着技术的进步,ELISA在临床诊断和流行病学调查中将会有更加广泛的应用.

  1. Promotion of N-methyl-N-nitrosourea-induced thyroid tumors by iodine deficiency in F344/NCr rats.

    Science.gov (United States)

    Ohshima, M; Ward, J M

    1984-07-01

    Six-week-old male F344 rats were each given an injection once iv of N-methyl-N-nitrosourea [(MNU) CAS: 684-93-5] at a dose of 41.2 mg/kg body weight. Two weeks later, groups of rats were placed on iodine-deficient (ID) or iodine-adequate (IA) diets and then sacrificed at 20 and 33 weeks. Other groups received ID or IA diets without MNU. For localizing thyroid-stimulating hormone (TSH) and prolactin, sections of pituitary glands were stained by the avidin-biotin-peroxidase complex technique with the use of anti-rat TSH or prolactin antibody. At 20 weeks, rats receiving MNU and ID diets had a 100% incidence of diffuse follicular goiter and multiple follicular adenomas of the thyroid. Focal proliferative thyroid follicular lesions including focal hyperplasias and adenomas per square centimeter of thyroid gland were significantly increased in rats given MNU and ID diets in comparison with rats given MNU and IA diets. At 33 weeks, all MNU rats on ID diets had a significantly increased incidence of thyroid carcinoma of the follicular or papillary types and diffuse pituitary thyrotroph hyperplasia, hypertrophy, and vacuolar degeneration. Rats fed ID diets without MNU had diffuse follicular goiter but no tumors at any time period. MNU given alone in rats fed IA diets induced a 10% incidence of single thyroid adenomas at 20 weeks and 70% at 33 weeks and a 10% incidence of thyroid carcinoma at 33 weeks. Tumors induced in other organs by MNU were not affected by the ID diets. Thus this experiment provided evidence that ID diets are potent promoters of thyroid tumors in this system, but the ID diet itself without carcinogen was not carcinogenic under the conditions of the study.

  2. Significant association of insulin and proinsulin with clustering of cardiovascular risk factors

    Institute of Scientific and Technical Information of China (English)

    En-Zhi Jia; Xin-Li Li; Hai-Yan Wang; Wen-Zhu Ma; Zhi-Jian Yang; Shi-Wei Chen; Guang-Yao Qi; Chun-Fa You; Jian-Feng Ma; Jing-Xin Zhang; Zhen-Zhen Wang; Wei-Chong Qian

    2005-01-01

    AIM: To investigate the association between true insulin and proinsulin and clustering of cardiovascular risk factors.METHODS: Based on the random stratified sampling principles, 1196 Chinese people (533 males and 663 females,aged 35-59 years with an average age of 46.69 years) were recruited. Biotin-avidin based double monoclonal antibody ELISA method was used to detect the true insulin and proinsulin, and a risk factor score was set to evaluate individuals according to the number of risk factors.RESULTS: The median (quartile range) of true insulin and proinsulin was 4.91 mIu/L (3.01-7.09 mIu/L) and 3.49 pmol/L (2.14-5.68 pmol/L) respectively, and the true insulin level of female subjects was significantly higher than that of male subjects (P = 0.000), but the level of proinsulin displayed no significant difference between males and females (P = 0.566). The results of covariate ANOVA after age and sex were controlled showed that subjects with any of the risk factors had a significantly higher true insulin level (P = 0.002 for hypercholesterolemia, P = 0.021 for high low-density lipoprotein cholesterol, P = 0.003 for low high-density lipoprotein cholesterol, and P = 0.000 for other risk factors) and proinsulin level (P = 0.001 for low high-density lipoprotein cholesterol, and P = 0.000 for other risk factors)than those with no risk factors. Furthermore, subjects with higher risk factor scores had a higher true insulin and proinsulin level than those with lower risk factor scores (P = 0.000). The multiple linear regression models showed that true insulin and proinsulin were significantly related to cardiovascular risk factor scores respectively (P = 0.000).CONCLUSION: True insulin and proinsulin are significantly associated with the clustering of cardiovascular risk factors.

  3. Pharmacokinetics of Genetically Engineered Antibody Forms Using Positron Emission Tomography

    Energy Technology Data Exchange (ETDEWEB)

    Steven M. Larson, M.D. Nai-Kong Cheung, M.D., Ph.D.

    2004-08-31

    In the last grant period we have focused on multi-step targeting methodologies (MST), as a method for delivery of high dose to the tumor, with low dose to the bone marrow. We have explored uptake in colorectal, pancreatic and prostate cancer, using an special preparation, developed in collaboration with NeoRex A high tumor/bone marrow ratio is clearly achieved with MST, but with a cost, namely the higher dose to normal kidney. For this reason, we have in particular, (a) looked dosimetry for both tumor and normal organ, and especially renal dosimetry, which appears to be the target organ, for Y-90. (b) In parallel with this we have explored the dosimetry of very high dose rate radionuclides, including Holmium-166. (c) In addition, with NaiKong Cheung, we have developed a new MST construct based on the anti-GD2 targeting 5F11; (d) we have successfully completed development of s-factor tables for mice. In summary, renal dosimetry is dominated by about 4-5% of the injected dose being held long-term in the renal cortex, probably in the proximal tubule, due to the universal uptake of small proteins. This appears to be a function of a biotynlated protein binding of the strept-avidin construct, to HSP70. This cortical uptake has caused us to reconsider renal dosimetry as a whole, with the smaller mass of the cortex, rather than the whole kidney, as the target organ. These insights into dosimetry will be of great importance as MST, becomes more common in clinical practice.

  4. Potato lectin activates basophils and mast cells of atopic subjects by its interaction with core chitobiose of cell-bound non-specific immunoglobulin E.

    Science.gov (United States)

    Pramod, S N; Venkatesh, Y P; Mahesh, P A

    2007-06-01

    A major factor in non-allergic food hypersensitivity could be the interaction of dietary lectins with mast cells and basophils. Because immunoglobulin E (IgE) contains 10-12% carbohydrates, lectins can activate and degranulate these cells by cross-linking the glycans of cell-bound IgE. The present objective focuses on the effect of potato lectin (Solanum tuberosum agglutinin; STA) for its ability to release histamine from basophils in vitro and mast cells in vivo from non-atopic and atopic subjects. In this study, subjects were selected randomly based on case history and skin prick test responses with food, pollen and house dust mite extracts. Skin prick test (SPT) was performed with STA at 100 microg/ml concentration. Histamine release was performed using leucocytes from non-atopic and atopic subjects and rat peritoneal exudate cells. SPT on 110 atopic subjects using STA showed 39 subjects positive (35%); however, none showed STA-specific IgE; among 20 non-atopic subjects, none were positive by SPT. Maximal histamine release was found to be 65% in atopic subjects (n = 7) compared to 28% in non-atopic subjects (n = 5); the release was inhibited specifically by oligomers of N-acetylglucosamine and correlates well with serum total IgE levels (R(2) = 0.923). Binding of STA to N-linked glycoproteins (horseradish peroxidase, avidin and IgG) was positive by dot blot and binding assay. As potato lectin activates and degranulates both mast cells and basophils by interacting with the chitobiose core of IgE glycans, higher intake of potato may increase the clinical symptoms as a result of non-allergic food hypersensitivity in atopic subjects.

  5. Vagal Intramuscular Arrays: The Specialized Mechanoreceptor Arbors That Innervate the Smooth Muscle Layers of the Stomach Examined in the Rat.

    Science.gov (United States)

    Powley, Terry L; Hudson, Cherie N; McAdams, Jennifer L; Baronowsky, Elizabeth A; Phillips, Robert J

    2016-03-01

    The fundamental roles that the stomach plays in ingestion and digestion notwithstanding, little morphological information is available on vagal intramuscular arrays (IMAs), the afferents that innervate gastric smooth muscle. To characterize IMAs better, rats were given injections of dextran biotin in the nodose ganglia, and, after tracer transport, stomach whole mounts were collected. Specimens were processed for avidin-biotin permanent labeling, and subsets of the whole mounts were immunohistochemically processed for c-Kit or stained with cuprolinic blue. IMAs (n = 184) were digitized for morphometry and mapping. Throughout the gastric muscle wall, IMAs possessed common phenotypic features. Each IMA was generated by a parent neurite arborizing extensively, forming an array of multiple (mean = 212) branches averaging 193 µm in length. These branches paralleled, and coursed in apposition with, bundles of muscle fibers and interstitial cells of Cajal. Individual arrays averaged 4.3 mm in length and innervated volumes of muscle sheet, presumptive receptive fields, averaging 0.1 mm(3) . Evaluated by region and by muscle sheet, IMAs displayed architectural adaptations to the different loci. A subset (32%) of circular muscle IMAs issued specialized polymorphic collaterals to myenteric ganglia, and a subset (41%) of antral longitudinal muscle IMAs formed specialized net endings associated with the serosal boundary. IMAs were concentrated in regional patterns that correlated with the unique biomechanical adaptations of the stomach, specifically proximal stomach reservoir functions and antral emptying operations. Overall, the structural adaptations and distributions of the IMAs were consonant with the hypothesized stretch receptor roles of the afferents.

  6. VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.

    Science.gov (United States)

    Leblanc, Pierre; Moise, Leonard; Luza, Cybelle; Chantaralawan, Kanawat; Lezeau, Lynchy; Yuan, Jianping; Field, Mary; Richer, Daniel; Boyle, Christine; Martin, William D; Fishman, Jordan B; Berg, Eric A; Baker, David; Zeigler, Brandon; Mais, Dale E; Taylor, William; Coleman, Russell; Warren, H Shaw; Gelfand, Jeffrey A; De Groot, Anne S; Brauns, Timothy; Poznansky, Mark C

    2014-01-01

    Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.

  7. Immunoperoxidase detection of neuronal antigens in full-thickness whole mount preparations of hollow organs and thick sections of central nervous tissue.

    Science.gov (United States)

    Llewellyn-Smith, Ida J; Gnanamanickam, Greta J E

    2011-03-15

    Immunofluorescently stained whole mounts have proved useful for defining the innervation of the gut and large blood vessels. Nerves supplying other hollow organs are usually studied in sections, which provide much less information. Aiming to describe the entire innervation of rat uterus, we developed a method for immunoperoxidase staining of full-thickness whole mounts that allowed us to visualize all immunoreactive axons. Uterine horns were dissected out, slit open, stretched, pinned flat and fixed. Entire horns were treated with methanol/peroxide, buffered Triton X-100 and normal serum and then incubated in primary antibodies, biotinylated secondary antibodies and avidin-horseradish peroxidase (HRP), each for at least 3 days. Peroxidase reactions revealed immunoreactivity. Immunostained horns were dehydrated, infiltrated with epoxy resin, mounted on slides under Aclar coverslips and polymerized. We treated bladders, gut, major pelvic ganglia and thick sections of perfused medulla oblongata similarly to assess the applicability of the method. Using this method, we could map the entire uterine innervation provided by axons immunoreactive for a variety of antigens. We could also assess the entire tyrosine hydroxylase-immunoreactive innervation in all layers of bladder, gut and ganglia whole mounts and throughout 300 μm sections of medulla. These observations show that this method for immunoperoxidase staining reliably reveals the complete innervation of full-thickness whole mounts of hollow organs and thick sections of central nervous tissue. The method has several advantages. The resin-embedded tissue does not degrade; the immunostaining is non-fading and permanent and neurochemically defined features can be mapped at large scale without confocal microscopy.

  8. Loss of Aβ-nerve endings associated with the Merkel cell-neurite complex in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis.

    Science.gov (United States)

    Carrión, Daniela Calderón; Korkmaz, Yüksel; Cho, Britta; Kopp, Marion; Bloch, Wilhelm; Addicks, Klaus; Niedermeier, Wilhelm

    2016-03-30

    The Merkel cell-neurite complex initiates the perception of touch and mediates Aβ slowly adapting type I responses. Lichen planus is a chronic inflammatory autoimmune disease with T-cell-mediated inflammation, whereas hyperkeratosis is characterized with or without epithelial dysplasia in the oral mucosa. To determine the effects of lichen planus and hyperkeratosis on the Merkel cell-neurite complex, healthy oral mucosal epithelium and lesional oral mucosal epithelium of lichen planus and hyperkeratosis patients were stained by immunohistochemistry (the avidin-biotin-peroxidase complex and double immunofluorescence methods) using pan cytokeratin, cytokeratin 20 (K20, a Merkel cell marker), and neurofilament 200 (NF200, a myelinated Aβ- and Aδ-nerve fibre marker) antibodies. NF200-immunoreactive (ir) nerve fibres in healthy tissues and in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis were counted and statistically analysed. In the healthy oral mucosa, K20-positive Merkel cells with and without close association to the intraepithelial NF200-ir nerve fibres were detected. In the lesional oral mucosa of lichen planus and hyperkeratosis patients, extremely rare NF200-ir nerve fibres were detected only in the lamina propria. Compared with healthy tissues, lichen planus and hyperkeratosis tissues had significantly decreased numbers of NF200-ir nerve fibres in the oral mucosal epithelium. Lichen planus and hyperkeratosis were associated with the absence of Aβ-nerve endings in the oral mucosal epithelium. Thus, we conclude that mechanosensation mediated by the Merkel cell-neurite complex in the oral mucosal epithelium is impaired in lichen planus and hyperkeratosis.

  9. Morphological and laminar distribution of cholescystokinine - immunoreactive neurons in cortex of human inferior parietal lobule and their clinical significance

    Directory of Open Access Journals (Sweden)

    Puškaš Laslo

    2008-01-01

    Full Text Available Introduction. Cholecystocinine is a neuropeptide whose function in the cortex has not yet been clarified, although its relation with some psychic disorders has been noticed. Previous studies have not provided detailed data about types, or arrangement of neurons that contain those neuropeptide in the cortex of human inferior parietal lobe. The aim of this study was to examine precisely the morphology and typography of neurons containing cholecytocinine in the human cortex of inferior parietal lobule. Material and methods. There were five human brains on which we did the immunocystochemical research of the shape and laminar distribution of cholecystocinine immunoreactive neurons on serial sections of supramarginal gyrus and angular gyrus. The morphological analysis of cholecystocinine-immunoreactive neurons was done on frozen sections using avidin-biotin technique, by antibody to cholecystocinine diluted in the proportion 1:6000 using diamine-benzedine. Results. Cholecystocinine immunorective neurons were found in the first three layers of the cortex of inferior parietal lobule, and their densest concentration was in the 2nd and 3rd layer. The following types of neurons were found: bipolar neurons, then its fusiform subtype, Cajal-Retzius neurons (in the 1st layer, reverse pyramidal (triangular and unipolar neurons. The diameters of some types of neurons were from 15 to 35 µm, and the diameters of dendritic arborization were from 85-207 µm. A special emphasis is put on the finding of Cajal-Retzius neurons that are immunoreactive to cholecystocinine, which demands further research. Conclusion. Bearing in mind numerous clinical studies pointing out the role of cholecystokinine in the pathogenesis of schizophrenia, the presence of a great number of cholecystokinine immunoreactive neurons in the cortex of inferior parietal lobule suggests their role in the pathogenesis of schizophrenia.

  10. Ultrasensitive electrochemical sensing platform for microRNA based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification.

    Science.gov (United States)

    Shuai, Hong-Lei; Huang, Ke-Jing; Xing, Ling-Li; Chen, Ying-Xu

    2016-12-15

    An ultrasensitive electrochemical biosensor for microRNA (miRNA) is developed based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification. WO3-Gr is prepared by a simple hydrothermal method and then coupled with gold nanoparticles to act as a sensing platform. The thiol-terminated capture probe H1 is immobilized on electrode through Au-S interaction. In the presence of target miRNA, H1 opens its hairpin structure by hybridization with target miRNA. This hybridization can be displaced from the structure by another stable biotinylated hairpin DNA (H2), and target miRNA is released back to the sample solution for next cycle. Thus, a large amount of H1-H2 duplex is produced after the cyclic process. At this point, a lot of signal indicators streptavidin-conjugated alkaline phosphatase (SA-ALP) are immobilized on the electrode by the specific binding of avidin-biotin. Then, thousands of ascorbic acid, which is the enzymatic product of ALP, induces the electrochemical-chemical-chemical redox cycling to produce a strongly electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under the optimal experimental conditions, the established biosensor can detect target miRNA down to 0.05fM (S/N=3) with a linear range from 0.1fM to 100pM, and discriminate target miRNA from mismatched miRNA with a high selectivity.

  11. Characterization of the specificity by immunohistology of a monoclonal antibody to a novel epithelial antigen of ovarian carcinomas.

    Science.gov (United States)

    Mariani-Costantini, R; Agresti, R; Colnaghi, M I; Ménard, S; Andreola, S; Rilke, F

    1985-08-01

    An immunohistological study, using the avidin-biotin-peroxidase complex method, was carried out to define the reactivity profile of a murine monoclonal antibody, MOv2, which recognizes a novel glycoprotidic antigen associated with ovarian epithelial tumors. Among the primary ovarian tumors tested, MOv2 immunostained 93% of mucinous and 75% of serous cystadenomas, 100% of mucinous, 81% of serous and 73% of endometrioid carcinomas. Undifferentiated and clear cell tumors revealed more limited reactivity with the antibody, whereas ovarian sex cord-stromal and germinal tumors were immunonegative. Positive reactions were also documented in omental metastases from primary ovarian carcinomas. No immunoreactivity was detected in normal ovarian epithelium, whereas the cells lining Walthard's nests adjacent to the fallopian tubes and a variety of normal epithelia were consistently immunolabeled. These included the lining epithelia of the gastrointestinal tract, bronchi and endocervix, and the epithelium of salivary, biliary and pancreatic ducts and sweat glands. To a lesser extent, positive reactions were detected in other surface epithelia, such as squamous and transitional epithelia. Among tumors other than ovarian, MOv2 consistently reacted with adenocarcinomas and squamous cell carcinomas from different sites, most notably breast, lung and gastrointestinal tract, and with transitional cell carcinomas. In contrast, no staining was demonstrated in non-epithelial malignancies. The antigen defined by MOv2 may be operationally useful as a marker of epithelial lineage in tumor histopathology. Its pattern of immunohistochemical distribution indicates that an antigenic phenotype shared by normal surface epithelia and non-ovarian carcinomas is strongly associated with common epithelial neoplasms of the ovaries.

  12. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein.

    Science.gov (United States)

    Senis, Yotis A; Tomlinson, Michael G; García, Angel; Dumon, Stephanie; Heath, Victoria L; Herbert, John; Cobbold, Stephen P; Spalton, Jennifer C; Ayman, Sinem; Antrobus, Robin; Zitzmann, Nicole; Bicknell, Roy; Frampton, Jon; Authi, Kalwant S; Martin, Ashley; Wakelam, Michael J O; Watson, Stephen P

    2007-03-01

    The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase

  13. Immunohistochemical localization of androgen receptor in rat caput epididymis during postnatal development

    Directory of Open Access Journals (Sweden)

    Sema Timurkaan

    2011-09-01

    Full Text Available Objectives: The aim of this study was to investigate the developmental pattern of androgen receptor (AR in caput epididymis.Materials and methods: In this study three randomly selected rats were sacrificed at ages 21, 56, 90 and 120 days old. All male rats were anesthetized with ethyl ether before killing. Then, the caput epididymides were removed and fixed in Bouin’s fixative at +4°C for 36 hour. Afterwards the tissue samples were embedded in paraffin for routine histological methods. Later the tissues were sectioned at 5μm and mounted on poly-L-lysin-coated slides. To solve the antigen masking problem, we performed microwave stimulated antigen retrieval technique before the immunohistochemical staining. Avidin-Biotin-Peroxidase Complex (ABC method was applied for immunohistochemical staining.Results: In all age groups of rats studied, positive immunohistochemical staining for the AR appeared in nuclei of epididymal cells. The staining intensity of AR positive cells did not change depending on age. In caput epididymis, immunostainable AR was found in tubular epithelial cells (principal cells, basal cells and apical cells and peritubular smooth muscle cells. The AR staining in the epithelial cells appeared to be stronger than in the peritubular smooth muscle cells. In the epithelial cells; staining intensity was stronger in principal cells than in basal cells and apical cells.Conclusion: Staining intensity of AR positive epididymal cells irrespective of age indicated the necessity of androgens for postnatal differentiation and maintaining the structure of the epididymis. Stronger staining intensity in principal cells suggested that principal cells are more sensitive to androgen stimulation. J Clin Exp Invest 2011; 2 (3: 260-266.

  14. Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip.

    Directory of Open Access Journals (Sweden)

    Kaori Abe

    Full Text Available Sandwich enzyme-linked immunosorbant assay (ELISA using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6 or tumor necrosis factor-α (TNF-α, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R(2 = 0.9994, TNF-α: R(2 = 0.9977, and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R(2 = 0.9954, TNF-α: R(2 = 0.9928. The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and

  15. Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp (Carassius auratus) hepatocytes

    Institute of Scientific and Technical Information of China (English)

    LIANG Yong; C. K. C. Wong; XU Ying; M. H. Wong

    2004-01-01

    Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in vitro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estro-genic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL) seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1 μmol/L), and β-naphtho-flavone (β-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with β-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for β-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.

  16. The role of interleukin 17 in the pathogenesis of eczema%白细胞介素17在湿疹发病机制中的作用

    Institute of Scientific and Technical Information of China (English)

    高金銮

    2015-01-01

    目的:探讨白细胞介素17(IL-17)在湿疹发病机制中的作用.方法:从2013-06/2014-06我院收治的急性湿疹患者和慢性湿疹患者中各抽取20例,并选取10例正常者作为研究对象,采用链霉素抗生物素-过氧化物酶连接(SP)的方法,检测患者表皮和真皮细胞质中的表达.结果:IL-17在正常皮肤组织细胞中无表达,在湿疹患者表皮角质形成细胞和真皮浸润淋巴细胞中均有表达,且在急性湿疹中的表达高于慢性湿疹中的表达(P <0.05).结论:IL-17在湿疹患者发病初级阶段发挥作用.%AIM:To investigate the role of interleukin 17 (IL-17)in the pathogenesis of eczema.METHODS:From 2013 June to 2014 June in our hospital 20 patients with acute eczema and 20 patients with chronic eczema,along with 10 cases of nor-mal persons were selected as the research object,using the avidin biotin-peroxidase connection (SP)method,detecting the expres-sion of epidermal and dermal cytoplasm in patients.RESULTS:IL-17 expression was not detected in normal skin tissue cells,but the formation of lymphocytes infiltrating cells expressed in the epi-dermis and dermis of patients with eczema horny,and expression level in acute eczema was higher than that of chronic eczema (P <0.05).CONCLUSION:IL-17 plays a role in the patho-genesis of primary stage in patients with eczema.

  17. BetaSearch: a new method for querying β-residue motifs

    Directory of Open Access Journals (Sweden)

    Ho Hui

    2012-07-01

    -residue motif between an entirely synthetic (Top7 and a naturally-occurring protein (Charcot-Leyden crystal protein, as well as identifying structural similarities between biotin-binding domains of avidin, streptavidin and the lipocalin gamma subunit of human C8.

  18. Active targeting behaviors of biotinylated poly(ethylene oxide)/poly(lactic acid) nanoparticles in vitro%生物素化聚乙二醇/聚乳酸纳米粒子的体外主动靶向行为

    Institute of Scientific and Technical Information of China (English)

    龚妍春; 熊向源; 李资玲; 李玉萍; 郭亮

    2010-01-01

    背景:目前研究的大部分高分子药物载体没有靶向性,在应用上有局限性,只有几个国外课题组报道生物素化聚乙二醇/聚乳酸(Biotin-PEO-PLA)纳米粒子的体外靶向行为,国内没有这方面的研究报道.目的:分析Biotin-PEO-PLA纳米粒子作为靶向药物载体的可行性.方法:透析法制备包埋紫杉醇的Biotin-PEO-PLA纳米粒子并表征;通过高效液相色谱研究包埋紫杉醇的Biotin-PEO-PLA纳米粒子的体外释放行为;利用细胞毒性法比较研究生物素-亲和素三步法实施的包埋紫杉醇的Biotin-PEO-PLA纳米粒子对OVCAR-3(表面表达CA-125抗体)和SKOV-3(表面不表达CA-125抗体)细胞的体外靶向行为.结果与结论:包埋在Biotin-PEO-PLA纳米粒子中的紫杉醇的释放呈现初期的快速释放以及随后的缓慢释放.利用三步法处理的OVCAR-3细胞存活率明显低于SKOV-3细胞,表明通过Biotin-PEO-PLA/avidin/biotinylated MAB X306与OVCAR-3细胞表面CA-125抗原的特异性相互作用,包埋紫杉醇的Biotin-PEO-PLA纳米粒子被更为有效地传递进了OVCAR-3细胞.

  19. 光电化学竞争法检测生物素%Photoelectrochemical Competitive Detection of Biotin

    Institute of Scientific and Technical Information of China (English)

    刘世利; 陈守臻; 赵倩; 徐政虎; 李玉; 贾继辉; 郭良宏

    2013-01-01

    建立了光电化学系统竞争性检测生物素(Biotin)小分子浓度的方法.采用联吡啶钌[Tfis(2,2'-bipyridine) ruthenium,Ru-bpy)]作为标记物,以氧化锡纳米颗粒为电极,草酸盐为电子供体还原标记物.在470 nm光激发下,联吡啶钌的外层电子吸收能量后由基态变为激发态,注入半导体氧化锡纳米颗粒电极的导带,形成光电流信号;草酸盐还原失去电子的联吡啶钌使其恢复初始状态,从而可以再次作为电子供体受激发产生光电流信号.在竞争性检测生物素(Biotin)浓度时,亲和素(Avidin)吸附到氧化锡纳米颗粒电极表面作为识别元件,在浓度大于0.5 g/L时能够达到最大的电极表面覆盖率.1μmol/L Ru-bpy-biotin与不同浓度Biotin组成的混合溶液与电极表面的Avidin发生亲和反应,光激发后检测光电流大小;当溶液中Biotin的浓度增加时,致使与电极表面Avidin结合的Ru-bpy-biotin量减少,在光照射下光电流信号降低.这一竞争性光电检测方法检测Biotin时,检出限为8μg/L.本方法可进一步扩展,应用于有机化合物的竞争性免疫检测.

  20. A BIOTIN-DEPENDENT BACTERIUM BIO-5: ISOLATION, CHARACTERIZATIOIN AND ITS USE FOR DETERMINATION OF BIOTIN CONCENTRATION%一株生物素依赖型菌株bio-5:分离,初步鉴定及其用于生物素浓度测定

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A biotin-dependent bacterium bio-5 was isolated from a tomato rhizosphere soil sample. Its intrinsic antibiotic resistance (IAR) and fatty acid profile were characterized, and it was preliminary named as Pseudomonas sp. bio-5. This bacterium was very sensitive to lower biotin amount and a standard biotin disk assay was set up based on this bacterium. The biotin standard disk assay had very good linear equation between biotin amount and promoting growth area (diameter, mm) and had wide effective assay range. This bacterium could only use free biotin when biotin and avidin were mixed together. Biotin disk assay test showed that different kind of plants had different biotin concentration, similar to the reference papers. Using bio-5, stv gene transgenic tobacco plants were successfully screened out. Compared with the traditional biotin microbiological assay bacterium Lactobacillus plantarum ATCC8014, this bacterium was easy to obtain and cultivate, easy to perform biotin disk assay and more practical for biotin concentration test. Fig 7, Tab 2, Ref 22%从西红柿根圈土壤中分离纯化到一株生物素依赖型菌株bio-5,经测定其内源抗生素抗性和脂肪酸特征,初步定名为假单胞菌bio-5.这一菌株对生物素非常敏感.建立了以此菌株用于测定生物素浓度的标准方法.利用这一方法,测定了不同作物叶子中生物素的含量,并将这一方法成功用于筛选转stv基因烟草植物.与传统生物素测定菌株Lactobacillus plantarum ATCC8014比较,我们所分离的菌株具有易于获得,易于培养,快速用于生物素浓度测定的特点.图 7 表 2 参 22

  1. Mouse bronchiolar cell carcinogenesis. Histologic characterization and expression of Clara cell antigen in lesions induced by N-nitrosobis-(2-chloroethyl) ureas.

    Science.gov (United States)

    Rehm, S.; Lijinsky, W.; Singh, G.; Katyal, S. L.

    1991-01-01

    Female Swiss mice (Cr:NIH(S)) developed bronchiolar cell hyperplasia, dysplasia, metaplasia, and various morphologic types of bronchiolar cell tumors after topical (skin) application of N-nitroso-methyl-bis-chloroethylurea (NMBCU) or N-nitroso-tris-chloroethylurea (NTCU). These compounds are the first found to induce systemically bronchiolar cell tumors in mice in high incidence. Twice a week, with a 3-day interval, a 25-microliter drop of 0.04 mol/l (molar) NMBCU or NTCU in acetone was applied to the shaved interscapular integument for a maximum of 35 to 40 weeks. The earliest lung neoplasms were seen in mice that died after 23 weeks of treatment and affected 11 of 19 with NMBCU and 14 of 19 with NTCU treatment. Tumor growth pattern was nodular or the neoplastic tissue was frequently disseminated throughout the parenchyma, starting from multicentric peribronchiolar foci. The most common tumor types were squamous cell carcinomas and adenosquamous carcinomas, followed by adenocarcinomas with or without secretory cells, and a single ciliated-cell tumor. Histochemical and immunohistochemical studies were carried out on paraffin-embedded lungs using the avidin-biotin immunoperoxidase complex procedure and antisera against keratin, Clara cell antigen, surfactant apoprotein, neuron-specific enolase, bombesin, and chromogranin A. In several mice from both groups, hyperplasias and tumors were composed of cells expressing Clara cell antigen. No tumor cells were found expressing alveolar type II or neuroendocrine cell markers. It appeared that bronchiolar cells, in particular Clara cells, had migrated from terminal bronchioles or invaded bronchiolar walls to extend into the alveolar parenchyma. Squamous cell metaplasia with keratin expression was seen within airways or associated with glandular tumors, especially at the periphery. A unique cell type, with large eosinophilic globules and associated eosinophilic crystals, was seen lining airways or forming hyperplastic and

  2. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    Directory of Open Access Journals (Sweden)

    Annamari Paino

    Full Text Available Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI, was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control

  3. CdTe量子点荧光探针在生物分析中的应用%Application of Quantum Dots as Fluoresent Probes in Biological Analysis

    Institute of Scientific and Technical Information of China (English)

    胡兵; 韩崇旭

    2012-01-01

    目的 探讨巯基丙酸包被的CdTe量子点在生物分析中应用价值.方法 水相中合成CdTe量子点并进行巯基丙酸包被,并对其进行了荧光发射光谱及透射电子显微镜成像表征;将量子点与亲和素连接、制备成量子点荧光探针;应用激光扫描共聚焦显微术观察量子点荧光探针标记小鼠腹腔巨噬细胞(peritoneal macrophage,PMΦ)MHCⅡ抗原的表达;以H22肝癌细胞为靶细胞,MTT法观察量子点的生物相容性.结果 CdTe量子点粒径均匀,具备良好的光学性能;量子点荧光探针标记细胞具有较强的荧光表达;量子点在一定浓度范围内对细胞的毒性小.结论量子点荧光探针能对固定的组织细胞进行标记,同时具有较好的生物相容性,可对活细胞进行直接或动态标记.%Objective To investigate the application of CdTe quantum dots as fluoresent probes in Biological analysis. Methods Prepared CdTe quantum dots in aqueous phase and coating with MPA. Exosyndrome QDs with TEM and Fluorspectro-photometer. Binded QDs to avidin, purified the connective and prepared it as flurescent probe. LSCM was used to observe the QDs fluorescent probe label expression of MHCⅡ antigen on PMΦ and used MTT to observe the biocompatibility of CdTe quantum dots coated with MPA using the H22 cells as target cells. Results The particle diameter of CdTe quantum dots prepared in aqueous phase was well-distributed and it had good photology performance. QDs had little cytotoxicity within some concentration range. Conclusion CdTe QDs has good biocompatibility. It's not only fit for immunofluorescence label of fixed histiocyte but also can satisfy the experiment desire of direct or dynamic label of live cells.

  4. Prostate-specific membrane antigen is undetectable in choroidal neovascular membrane

    Directory of Open Access Journals (Sweden)

    Godeiro Katyanne

    2006-08-01

    Full Text Available Abstract Background Choroidal neovascular membrane (CNVM is one of the leading causes of severe visual loss and is often associated with age-related macular degeneration (AMD. Various modalities of treatment, including photocoagulation and surgery, are being considered as options, but with limited success. Prostate-specific membrane antigen (PSMA is a type II membrane glycoprotein expressed in benign and malignant prostatic tissues, in some non-prostatic tissues, and in the endothelium of tumor-associated neovasculature of non-prostatic neoplasm. Some studies have suggested that the expression of PSMA is restricted to endothelium from tumor-associated neovasculature and might be stimulated by some tumor-secreted angiogenic factors. However, no previous study demonstrating PSMA expression in non-related tumor neovasculature, such as CNVM, has been performed to date. Furthermore, demonstration of PSMA expression in CNVM in AMD patients could reveal a novel target for antineovascular therapy. The purpose of this study was to evaluate the immunohistochemical expression of PSMA in CNVM from AMD. Methods Immunohistochemical analysis, with a standard avidin-biotin complex technique, was performed using an anti-PSMA mouse monoclonal antibody in 30 specimens of surgically excised CNVM from AMD patients. Antibody to an endothelial cell specific marker, factor VIII, was used to confirm the location of the endothelial cells. Results The angiogenic microvessels of the 30 cases demonstrated negative staining to PSMA while factor VIII was expressed in all cases. Seventy-five percent of the secretory-acinar epithelium of the prostatic hyperplasia specimen stained positive, confirming that the immunohistochemical technique was correctly performed. Conclusion The absence of PSMA expression in non-tumoral neovasculature supports the theory, previously suggested, that endothelial cell PSMA expression may be stimulated by one or more tumor-secreted angiogenic

  5. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  6. Human papilloma virus early proteins E6 (HPV16/18-E6) and the cell cycle marker P16 (INK4a) are useful prognostic markers in uterine cervical carcinomas in Qassim Region--Saudi Arabia.

    Science.gov (United States)

    Omran, O M; AlSheeha, M

    2015-01-01

    Cervical cancer is a common and an important public health problem for adult women in developing countries. In contrast, cervical cancer incidence is low in Saudi Arabia. High-risk types of human papilloma viruses (HPV16 and HPV18) are the most significant risk factors for cervical cancer. HPV16/18-E6 oncoprotein is associated with HPV etiology, viral persistence and epithelial transformation. Cell cycle protein p16 INK4a (p16) plays an important role in the pathophysiology of cervical carcinomas. The aims of this study were to investigate the expression of HPV16/18-E6 and p16 in uterine cervical carcinomas in Qassim Region--Saudi Arabia, and to relate the results to the established clinicopathological prognostic parameters (age of the patient, educational level, birth control methods, number of pregnancy, smoking status, degree of histological differentiation, clinical stage, and lymph node metastasis) The study included 40 specimens of uterine cervical squamous cell carcinomas diagnosed and confirmed by biopsy. Histopathological classification of cervical tumors cases was performed according to the International Federation of Gynecology and Obstetrics (FIGO). Immunohistochemical analysis for HPV16/18-E6 and p16 were carried out on formalin-fixed paraffin-embedded sections of cervical tissues using avidin-biotin peroxidase method. There was a significant statistical correlation between HPV16/18-E6 expression in cervical carcinoma and nationality, smoking status and size of the tumor. HPV16/18-E6 oncoprotein expression in normal lymphocytes and endothelial cells in the tumor tissues and the adjacent normal cervical tissues suggest the possibility that HPV infection might spread to other organs through blood circulation. P16 expression has been correlated with high grade, stage of cervical SCC and HPV16/18-E6 expression. The current study supports the critical function of p16 and HPV16/18-E6 as specific markers for cervical carcinoma. However the potential for usage

  7. Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Alice Kar Lai, E-mail: s0907465@cuhk.mail.serv.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Lu, Haifei, E-mail: hflu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Wu, Shu Yuen, E-mail: sywu@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Kwok, Ho Chin, E-mail: hckwock@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Ho, Ho Pui, E-mail: hpho@ee.cuhk.edu.hk [Center for Advanced Research in Photonics, Department of Electronic Engineering, The Chinese University of Hong Kong (Hong Kong); Yu, Samuel, E-mail: samscyu@gmail.com [The MacDiarmid Institute for Advanced Materials and Nanotechnology, Christchurch (New Zealand); Izon Science, PO Box 39-168, Harewood, Christchurch 8545 (New Zealand); Cheung, Anthony Ka Lun, E-mail: kalun2004@hotmail.com [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong); Kong, Siu Kai, E-mail: skkong@cuhk.edu.hk [Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong (Hong Kong)

    2013-06-11

    Graphical abstract: -- Highlights: •A novel diagnostic assay is developed to detect the MRSA's Panton-Valentine Leukocidin toxin. •Detection is based on target DNA amplification at one single temperature at 65 °C by LAMP. •Amplicons are then hybridized with 2 Au-nanoparticles with specific DNA probes for sensing. •The supra-assemblies are subsequently sensed by resistive pulse sensing. •Detection limit: ∼200 copies of DNA; time for detection: completed within 2 h. -- Abstract: This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55 ± 2 nm) through biotin–avidin binding. A second AuNP2 (30 ± 1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.

  8. Expression of MMP-2,TIMP-2 protein and the ratio of MMP-2/TIMP-2 in gallbladder carcinoma and their significance

    Institute of Scientific and Technical Information of China (English)

    Yue-Zu Fan; Jing-Tao Zhang; Hu-Chuan Yang; Yao-Qin Yang

    2002-01-01

    AIM: To study the correlation between expression of MMP-2, TIMP-2 protein and the ratio of MMP-2/TIMP-2 and clinicalpathological parameters of patients with gallbladder carcinoma.METHODS: Carcinomas (n=45) and polypoid lesions (n=15) of the gallbladder were studied for the expression of MMP-2 and TIMP-2 protein by immunohistochemical avidin-biotin-complex method and image analysis. Clinicalpathological data of patients with gallbladder carcinoma such as histological type, grade of differentiation, level of infiltration, liver invasion and lymph node involvement, etc, were recorded.RESULTS: There was significant difference between the average level (1.123±0.108 VS 1.030±0.054, P=0.002) of MMP-2, the ratio (1.050±0.013 VS0.937±0.078, P=0.003) of MMP-2/TIMP-2 in gallbladder carcinomas and in polypoid lesions of the gallbladder. Significant difference was found between the expression of MMP-2 in early stage and advanced tumors, but there was no correlation between MMP-2 protein expression and histological type, differentiation degree, infiltration level, lymph node involvement or liver invasion. Although no difference was observed between TIMP-2 expression and histological type or differentiation degree, signific ant difference was found between TIMP-2 expression and different Nevin stage, infiltration level, local lymph node involvement or liver invasion (1.168±0.067 VS1.048±0.075, 1.170±0.062 vs 1.039±0.06g, 1.039±0.076 VS1.147±0.083, 1.048±0.074 vs 1.103±0.095, P<0.05). MMP-2/TIMP-2 ratio did not correlate with histological type, grade of differentiation and liver invasion, but significant differences were found between MMP-2/TIMP-2 ratio and different Nevin stage, infiltration level and lymph node involvement in patients with carcinoma of gallbladder.CONCLUSION: TIMP-2 and MMP-2/TIMP-2 ratio could reflect more accurately biological characteristic of gallbladder carcinoma and MMP-2/TIMP-2 ratio might be a new significant marker in early diagnosis, in

  9. Montelukast treatment (cysteinyl leukotriene receptor antagonist in a model of food allergy: modifications in lymphatic cell population from rectal mucosa

    Directory of Open Access Journals (Sweden)

    M. Vinuesa

    Full Text Available Objective: the aim is to determine immunopathological modifications in rectal mucosa from rabbits after local challenge in ovalbumin (OVA sensitized animals previously treated with montelukast. Material and methods: experimental design: thirty two rabbits divided into four groups: G1: normal; G2: subcutaneously OVA sensitized; G3: sensitized, locally OVA challenged and sampled 4 hours after challenge; and G4: sensitized, locally OVA challenged and treated 4 hours before challenge with montelukast (0.15 mg/kg. Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA. In each group 200 high microscopical power fields (HPF were counted. Results were expressed as arithmetic mean and SE. Anti -CD4, CD5, µ chain monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. Results: CD 4: G1: 8.3 ± 0.06; G2: 13.4 ± 0.08, G3: 8.25 ± 0.06, G4: 11.8 ± 0.02. CD 5: G1: 7.3 ± 0.05; G2: 9.4 ± 0.05, G3: 11.3 ± 0.06, G4: 8.1 ± 0.06. μ chain: G1: 10.4 ± 0.06; G2: 3.8 ± 0.02, G3: 6.0 ± 0.10, G4: 2.2 ± 0.10. In all cases, experimental groups (G3 vs. G4 presented statistical significant differences (p < 0.05. CD4+, CD5+ cells and μ chain+ decrease in experimental group (G4, probably due to lymphocyte migration inhibition to challenged mucosa. μ chain+ cell decrease could be based on B cell activation and expression of different surface immunoglobulins. Cells expressing μ chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. Conclusions: we conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals after treatment with montelukast.

  10. iRGD靶向载药脂质体-微泡复合物的制备及其靶向性研究%Preparation and targeting effect of iRGD modified liposome-microbubble complex

    Institute of Scientific and Technical Information of China (English)

    张婧; 严飞; 李莉

    2016-01-01

    目的:制备iRGD靶向载药脂质体-微泡复合物,研究其靶向性.方法:采用薄膜-超声分散法制备生物素化的iRGD靶向载药脂质体和生物素化的超声微泡.利用生物素-亲和素系统(Biotin-avidin-system,BAS)连接脂质体与微泡,构建并表征iRGD靶向载药脂质体-微泡复合物.细胞黏附实验验证复合物的体外靶向结合性能;构建小鼠乳腺癌移植瘤模型,通过靶组织的药物荧光强度验证复合物的体内靶向性.结果:iRGD靶向载药脂质体的粒径为(165.07±4.01)nm,电位为(-12.92±0.26)mv,复合物的载药量为每108个复合物载紫杉醇(46.22±1.95)μg;黏附实验表明靶向组复合物与血管内皮细胞结合数量明显多于非靶向组复合物(7.8±1.1,0.2±0.45,P<0.01);荷瘤小鼠活体成像实验显示靶向组复合物的肿瘤组织荧光明显强于非靶向组复合物.结论:iRGD靶向载药脂质体-微泡复合物,作为一种靶向给药系统,可以实现超声分子成像与超声给药的有机结合,显著提高药物靶向递送的效率.

  11. TrkA、TrkB和TrkC受体在大鼠三叉神经本体觉中枢通路上的表达%XPRESSION OF TrkA, TrkB and TrkC RECEPTORS IN THE CENTRAL PATHWAY OF TRIGEMINAL PROPRIOCEPTION IN THE RAT

    Institute of Scientific and Technical Information of China (English)

    张富兴; 黎振航; 李金莲; 岑國欣; 陳應城

    2004-01-01

    Objective To examine the distribution of high-affinity neurotrophin receptors, viz. TrkA, TrkB and TrkC receptors, in the four-order central pathway of trigeminal proprioception in the rat. Methods Avidin-biotin-peroxidase complex (ABC) method of immunohistochemistry with diaminobenzidine (DAB) as the chromogen was employed to identify the immunoreactive cells. Results All three receptors were detected in the mesencephalic trigeminal nucleus, the second-and third order nuclei iu the central pathway of trigeminal proprioception. Within each of these nuclei, cells immunostained for the three receptors differed in density. TrkA-and TrkB-immunostaining was observed on the cell bodies and processes; TrkB, however,labelled much longer processes. TrkC staining was mainly restricted to the cell bodies. Conclusion In the central pathway of the trigeminal proprioception, each of the three receptors may play a specific role in maintaining the survival of neurons and in regulating the neurotransmitter release.%目的检查高亲和性的神经营养物质受体(TrkA、TrkB和TrkC)在大鼠三叉神经本体觉中枢四级通路上的分布.方法采用免疫组织化学的抗生物素-生物素-过氧化物酶复合物(ABC)方法并以二氨基联苯胺(DAB)为呈色剂,检查免疫反应细胞.结果 3种受体在三叉神经本体觉中枢四级通路的三叉神经中脑核、二级和三级核团均有表达.同一核团内3种受体的免疫阳性细胞密度不同.在胞体和突起上均观察到TrkA和TrkB免疫反应染色,但TrkB标记的突起较长,TrkC染色多局限于细胞体部分.结论在三叉神经本体觉中枢通路中,3种神经营养物质受体都可能在维持神经元存活和调节递质释放等方面发挥特定的作用.

  12. Radiation-induced apoptosis in SCID mice spleen after low dose irradiation

    Science.gov (United States)

    Takahashi, A.; Kondo, N.; Inaba, H.; Uotani, K.; Kiyohara, Y.; Ohnishi, K.; Ohnishi, T.

    To assess the radioadaptive response of the whole body system in mice, we examined the temporal effect of low dose priming as an indicator of challenging irradiation-induced apoptosis through a p53 tumor suppressor protein- mediated signal transduction pathway. The p53 protein also plays an important role both in cell cycle control and DNA repair through cellular signal transduction. Using severe combined immunodeficiency mice defective in DNA-dependent protein kinase catalytic subunit, we examined the role of DNA-dependent protein kinase activity in radioadaptation induced by low dose irradiation. Specific pathogen free 5-week-old female severe combined immunodeficiency mice and the parental mice (CB-17 Icr +/ + were irradiated with X-ray at 3.0 C3y at 1, 2, 3 or 4 weeks after the conditioning irradiation at 0.15, 0.30, 0.45 or 0.60 Gy. The mice spleens were fixed for immunohistochemistry 12 h after the challenging irradiation. The p53-dependent apoptosis related Bax proteins on formalin-fixed paraffin-embedded sections were stained by the avidin-biotin peroxidase complex method The apoptosis incidence in the sections was measured by hematoxylin-eosin staining. The frequency of Bax- and apoptosis-positive cells increased up to 12 h after the challenging irradiation in the spleen of both mice. However, these cells were not observed after a low dose irradiation at 0.15-0.60 Gy When pre-irradiation at 0.45 Gy 2 weeks before the challenging irradiation at 3.0 Gy was performed, Bax accumulation and apoptosis induced by challenging irradiation were depressed in the spleens of CB-17 Icr +/ + mice, but not in severe combined immunodeficiency mice. These data suggest that DNA-dependent protein kinase might play a major role in radioadaptation induced by pre-irradiation with a low dose in mice spleen. We expect that the present findings will provide useful information in the health care of space crews.

  13. Evidence for the involvement of zinc in the association of CRISP1 with rat sperm during epididymal maturation.

    Science.gov (United States)

    Maldera, Julieta A; Vasen, Gustavo; Ernesto, Juan I; Weigel-Muñoz, Mariana; Cohen, Débora J; Cuasnicu, Patricia S

    2011-09-01

    Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of

  14. Expression of apoptosis-regulating genes in the rat prostate following botulinum toxin type a injection

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    Gorgal Tiago

    2012-01-01

    Full Text Available Abstract Background Onabotulinumtoxin A (OnabotA injection has been investigated as a novel treatment for benign prostatic enlargement caused by benign prostatic hyperplasia. An OnabotA - induced volume reduction caused by sympathetic fibers impairment has been proposed as a potential mechanism of action. Our aim was to investigate the expression of apoptosis-regulating proteins in the rat prostate following OnabotA intraprostatic injection. Methods Adult Wistar rats were injected in the ventral lobes of the prostate with 10 U of OnabotA or saline. A set of OnabotA-injected animals was further treated with 0.5 mg/kg of phenylephrine (PHE subcutaneously daily. All animals were sacrificed after 1 week and had their prostates harvested. Immunohistochemical staining was performed for Bax, Bcl-xL and caspase-3 proteins and visualized by the avidin-biotin method. The optical density of the glandular cells was also determined, with measurement of differences between average optical densities for each group. Results Saline-treated animals showed intense epithelial staining for Bcl-xL and a faint labelling for both Bax and Caspase-3. OnabotA-treated rats showed a reduced epithelial staining of Bcl-xL and a consistently increased Bax and Caspase-3 staining when compared with saline-treated animals. PHE-treated animals showed a stronger Bcl-xL staining and reduced staining of both Bax and Caspase-3 when compared to the OnabotA group. Mean signal intensity measurements for each immunoreaction confirmed a significant decrease of the signal intensity for Bcl-xL and a significant increase of the signal intensity for Bax and Caspase 3 in OnabotA-injected animals when compared with the control group. In OnabotA+PHE treated animals mean signal intensity for Bcl-xL, Bax and Caspase 3 immunoreactions was identical to that of the control animals. Conclusions These results support the hypothesis that OnabotA activates apoptotic pathways in the rat prostate through a

  15. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    Science.gov (United States)

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  16. Montelukast treatment (cysteinyl leukotriene receptor antagonist in a model of food allergy: modifications in lymphatic cell population from rectal mucosa Tratamiento con Montelukast (antagonista cisteinílico del receptor de leucotrienos en un modelo de alergia alimentaria: cambios en la población linfocítica de la mucosa renal

    Directory of Open Access Journals (Sweden)

    M. Vinuesa

    2010-07-01

    Full Text Available Objective: the aim is to determine immunopathological modifications in rectal mucosa from rabbits after local challenge in ovalbumin (OVA sensitized animals previously treated with montelukast. Material and methods: experimental design: thirty two rabbits divided into four groups: G1: normal; G2: subcutaneously OVA sensitized; G3: sensitized, locally OVA challenged and sampled 4 hours after challenge; and G4: sensitized, locally OVA challenged and treated 4 hours before challenge with montelukast (0.15 mg/kg. Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA. In each group 200 high microscopical power fields (HPF were counted. Results were expressed as arithmetic mean and SE. Anti -CD4, CD5, µ chain monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. Results: CD 4: G1: 8.3 ± 0.06; G2: 13.4 ± 0.08, G3: 8.25 ± 0.06, G4: 11.8 ± 0.02. CD 5: G1: 7.3 ± 0.05; G2: 9.4 ± 0.05, G3: 11.3 ± 0.06, G4: 8.1 ± 0.06. μ chain: G1: 10.4 ± 0.06; G2: 3.8 ± 0.02, G3: 6.0 ± 0.10, G4: 2.2 ± 0.10. In all cases, experimental groups (G3 vs. G4 presented statistical significant differences (p < 0.05. CD4+, CD5+ cells and μ chain+ decrease in experimental group (G4, probably due to lymphocyte migration inhibition to challenged mucosa. μ chain+ cell decrease could be based on B cell activation and expression of different surface immunoglobulins. Cells expressing μ chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. Conclusions: we conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals after treatment with montelukast.

  17. Comparison of cardiomyocyte apoptosis and early postoperative recovery between propofol-and midazolam-combined anesthesia in patients undergoing cardiac valve replacement%异丙酚或咪达唑仑复合麻醉下心脏手术患者心肌细胞凋亡及术后早期恢复的比较

    Institute of Scientific and Technical Information of China (English)

    诸绍君; 周燕丰; 祝胜美

    2010-01-01

    Objective To compare the cardiomyocyte apoptosis and early postoperative recovery in patients undergoing cardiac valve replacement under propofol-or midazolam-combined anesthesia.Methods Forty NYHA class Ⅱ or Ⅲ patients (aged 48-64 yr and weighing 45-78 kg) undergoing cardiac valve replacement with cardiopulmonary bypass (CPB) were randomly divided into midazolam group (Group M) and propofol group (Group P) (n=20each). The patients were premedicated with morphine 0.1 mg/kg i.v. and scopolamine 0.3 mg i.v. Anesthesia was induced with midazolam 0.2 mg/kg (in Group M) or propofol 2 mg/kg (in Group P) combined with fentanyl 10 μg/kg and vecuronium 0.1 mg/kg, and maintained with propofol 5 mg. kg-1·h-1 (in Group P) or midazolam 0.1 mg·kg-1·h-1(in Group M) and intermittent i.v. boluses of fentanyl and vecuronium after tracheal intubation. The patients were mechanically ventilated with PETCO2 maintained at 35-45 mm Hg. Myocardial tissues were obtained from the right atrium before and after CPB for determination of apoptosis in cardiomyocytes (by TUNEL). The apoptotic index was calculated. The expression of caspase-3 and caspase-9 was determined by immunohistochemical avidin-biotin-peroxidase complex (ABC) technique staining. The mean airway pressure (MAP) and heart rate (HR) were monitored. Aortic cross-clamping time, surgical and CPB times, spontaneous recovery of normal heart beat, emergence from anesthesia, extubation time and duration of ICU stay were recorded and compared between the two groups.Results The percentage of spontaneous recovery of normal heart beat after release of aortic cross clamp was significantly higher and the need for dobutamine support was significantly less in Group P than in Group M ( P < 0.05). The emergence from anesthesia was significantly more rapid, the extubation time and the ICU stay were significantly shorter in Group P than in Group M (P<0.05). There were no significant differences in apoptosis index and expression of

  18. Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Xuewei Xie; Zhouping Tang; Feng Xu; Na Liu; Zaiwang Li; Suiqiang Zhu; Wei Wang

    2009-01-01

    BACKGROUND: Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors.OBJECTIVE: To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells.DESIGN, TIME AND SETrlNG: Cytology was performed at the Department of Neurology, Tongji Medical College, Huazhong University of Science and Technology, China, from September 2007 to October 2008.MATERIALS: Mouse anti-nestin polyclonal antibody (Chemicon, USA), mouse anti-glial fibrillary acidic protein (GFAP) IgG1, mouse anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) IgG1, mouse anti-Tubulin Class-Ill IgG1 (Neo Markers, USA), Avidin-labeled Cy3 (KPL, USA), and goat anti-mouse IgG1: fluorescein isothiocyanate (FITC) (Serotec, UK) were used in this study.METHODS: Tissues were isolated from the embryonic olfactory bulb and subependymal region of Wistar rats. Serum-free DMEM/F12 culture media was used for co-culture experiments. Neural stem cells were incubated in serum-free or 5% fetal bovine serum-containing DMEM/F12 as controls.MAIN OUTCOME MEASURES: After 7 days of co-culture, neural stem cells and olfactory ensheathing cells underwent immunofluorescent staining for nestin, tubulin, glial fibrillary acidic protein, and CNPase.RESULTS: Olfactory ensheathing cells promoted proliferation and differentiation of neural stem cells into neuron-like cells, astrocytes and oligodendrocytes. The proportion of neuron-like cells was 78.2%, but the proportion of neurons in 5% fetal bovine serum DMEM/F12 was 48.3%. In the serum-free DMEM/F12, neural stem cells contracted, unevenly adhered to the glassware wall, or underwent apoptosis at 7 days.CONCLUSION: Olfactory ensheathing cells promote differentiation of neural stem cells mainly into neuron-like cells, and accelerate proliferation of neural stem cells. The outcome is better compared with serum-free medium or medium containing 5% fetal bovine

  19. Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    Peng Huang; Chun-You Wang; Shan-Miao Gou; He-Shui Wu; Tap Liu; Jiang-Xin Xiong

    2008-01-01

    AIM: To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1.METHODS: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 (1:1 volume)(DMEM-F12) supplemented with 20% fetal bovine serum (FBS).Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry.The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) method.And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied,and expression of CD44 and CD24 of the CD44+CD24+ cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex (ABC) immunohistochemical staining.RESULTS: The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44,57.8% -70.1% expressed CD24,only 2.1%-3.5% of cells were CD44+ CD24+.Compared with CD44-CD24- cells,CD44+CD24+ cells had a lower growth rate in vitro.Implantation of 104 CD44 CD24- cells in nude mice showed no evident tumor growth at wk 12.In contrast,large tumors were found in nude mice implanted with 103 CD44+CD24+ cells at wk 4 (2/8),a 20-fold increase in tumorigenic potential (P<0.05 or P<0.01).There was no obvious histological difference between the cells of the CD44+CD24+ cell-formed nodules and PANC-1 cells.CONCLUSION: CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1.Subpopulation cells CD44+CD24+ have properties of tumor stem cells.Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy,it may be a very promising target for new drug development.

  20. Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling

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    Brasseur Robert

    2008-01-01

    subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

  1. Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers

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    Goldenberg David M

    2007-01-01

    Full Text Available Abstract Background Many breast, pancreatic, colonic and non-small-cell lung carcinoma lines express CEACAM6 (NCA-90 and CEACAM5 (carcinoembryonic antigen, CEA, and antibodies to both can affect tumor cell growth in vitro and in vivo. Here, we compare both antigens as a function of histological phenotype in breast, pancreatic, lung, ovarian, and prostatic cancers, including patient-matched normal, primary tumor, and metastatic breast and colonic cancer specimens. Methods Antigen expression was determined by immunohistochemistry (IHC using tissue microarrays with MN-15 and MN-3 antibodies targeting the A1B1- and N-domains of CEACAM6, respectively, and the MN-14 antibody targeting the A3B3 domain of CEACAM5. IHC was performed using avidin-biotin-diaminobenzide staining. The average score ± SD (0 = negative/8 = highest for each histotype was recorded. Results For all tumors, the amount of CEACAM6 expressed was greater than that of CEACAM5, and reflected tumor histotype. In breast tumors, CEACAM6 was highest in papillary > infiltrating ductal > lobular > phyllodes; in pancreatic tumors, moderately-differentiated > well-differentiated > poorly-differentiated tumors; mucinous ovarian adenocarcinomas had almost 3-fold more CEACAM6 than serous ovarian adenocarcinomas; lung adenocarcinomas > squamous tumors; and liver metastases of colonic carcinoma > primary tumors = lymph nodes metastases > normal intestine. However, CEACAM6 expression was similar in prostate cancer and normal tissues. The amount of CEACAM6 in metastatic colon tumors found in liver was higher than in many primary colon tumors. In contrast, CEACAM6 immunostaining of lymph node metastases from breast, colon, or lung tumors was similar to the primary tumor. Conclusion CEACAM6 expression is elevated in many solid tumors, but variable as a function of histotype. Based on previous work demonstrating a role for CEACAM6 in tumor cell migration, invasion and adhesion, and formation of distant

  2. Intracellular expression of the proliferative marker Ki-67 and viral proteins (NS3, NS5A and C in chronic, long lasting hepatitis C virus (HCV infection.

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    Karolina Olejniczak

    2008-01-01

    Full Text Available Hepatitis C virus (HCV continues to represent the main causative agent of the hepatitis, which leads to chronic transformation of the process in 60-80% individuals. It remains unclear how far cellular expression of HCV proteins in vivo may represent an index of progression of the disease and of proliferative activity in the liver in chronic hepatitis C. Aim of the studies included detection and subcellular localization of three HCV proteins (NS3, NS5A and C in liver biopsies from adults (n=19 with chronic, long lasting hepatitis C as related to hepatocyte proliferative activity. The immunocytochemical ABC (avidin biotin-peroxidase complex technique was applied, alone or associated with the ImmunoMax technique. Results of the immunocytochemical tests were compared to histological alterations in liver biopsies, proliferation index and with selected clinical data. A significantly higher expression of NS3 protein was noted, as compared to expressions of NS5A and C proteins. In all the patients, cytoplasmic localization of all proteins dominated over nuclear localization (p0.05. At the level of electron microscopy, protein localization in endoplasmic reticulum (ER membranes, mitochondria, perinuclear region and/or in hepatocyte cell nucleus was observed. No direct relationships could be demonstrated between expressions of HCV proteins and of Ki-67 antigen. No correlations could also be demonstrated between cellular expression of any HCV protein on one hand and grading or staging, alanine transaminase (ALT, serum level of HCV RNA or alpha-fetoprotein (AFP on the other. However, positive correlations were disclosed between proliferative activity of hepatocytes on one hand and patient's age, grading and staging on the other. Advanced hepatic fibrosis correlated also with serum levels of AFP. The studies were supplemented with data on subcellular localization of HCV proteins. Moreover, they indicated that in HCV infection grading and staging

  3. Proliferative vitreo-retinal disorders: experimental models in vivo and in vitro.

    Science.gov (United States)

    Martini, B

    1992-01-01

    The aim of the present thesis was to develop, refine, and assess experimental models for the study of proliferative vitreo-retinal disorders. An intravitreal injection of a colloidal solution of microparticles was used in the primate eye to produce pathologic changes including intraocular cell invasion, cell proliferation, neovascularization, collagen synthesis, and tractional retinal detachment. In a separate primate model for laser-induced subretinal neovascularization, the origin and the occurrence of macrophages was evaluated. Examinations were performed using ophthalmoscopy, slit-lamp microscopy, light microscopy, and transmission electron microscopy. Cell cultures were employed to study the effects of vitreous humor and macrophages on the proliferation of cultured retinal pigment epithelial (RPE) cells and cultured fibroblasts using a Coulter counter. Morphologic changes were documented by phase micrography. A quantitative estimation of the extracellular matrix deposition of fibrous proteins by macrophage-modulated RPE cells as well as by vitreous-modulated RPE cells was done using enzymatic digestion and radioactive labeling techniques. A qualitative analysis of the types of collagen that was deposited in the extracellular matrices by vitreous modulated cultures was also made using indirect immunofluorescence. Using a newly developed RPE cell specific monoclonal antibody, the avidin-biotin-peroxidase labeling technique was finally employed to test the phenotypic epitope expression of macrophage-modulated and non-modulated RPE cells. A new experimental in vivo model for pathologic changes that characterize proliferative vitreo-retinal disorders was developed in the primate eye. In the model for laser-induced subretinal neovascularization, macrophages were shown to be principally recruited from the systemic circulation. Using cell cultures, it was found that both macrophage-conditioned medium and vitreous humor, separately or combined, exert mitogenic effects

  4. 大鼠Scarpa神经节中Trk受体的免疫组织化学研究%IMMUNOHISTOCHEMISTRY FOR Trk RECEPTORS IN RAT SCARPA'S GANGLION

    Institute of Scientific and Technical Information of China (English)

    陈宇; 董玉琳; 刘涛; 张富兴; 李金莲; 李云庆

    2007-01-01

    采用免疫组织化学的卵白素-生物素-HRP复合物(ABC)法,以二氨基联苯胺(DAB)为呈色剂,观察高亲和性的神经营养物质受体(酪氨酸激酶受体),即TrkA,TrkB和TrkC在大鼠Scapa神经节(前庭神经节,VG)中的分布.结果显示,许多VG神经元分别对3种Trk受体呈阳性反应,受体位于神经元胞体.每一种Trk受体的反应强度在不同神经元间有区别,有弱、中等和强反应之分.对同一种Trk受体而言,阳性神经元的大小不等;统计结果显示:TrkA,TrkB和TrkC阳性神经元平均面积分别为330.8±7.6,303.89±10.6和355.05±8.3 μm2.该研究结果为Trk受体在VG内发挥维持神经元存活、保持其形态特征等的作用提供了形态学支持.%With the aim to examine the distribution of high-affinity neurotrophin receptors (tyrosine kinase receptors, TrkA, TrkB and TrkC)in the rat Scapa's ganglion ( vestibular ganglion, VG), Avidin-Biotin-peroxidase Complex ( ABC ) method of immunohistochemistry with diaminobenzidine (DAB) as the chromogen to identify the immunoreactivities was employed in the present study. The results showed that many VG neurons were immunoreactive to each Trk isoform. The receptors were localized in the neuronal somata. The intensity of immunoreactivity for each Trk receptor was different among neurons, ranging from weak, moderate to intense. For each individual Trk receptors, the labelled neurons were of different size; the result sfatistical of analysis showed that the mean areas for neurons immunoreative to TrkA, B and C were 330.8 ± 7.6, 303.89 ± 10.6 and 355.05 ± 8.3 μm2 , respectively. The present study provids morphological substrate for the important roles played by Trk receptors in maintaining the survival and stabilizing the phenotype of VG neurons.

  5. HCV游离抗原BA-ELISA检测方法的临床应用评价%Evaluation on clinical application of HCV free antigen detection methods of BA-ELISA

    Institute of Scientific and Technical Information of China (English)

    龙润乡; 陈红英; 朱祥明; 沈云松; 孙翳; 苏品璨; 王俊; 谢忠平

    2013-01-01

    目的 根据国家相关法规对HCV游离抗原BA-ELISA检测方法进行临床应用效果评价.方法 生物素标记丙型肝炎病毒抗体(HCV-Ab)与辣根过氧化物酶标记亲和素联合应用建立HCV游离抗原BA-ELISA检测法,分别在3个省级医疗卫生机构进行临床试验,同时使用HCV-cAg、HCV-Ab、HCV-RNA荧光定量检测试剂盒进行对比同步检测.结果 临床试剂与HCV-Ab检测结果相比一致性66.67%,特异性100%;与荧光定量PCR相比结果一致性为80.85%、特异性为98.87%;同类市售产品HCV核心抗原检测试剂检测结果与之接近.结论 临床试剂特异性较好,灵敏度有待提高;临床研究试剂比市售同类产品阳性检出率略高,但两种试剂检出率均低于核酸及抗体检测试剂.%OBJECTIVE According to the national relative regulations, to evaluate effect of clinical application of HCV free antigen BA-ELISA detection method. METHODS Used biotin-labeled hepatitis C virus antibody (HCV-Ab) and horseradish peroxidase -conjugated avidin system to establish of free HCV antigen detection methods of BA-ELISA. Respectively in the three provincial-level medical and health institutions for clinical trials, meanwhile HCV-cAg, HCV-Ab, HCV-RNA fluorescence quantitative detection kit synchronous detection were compared. RESULTS Results of clinical study reagent compared with HCV-Ab test results, coincidence rate was 66.67%, specificity 100%; And compared with HCV-PCR, coincidence rate was 80.85%, specificity was 98.87%; The results were analogue by the commercial product HCV-Ag detection reagents and HCV-Ag detection reagents. CONCLUSION Better clinical study reagent specificity, sensitivity need to be improved; clinical study reagents than similar products commercially, positive rate is slightly higher, but both are lower than nucleic acid detection reagent and antibody detection kit.

  6. Distribution of Foxp3(+) T cells in the liver and hepatic lymph nodes of goats and sheep experimentally infected with Fasciola hepatica.

    Science.gov (United States)

    Escamilla, A; Zafra, R; Pérez, J; McNeilly, T N; Pacheco, I L; Buffoni, L; Martínez-Moreno, F J; Molina-Hernández, V; Martínez-Moreno, A

    2016-10-30

    Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola hepatica infection; however, the distribution and role of Foxp3(+) Tregs cells have not been investigated in F. hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3(+) Tregs in the liver and hepatic lymph nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n=6) and three groups of sheep (n=6) were used in this study. Goats in groups 1-2 and sheep in groups 4-5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin-biotin-complex method was used for the immunohistochemical study. At nine dpi in acute hepatic lesions, the number of both Foxp3(+) and CD3(+) T lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15-19wpi), the number of Foxp3(+) and CD3(+) T lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3(+) Tregs were increased in the cortex of hepatic lymph nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and hepatic lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3(+) Tregs in acute and

  7. MGMT和Survivin在乳腺癌中的表达及其临床意义%Expression of MGMT and Survivin in Breast Cancer and Its Clinical Significance

    Institute of Scientific and Technical Information of China (English)

    王发亮; 薄爱华; 芦广萍; 侯金超; 吴荣薇

    2008-01-01

    Objective:To investigate the expression of MGMT and Survivin in breast cancer and its clinical significance.Methods:Specimens from breast adenoma and breast cancer were taken and had been Formalin-fixed,paraffin-embedded.With Strept Avidin-Biotin Complex(SABC) immunohistochemical technique,the expression of MGMT and Survivin in these specimens was examined.Results:It was found that there were significant differences between MGMT and Survivin in breast adenoma and breast lymph node metastasis,while the expression of Survivin was associated with lymph node metastasis only.Meanwhile,the expression of MGMT was correlated with that of Survivin(r=0.34,P<0.01).Conclusion:It was concluded that the abnormal expressions of MGMT and Survivin were associated with lymph node metastasis.They possibly could be useful indexes for the primary screening and the prognosis of breast cancer.The examination of them may have an important guiding significance in the chemotherapy strategy.%目的:研究MGMT和Survivin在乳腺癌中的表达及其临床意义.方法:福尔马林固定,石蜡包埋乳腺癌和腺瘤标本,采用SABC免疫组织化学方法检测MGMT和Survivin在这些组织中的表达.结果:MGMT和Survivin在乳腺癌和乳腺腺瘤中的表达有显著性差异.MGMT在乳腺癌中的表达与患者的年龄、淋巴结转移有关,而Survivin仅与淋巴结转移有关.另外,MGMT和Survivin之间具有相关性(r=0.48,P<0.01).结论:MGMT和Survivin的异常表达与乳腺癌的淋巴结转移有关;MGMT和Survivin可以作为判断乳腺癌发生和预后的重要指标;检测它们的表达可以指导临床上化疗方案的制定.

  8. Multifunctional, chitosan-based nano therapeutics: design and application for two- and three-dimensional cell culture systems

    Science.gov (United States)

    Suarato, Giulia

    There is a constant demand for sensitive and effective anti-cancer drug delivery systems, capable of detecting early-stage pathological conditions and increasing patient survival. Recently, chitosan-based drug delivery nanocomplexes have shown to smartly respond to the distinctive features of the tumor microenvironment, a complex network of extracellular molecules, stromal and endothelial cells, which supports the tumor formation and its metastatic invasion. Due to biocompatibility, easy chemical tailorability, and pH-responsiveness, chitosan has emerged as a promising candidate for the formulation of supramolecular multifunctional materials. The present study focuses on the design, fabrication and characterization of fluorescently labelled, hydrophobically modified glycol chitosan nano-micelles (HGC NPs), suitably tailored for the delivery of anti-neoplastic compounds to various tumor models. Doxorubicin-loaded HGC NPs have been delivered to a bone cancer model, both in monolayer and in 3D spheroid configuration, to assess for differences in the delivery profiles and in the therapeutic efficacy. Compared to the free drug, nanocomplexes showed rapid uptake and a more homogeneous distribution in 3D spheroids, a powerful cellular tool which recapitulates some of the in vivo tumor microenvironment features. In a second part of this thesis work, with the purpose of designing an active targeting tumor-homing nano-therapeutic system, HGC NPs have been linked, via avidin-biotin interaction, with a IVS4 peptide, a small molecule with inhibitory activity on MMP-14-mediated functions. An extensive study conducted on triple negative breast cancer cells in monolayer revealed the MMP-14-IVS4-HGC association at the cancer cell membrane, the preferential uptake, and the consequent impairment of protease-associated migratory ability. As an additional application of our engineered construct, HGC micelles have been decorated with a liver kinase B1 (LKB1), a critical kinase involved

  9. Quantitative analysis of expression of cyclin D1 and CDK4 in normal, inflammatory and malignant epithelia of cheek mucosa%cyclin D1和CDK4在口腔正常上皮、炎症及鳞癌中表达的定量分析

    Institute of Scientific and Technical Information of China (English)

    马妍; Tipoe,GL; 等

    2001-01-01

    AIM To evaluate the expression and significance of cyclinD1/CDK4in normal epithelia (N), inflammatory (IF) and squamous cell cacinoma (SCC) of cheek mucosa. METHODS Oringinal pathology specimens were collected cut and immunostained by cyclin D1 monoclonal antibody and CDK4 polyclonal antibody, using the avidin-biotin peroxidase complex technique (ABC). RESULTS There was statistical significance between the N group and SCC group, but not between the N group and IF group. Cyclin D1 and CDK4 were overexpressed in SCC. CONCLUSION Overexpression of cyclinD1 and CDK4 in SCC may be due to the gene amplification and/or other related factors. The variations of cyclin D1and CDK4 in different subgroups of SCC may be a helpful indicator for tumor grading.%目的 通过检测口腔粘膜的正常上皮、非特异性炎症上皮及鳞癌中cyclinD1与CDK4的表达,探讨其在上皮组织不同状态中的变化及意义.方法 用临床病理标本,设立对照,选择cyclinD1和CDK4抗体免疫组化染色.结果 cyclinD1和CDK4在正常口腔上皮与口腔鳞癌上皮、非特异性炎症与口腔鳞癌上皮中的表达间有统计学差别(P<0.05),正常组与炎症组间无差别,cyclinD1及CDK4在口腔鳞癌上皮中过表达(P<0.05).结论 cyclinD1与CDK4过表达的机制可能由于基因扩增或其他因素使其蓄积,口腔鳞癌组分级间的差异性提示其可作为肿瘤分级的评价指标之一.

  10. Cystic Fibrosis Transmembrane Conductance Regulator and H+ Permeability in Regulation of Golgi pH

    Directory of Open Access Journals (Sweden)

    Machen TE

    2001-07-01

    Full Text Available This paper reviews experiments from this lab that have tested the hypothesis that pH of the Golgi (pH(G of cystic fibrosis (CF airway epithelial cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other Golgi enzymes controlling biochemical composition of the plasma membrane and that altered surface biochemistry increases bacterial binding. We generated a plasmid encoding a modified green fluorescence protein-sialyltransferase (GFP-ST chimera protein that was pH-sensitive and localized to the Golgi when transfected into HeLa cells and also CF and normal or cystic fibrosis transmembrane conductance regulator- (CFTR-corrected airway epithelial cells. Digital imaging microscopy of these Golgi-localized probes showed that there was no correlation between pH(G (6.4-7.0 and the presence of CFTR, whether cells were in HCO(3(-/CO(2-containing or in HCO(3(-/CO(2-free solutions. Activation of CFTR by raising cell [cAMP] had no effect on pH(G. Thus, CFTR seemed not to be involved in controlling pH(G. Experiments on HeLa cells using an avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent biotin indicated that even in cells that do not express CFTR, Cl(- and K(+ conductances of the Golgi and other organelle membranes were large and that pH(G was controlled solely by the H(+ v-ATPase countered by a H(+ leak. A mathematical model was applied to these and other published data to calculate passive H(+ permeability (P(H+ of the Golgi, endoplasmic reticulum, trans-Golgi network, recycling endosomes and secrety granules from a variety of cells. An organelle's acidity was inversely correlated to its calculated P(H+. We conclude that the CFTR plays a minor role in organelle pH regulation because other (Cl(- and K(+ channels are present in sufficient numbers to shunt voltages generated during H(+ pumping. Acidity of the Golgi (and perhaps other organelles appears to be determined by the activity of H

  11. Multifunctional pH-sensitive polymeric nanoparticles for theranostics evaluated experimentally in cancer

    Science.gov (United States)

    Liu, Yongjun; Feng, Lixia; Liu, Tingxian; Zhang, Li; Yao, Yao; Yu, Dexin; Wang, Linlin; Zhang, Na

    2014-02-01

    A multifunctional pH-sensitive polymeric nanoparticle system was developed for simultaneous tumor magnetic resonance imaging (MRI) and therapy. The nanoparticles were self-assembled using the multi-block polymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)-diethylenetriamine pentaacetic acid (PLA-PEG-PLL-DTPA) and the pH-sensitive material poly(l-histidine)-poly(ethylene glycol)-biotin (PLH-PEG-biotin). The anti-hepatocellular carcinoma (HCC) drug sorafenib was encapsulated inside the nanoparticles. Gd ions were chelated to the DTPA groups which were distributed on the nanoparticle surface. Biotinylated vascular endothelial growth factor receptor (VEGFR) antibodies were linked to the surface biotin groups of nanoparticles through the avidin linker to form the target pH-sensitive theranostic nanoparticles (TPTN). TPTN exhibited spherical or ellipsoidal shapes, uniform particle size distribution (181.4 +/- 3.4 nm), positive zeta potential (14.95 +/- 0.60 mV), high encapsulation efficiency (95.02 +/- 1.47%) and drug loading (2.38 +/- 0.04%). The pH-sensitive sorafenib release from TPTN was observed under different pH values (47.81% at pH = 7.4 and 99.32% at pH = 5.0, respectively). In cell cytotoxicity studies, TPTN showed similar antitumor effect against HepG2 cells compared to solubilized sorafenib solution after pre-incubation in acid PBS (pH = 5.0) for 1 h in vitro (P > 0.05). In in vivo anti-tumor studies, TPTN showed significantly higher antitumor effect in H22 tumor (VEGFR overexpressed cell line) bearing mice compared to the solubilized sorafenib solution (oral or i.v. administration) group (P PLH-PEG-biotin). The anti-hepatocellular carcinoma (HCC) drug sorafenib was encapsulated inside the nanoparticles. Gd ions were chelated to the DTPA groups which were distributed on the nanoparticle surface. Biotinylated vascular endothelial growth factor receptor (VEGFR) antibodies were linked to the surface biotin groups of nanoparticles through the avidin

  12. 皮层电刺激联合康复锻炼对大鼠局灶性脑缺血模型前肢运动功能及运动区突触可塑性相关蛋白表达的影响%Cortical electrical Stimulation Combined with Rehabilitative Training Enhance Forelimb Motor Function and Synaptic Plasticity Following Focal Cortical Ischemia in Rats

    Institute of Scientific and Technical Information of China (English)

    郑建; 杨力军; 谢瑞禄; 赵兰峰; 薛晓伟; 王硕; 赵继宗; 曹勇

    2011-01-01

    Objective To assess the behavioral and synaptic plasticity effects of combining epidural cortical electrical stimulation with motor skills training following unilateral sensorimotor cortex (SMC)lesions in adult male rats.Methods Prior to lesion/electrode implantation surgeries, rats were pre-trained on the 'single pellet retrieval task' to a minimum criterion of 30% success rate for two consecutive days. Then these rats received partial unilateral SMC lesions and implantation of electrodes over the remaining SMC. Fourteen days later, rats received daily reach training concurrent with anodal or cathodal 100 Hz or no stimulation for 14 days. Performance was measured as the percent of successes out of the total number of reach attempts [(total successes/total reach attempts)*100]. Conventional avidin biotinylated enzyme complex (ABC) immunohistochemical method was used quantify the expression and distribution of microtubule-associated protein 2 (MAP-2) and growth associated protein 43 (GAP-43) in motor cortical area underlying the electrode.Results There was no statistical significance between the two groups on the 14th day of preoperative training (P=0.546). The stimulation group had significantly greater rates of improvement with the impaired forelimb in comparison to control group (49.12% vs 21.67%, P=0.004). The expression and distribution of MAP-2 and GAP-43 in the stimulating group were better than those in control group (GAP-43 : 0.3338 vs 0.3056. P=0.008; MAP-2: 0.4825 vs 0.4327. P=0.027).Conclusion These data indicate that cortical stimulation greatly improves the efficacy of rehabilitative reach training following SMC damage and raise the possibility that CS-induced functional improvements may be mediated by promoting the expression of MAP-2 and GAP-43 in perilesion cortex. and thus improve synaptic plasticity in cerebral ischemic rats.%目的 探讨皮层电刺激联合康复锻炼对大鼠局灶性脑缺血模型前肢运动功能恢复和运动区

  13. 靶向超声微泡对结肠癌新生血管分子成像的实验研究%Molecular imaging of tumor angiogenesis with VEGFR2 targeting microbubbles in colon cancer bearing nude mice

    Institute of Scientific and Technical Information of China (English)

    位红芹; 何洁; 杨莉; 纪丽景; 张霞; 王冬晓; 文戈; 谷英士; 李颖嘉

    2013-01-01

    Objective To evaluate the effect of tumor neovascularization imaging in a nude mouse model of colon cancer by contrast ultrasound molecular imaging (UMI) of VEGF receptor 2 (kinase insert domain receptor,KDR).Methods Targeted microbubbles (MBt) were built by conjugating K237,a small peptide with high affinity for KDR,to liposome microbubbles through a biotin-avidin bridge.Control microbubbles (MBc) with control peptide were prepared by the same method.Nude mice models of LS174T human colon cancer were established.MBt and MBc were injected intravenously in twelve mice in random order with an interval of 30 min.MBt were injected in another six mice after K237-peptide blocking.UMI was performed in all mice at 5 min postinjection to observe the imaging difference and measure the video intensity (Ⅵ) of tumor tissues in different groups.One-way analysis of variance and the least significant difference t test were performed to analyze the difference of tumor VI in the groups with MBt,MBc and K237 blocking.Immunohistochemistry was applied to detect the expression and distribution of KDR in tumor tissue and adjacent tumor tissues.Results K237 peptide was successfully conjugated to the surface of microbubbles through biotin-avidin mediation.Ultrasound imaging signal of the tumor was high in the MBt group,while there were no significant enhancement in the groups of K237 blocking and MBc.The VI in MBt,MBc and K237 blocking groups was significantly different (F =39.130,P < 0.01).There was a significant difference of VI in the MBt group compared to the MBc group (30.18 ± 9.56 vs 8.28 ± 4.74,t =6.91,P <0.01).In the K237 blocking group Ⅵ was significantly lower than that in the MBt group (9.23 ± 3.44 vs 30.18 ± 9.56,t =4.91,P < 0.01).Immunohistochemistry results showed that KDR was highy expressed in tumor tissue.Conclusions KDR-targeting liposome contrast microbubbles may specifically and efficiently link to tumor vascular endothelial cells in vivo.Thus it may be

  14. 靶向纳米液态氟碳球囊超声造影剂的制备及体外靶向研究%Preparation and in vitro-targeted study of liquid perfluorocarbons-filled nanocapsules ultrasound contrast agent

    Institute of Scientific and Technical Information of China (English)

    刘腾; 洪玉蓉; 董鑫; 陆世鎏; 康牧星; 张波; 吴育连

    2014-01-01

    目的 制备靶向人血管内皮细胞生长因子受体2(VEGFR2)的纳米液态氟碳球囊,进行体外靶向结合与超声显影的研究.方法 利用乳化/蒸发法制备生物素修饰的纳米液态氟碳球囊(LNCs),采用生物素-链霉亲和素链接法进一步偶联生物素修饰的anti-VEGFR2抗体,制备VEGFR2靶向纳米氟碳球囊(V2-LNCs).观察其形态、大小及结构,测量其粒径和表面电位.用免疫荧光法检测V2-LNCs与人血管内皮细胞(HUEVC)的靶向结合情况.观察纳米液态氟碳球囊体外超声显影效果,并进行统计学分析.结果 制备了平均粒径为(98.45±0.07)nm的V2-LNCs;V2-LNCs与配体结合后的荧光免疫染色试验为阳性;体外寻靶试验显示V2-LNCs能较好地黏附在HUEVC细胞上,反复洗涤不掉,用anti-VEGFR2抗体预封闭HUEVC细胞可成功阻断其靶向结合;而作为对照的LNCs经反复洗涤后纳米球囊与细胞完全分离,无结合;体外超声显影效果明显增强,在较低浓度(0.5 mg/ml)即可显著增强超声显影效果.结论 成功制备出VEGFR2靶向纳米液态氟碳球囊超声造影剂,该纳米球囊与体外HUEVC有较强结合力且能显著增强超声显影性能,为进一步的血管外超声靶向显影研究获得了实验室基础数据.%Objective To prepare human vascular endothelial growth factor receptor 2 (VEGFR2)-targeted liquid perfluorocarbons-filled nanocapsules ultrasound contrast agent,and to investigate its ability of targeting human umbilical endothelial vein cells(HUEVC) and the peculiarity of the enhancing ultrasound imaging in vitro.Methods Rotary evaporation/emulsion technique was used to prepare biotinylated liquid perfluorocarbons-filled nanocapsules (LNCs).VEGFR2-tageted LNCs (V2-LNCs) was further made through conjugating the ligand (biotinylated anti-VEGFR2 antibody) to the surface of LNCs by biotin-avidin system and its appearance,distribution,size and zeta-potential properties were assessed

  15. Expressão das proteínas BCL-2 e BAX em tumores astrocíticos humanos Expression of BCL-2 and BAX proteins in human astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2006-08-01

    Full Text Available INTRODUÇÃO: Os astrocitomas constituem os mais freqüentes tumores primários do sistema nervoso central (SNC. Admite-se que parte do crescimento tumoral seja resultante da inibição da morte celular programada: a apoptose. Tal fenômeno é basicamente regulado pelo equilíbrio entre moléculas antiapoptóticas (ex.: B-cell lymphoma protein 2 [BCL-2] e pró-apoptóticas (ex.: BCL-2 associated protein X [BAX]. OBJETIVO: O presente estudo objetivou avaliar a expressão de BCL-2 e BAX em tumores astrocíticos humanos. MATERIAL E MÉTODOS: Procedeu-se ao estudo imuno-histoquímico dessas proteínas utilizando-se o método da avidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do II, sete do III e 21 do grau IV e cinco amostras de tecido cerebral não-tumoral (grupo controle. RESULTADOS: Os índices de positividade para BCL-2 e BAX demonstraram propensão ao acréscimo, de acordo com a gradação tumoral, com positividade geral de 43,26% e 24,67%, respectivamente. Essas proteínas não foram detectadas entre os espécimes não-tumorais. Os escores de marcação para BCL-2 apresentaram tendência ao aumento conforme a progressão histológica, enquanto os para BAX mostraram-se semelhantes nas diversas graduações. A análise conjunta dessas proteínas demonstrou significativa correlação com a gradação tumoral (p BACKGROUND: Astrocytomas represent the most frequent primary tumors of the central nervous system. Admittedly, part of tumor growth is due to inhibition of programmed cell death: the apoptosis. This phenomenon is basically regulated by the balance between anti-apoptotic (e.g.: B-cell lymphoma protein 2 [BCL-2] and pro-apoptotic (e.g.: BCL-2 associated protein X [BAX] molecules. OBJECTIVE: The present study aimed to evaluate the expression of BCL-2 and BAX in human astrocytic tumors of different histopathological grades. MATERIAL AND METHOD: An immunohistochemical study of those proteins using the avidin

  16. Is the positive c-kit immunostaining associated with the presence of cells analogous to the intersticial cells of Cajal in the ciliary muscle? A imunomarcação positiva para c-kit está associada com a presença de células análogas às intersticiais de Cajal no músculo ciliar?

    Directory of Open Access Journals (Sweden)

    Jayter Silva Paula

    2009-02-01

    Full Text Available PURPOSE: Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes. METHODS: Eight eyes from four adult male new world monkeys (Cebus apella were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide. RESULTS: We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed. CONCLUSION: C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.OBJETIVO: As células intersticiais de Cajal estão presentes no trato gastrintestinal de diversas espécies animais, em íntima relação com o sistema nervoso entérico. Uma vez que as células intersticiais de Cajal expressam o produto do gene c-kit, realizou-se um ensaio imuno-histoquímico a fim de se verificar a marcação da proteína c-kit no músculo ciliar de amostras de olhos de macacos. MÉTODOS: Oito olhos de quatro macacos do novo mundo (Cebus apella foram estudados. Após bloqueio da peroxidase endógena e de ligação protéica não específica, os tecidos receberam aplicação de anticorpos de camundongos antioncoproteína c-kit humana (1:100. A reação antígeno-anticorpo foi verificada através da aplicação do complexo avidina-biotinilada-peroxidase em cada lâmina. RESULTADOS: Foram observados grupos de c

  17. 微量酶联杂交法定量检测HBV基因 竞争PCR扩增产物%An enzyme-linked hybridization on microplate for quantification of HBV gene amplicon after competitive PCR

    Institute of Scientific and Technical Information of China (English)

    缪晓辉; 戚中田; 孔宪涛

    2001-01-01

    目的 建立一种简便、敏感、精确的微反应板酶联杂交技术,以鉴定HBV基因的竞争PCR扩增产物。方法 设计了两种捕获探针,能分别与竞争PCR扩增产物中的野生片段和突变片段杂交。捕获探针通过3′-端修饰的氨基与微量DNA结合板孔表面的NOS基团化学结合而被“竖直”地包被在反应板上;将热变性后的产物加入两种捕获探针反应孔内,产物中带有生物素的野生或突变片段的一条单链与相应的捕获探针杂交;最后用链亲和素-碱性磷酸酶及底物检测杂交信号。结果 该方法检测PCR产物DNA的灵敏度为80ng/ml,大于琼脂糖凝胶电泳染色鉴定法。获得野生片段和突变片段杂交信号值后,可根据公式计算扩增前野生模板的初始量。结论 本方法操作简单、灵敏度高、结果数据化、特异性强,适用于竞争PCR产物分析。%Objective To develop a simple, sensitive, specific andquantitative method by competitive PCR for assay of hepatitis B virus amplicon produced. Methods Two capture probes, one for capturing wild fragments in PCR products and the other one for capturing mutant fragments, were designed. The capture probe can be coated to the surface of DNA binding well through the chemical binding between amino modified at 3′-end of probe and NOS group on wells. Heat-denatured products were then added to wells and hybridization occurs between capture probe and biotin-labeled strand of PCR amplicon. The hyb