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Sample records for protein microarray assay

  1. A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

    Directory of Open Access Journals (Sweden)

    Helene Andersson-Svahn

    2011-11-01

    Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  2. Creation of antifouling microarrays by photopolymerization of zwitterionic compounds for protein assay and cell patterning.

    Science.gov (United States)

    Sun, Xiuhua; Wang, Huaixin; Wang, Yuanyuan; Gui, Taijiang; Wang, Ke; Gao, Changlu

    2018-04-15

    Nonspecific binding or adsorption of biomolecules presents as a major obstacle to higher sensitivity, specificity and reproducibility in microarray technology. We report herein a method to fabricate antifouling microarray via photopolymerization of biomimetic betaine compounds. In brief, carboxybetaine methacrylate was polymerized as arrays for protein sensing, while sulfobetaine methacrylate was polymerized as background. With the abundant carboxyl groups on array surfaces and zwitterionic polymers on the entire surfaces, this microarray allows biomolecular immobilization and recognition with low nonspecific interactions due to its antifouling property. Therefore, low concentration of target molecules can be captured and detected by this microarray. It was proved that a concentration of 10ngmL -1 bovine serum albumin in the sample matrix of bovine serum can be detected by the microarray derivatized with anti-bovine serum albumin. Moreover, with proper hydrophilic-hydrophobic designs, this approach can be applied to fabricate surface-tension droplet arrays, which allows surface-directed cell adhesion and growth. These light controllable approaches constitute a clear improvement in the design of antifouling interfaces, which may lead to greater flexibility in the development of interfacial architectures and wider application in blood contact microdevices. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

    NARCIS (Netherlands)

    Joo, C.; Ozkumur, E.; Unlu, B.; de Boer, J.F.

    2009-01-01

    Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein-protein, protein-DNA and receptor-ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning

  4. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  5. Diagnostic and analytical applications of protein microarrays

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Christensen, C.B.V.

    2005-01-01

    DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the post...

  6. Protein microarray: sensitive and effective immunodetection for drug residues

    Directory of Open Access Journals (Sweden)

    Zer Cindy

    2010-02-01

    Full Text Available Abstract Background Veterinary drugs such as clenbuterol (CL and sulfamethazine (SM2 are low molecular weight ( Results The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g than the ci-ELISA (0.1 ng/g for detection of CL residues. Conclusions The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

  7. Printing Proteins as Microarrays for High-Throughput Function Determination

    Science.gov (United States)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  8. Microarray-based screening of heat shock protein inhibitors.

    Science.gov (United States)

    Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

    2014-06-20

    Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  10. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics

  11. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    International Nuclear Information System (INIS)

    Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

    2014-01-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

  12. Dynamic, electronically switchable surfaces for membrane protein microarrays.

    Science.gov (United States)

    Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

    2006-02-01

    Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

  13. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Science.gov (United States)

    Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

    2009-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

  14. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  15. Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

    International Nuclear Information System (INIS)

    Stempfer, René; Weinhäusel, Andreas; Syed, Parvez; Vierlinger, Klemens; Pichler, Rudolf; Meese, Eckart; Leidinger, Petra; Ludwig, Nicole; Kriegner, Albert; Nöhammer, Christa

    2010-01-01

    The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto

  16. Novel Protein Microarray Technology to Examine Men with Prostate Cancer

    National Research Council Canada - National Science Library

    Lilja, Hans

    2005-01-01

    The authors developed a novel macro and nanoporous silicon surface for protein microarrays to facilitate high-throughput biomarker discovery, and high-density protein-chip array analyses of complex biological samples...

  17. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    Energy Technology Data Exchange (ETDEWEB)

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  18. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  19. Development and Validation of Protein Microarray Technology for Simultaneous Inflammatory Mediator Detection in Human Sera

    Directory of Open Access Journals (Sweden)

    Senthooran Selvarajah

    2014-01-01

    Full Text Available Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL- 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the “gold standard” ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays.

  20. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Science.gov (United States)

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

  1. Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

    2016-12-01

    Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

  2. Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

    Science.gov (United States)

    Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

    2016-01-01

    A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

  3. Addressable droplet microarrays for single cell protein analysis.

    Science.gov (United States)

    Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

    2014-11-07

    Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

  4. Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2017-10-01

    Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

  5. Expanding the substantial interactome of NEMO using protein microarrays.

    LENUS (Irish Health Repository)

    Fenner, Beau J

    2010-01-01

    Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

  6. Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

    Science.gov (United States)

    Sitaraman, Kalavathy; Chatterjee, Deb K

    2011-01-01

    In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

  7. Label and Label-Free Detection Techniques for Protein Microarrays

    Directory of Open Access Journals (Sweden)

    Amir Syahir

    2015-04-01

    Full Text Available Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano‑biological events.

  8. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    Science.gov (United States)

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

    Science.gov (United States)

    Sachse, Konrad; Rahman, Kh Shamsur; Schnee, Christiane; Müller, Elke; Peisker, Madlen; Schumacher, Thomas; Schubert, Evelyn; Ruettger, Anke; Kaltenboeck, Bernhard; Ehricht, Ralf

    2018-03-16

    Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

  10. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  11. Identification of listeria species isolated in Tunisia by Microarray based assay : results of a preliminary study

    International Nuclear Information System (INIS)

    Hmaied, Fatma; Helel, Salma; Barkallah, Insaf; Leberre, V.; Francois, J.M.; Kechrid, A.

    2008-01-01

    Microarray-based assay is a new molecular approach for genetic screening and identification of microorganisms. We have developed a rapid microarray-based assay for the reliable detection and discrimination of Listeria spp. in food and clinical isolates from Tunisia. The method used in the present study is based on the PCR amplification of a virulence factor gene (iap gene). the PCR mixture contained cyanine Cy5labeled dCTP. Therefore, The PCR products were fluorescently labeled. The presence of multiple species-specific sequences within the iap gene enabled us to design different oligoprobes per species. The species-specific sequences of the iap gene used in this study were obtained from genBank and then aligned for phylogenetic analysis in order to identify and retrieve the sequences of homologues of the amplified iap gene analysed. 20 probes were used for detection and identification of 22 food isolates and clinical isolates of Listeria spp (L. monocytogenes, L. ivanovi), L. welshimeri, L. seeligeri, and L. grayi). Each bacterial gene was identified by hybridization to oligoprobes specific for each Listeria species and immobilized on a glass surface. The microarray analysis showed that 5 clinical isolates and 2 food isolates were identified listeria monocytogenes. Concerning the remaining 15 food isolates; 13 were identified listeria innocua and 2 isolates could not be identified by microarray based assay. Further phylogenetic and molecular analysis are required to design more species-specific probes for the identification of Listeria spp. Microarray-based assay is a simple and rapid method used for Listeria species discrimination

  12. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  13. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

    Science.gov (United States)

    Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

    2012-01-01

    Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

  14. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

    Directory of Open Access Journals (Sweden)

    Bettina Stieber

    Full Text Available S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

  15. Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigen microarray.

    Science.gov (United States)

    Matsudaira, Takahiro; Tsuzuki, Saki; Wada, Akira; Suwa, Akira; Kohsaka, Hitoshi; Tomida, Maiko; Ito, Yoshihiro

    2008-01-01

    Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes are characterized by the production of autoantibodies that serve as useful diagnostic markers, surrogate markers, and prognostic factors. We devised an in vitro system to detect these clinically pivotal autoantibodies using a photoimmobilized autoantigen microarray. Photoimmobilization was useful for preparing the autoantigen microarray, where autoantigens are covalently immobilized on a plate, because it does not require specific functional groups of the autoantigens and any organic material can be immobilized by a radical reaction induced by photoirradiation. Here, we prepared the microarray using a very convenient method. Aqueous solutions of each autoantigen were mixed with a polymer of poly(ethylene glycol) methacrylate and a photoreactive crosslinker, and the mixtures were microspotted on a plate and dried in air. Finally, the plate was irradiated with an ultraviolet lamp to obtain immobilization. In the assay, patient serum was added to the microarray plate. Antigen-specific IgG adsorbed on the microspotted autoantigen was detected by peroxidase-conjugated anti-IgG antibody. The chemical luminescence intensities of the substrate decomposed by the peroxidase were detected with a sensitive CCD camera. All autoantigens were immobilized stably by this method and used to screen antigen-specific IgG. In addition, the plate was covered with a polydimethylsiloxane sheet containing microchannels and automated measurement was carried out.

  16. Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

    Science.gov (United States)

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-10-10

    Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

  17. A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases.

    Directory of Open Access Journals (Sweden)

    Jia-Xu Chen

    Full Text Available BACKGROUND: Food-borne helminthiases (FBHs have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA. The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI: 95.3-98.7% to 100.0% (95% CI: 100.0% in the protein microarray and from 97.7% (95% CI: 96.2-99.2% to 100.0% (95% CI: 100.0% in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3% to 92.1% (95% CI: 83.5-100.0% in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6% to 92.1% (95% CI: 83.5-100.0%. Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. CONCLUSIONS/SIGNIFICANCE: The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

  18. An automated multiplex specific IgE assay system using a photoimmobilized microarray.

    Science.gov (United States)

    Ito, Yoshihiro; Moritsugu, Nozomi; Matsue, Takahisa; Mitsukoshi, Kiyomi; Ayame, Hirohito; Okochi, Norihiko; Hattori, Hideshi; Tashiro, Hideo; Sato, Sakura; Ebisawa, Motohiro

    2012-11-15

    An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 μL of serum within a period of 20 min. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Reverse phase protein microarray technology in traumatic brain injury.

    Science.gov (United States)

    Gyorgy, Andrea B; Walker, John; Wingo, Dan; Eidelman, Ofer; Pollard, Harvey B; Molnar, Andras; Agoston, Denes V

    2010-09-30

    Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology. Published by Elsevier B.V.

  20. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  1. Calling biomarkers in milk using a protein microarray on your smartphone

    NARCIS (Netherlands)

    Ludwig, S.K.J.; Tokarski, Christian; Lang, Stefan N.; Ginkel, Van L.A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, M.W.F.

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay

  2. Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays

    DEFF Research Database (Denmark)

    Poulsen, Lena; Søe, Martin Jensen; Moller, Lisbeth Birk

    2011-01-01

    Background: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions...

  3. A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

    Science.gov (United States)

    Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

    2018-04-01

    Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional

  4. Linearization of the bradford protein assay.

    Science.gov (United States)

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  5. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    DEFF Research Database (Denmark)

    Petersen, J.; Poulsen, Lena; Petronis, S.

    2008-01-01

    is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions......DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device...

  6. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  7. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  8. Development of a Novel Protein Microarray Method for Serotyping Salmonella enterica Strains

    OpenAIRE

    Cai, H. Y.; Lu, L.; Muckle, C. A.; Prescott, J. F.; Chen, S.

    2005-01-01

    An antibody microarray assay was developed for Salmonella serotyping based on the Kauffmann-White scheme. A model (8 by 15) array was constructed using 35 antibodies for identification of 20 common Salmonella serovars and evaluated using 117 target and 73 nontarget Salmonella strains. The assay allowed complete serovar identification of 86 target strains and partial identification of 30 target strains and allowed exclusion of the 73 nontarget strains from the target serovars.

  9. Twist on protein microarrays: layering wax-patterned nitrocellulose to create customizable and separable arrays of multiplexed affinity columns.

    Science.gov (United States)

    de Lange, Victoria; Vörös, János

    2014-05-06

    We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 μL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

  10. Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

    International Nuclear Information System (INIS)

    Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.; Selvapandiyan, Angamuthu; Hewlett, Indira; Duncan, Robert; Puri, Raj K.; Nakhasi, Hira L.; Kaplan, Gerardo G.

    2007-01-01

    Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients

  11. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrome

  12. Validation of the performance of a GMO multiplex screening assay based on microarray detection

    NARCIS (Netherlands)

    Leimanis, S.; Hamels, S.; Naze, F.; Mbongolo, G.; Sneyers, M.; Hochegger, R.; Broll, H.; Roth, L.; Dallmann, K.; Micsinai, A.; Dijk, van J.P.; Kok, E.J.

    2008-01-01

    A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct

  13. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Methods and devices for protein assays

    Science.gov (United States)

    Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  15. A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray

    Directory of Open Access Journals (Sweden)

    Qiao Yingjuan

    2008-01-01

    Full Text Available Abstract Background DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. Results We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray, provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. Conclusion This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

  16. Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

    Science.gov (United States)

    Hmaïed, F; Helel, S; Le Berre, V; François, J-M; Leclercq, A; Lecuit, M; Smaoui, H; Kechrid, A; Boudabous, A; Barkallah, I

    2014-02-01

    We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Parallel force assay for protein-protein interactions.

    Science.gov (United States)

    Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  18. Parallel force assay for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Daniela Aschenbrenner

    Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  19. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    International Nuclear Information System (INIS)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Yang, Guozhen; Li, Wei; Ruan, Kangcheng

    2010-01-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 10 4 µg ml −1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody–antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays

  20. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Glial fibrillary acidic protein assay... Glial fibrillary acidic protein assay. (a) Purpose. Chemical-induced injury of the nervous system, i.e... paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate...

  1. Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

    Science.gov (United States)

    Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

    2017-12-15

    A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Miniaturized Protein Microarray with Internal Calibration as Point-of-Care Device for Diagnosis of Neonatal Sepsis

    Directory of Open Access Journals (Sweden)

    Hedvig Toth-Székély

    2012-02-01

    Full Text Available Neonatal sepsis is still a leading cause of death among newborns. Therefore a protein-microarray for point-of-care testing that simultaneously quantifies the sepsis associated serum proteins IL-6, IL-8, IL-10, TNF alpha, S-100, PCT, E-Selectin, CRP and Neopterin has been developed. The chip works with only a 4 µL patient serum sample and hence minimizes excessive blood withdrawal from newborns. The 4 µL patient samples are diluted with 36 µL assay buffer and distributed to four slides for repetitive measurements. Streptavidin coated magnetic particles that act as distinct stirring detection components are added, not only to stir the sample, but also to detect antibody antigen binding events. We demonstrate that the test is complete within 2.5 h using a single step assay. S-100 conjugated to BSA is spotted in increasing concentrations to create an internal calibration. The presented low volume protein-chip fulfills the requirements of point-of-care testing for accurate and repeatable (CV < 14% quantification of serum proteins for the diagnosis of neonatal sepsis.

  3. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

    Science.gov (United States)

    Ludwig, Susann K. J.; Tokarski, Christian; Lang, Stefan N.; van Ginkel, Leendert A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W. F.

    2015-01-01

    Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

  4. Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone.

    Directory of Open Access Journals (Sweden)

    Susann K J Ludwig

    Full Text Available Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1. Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this 'protein microarray on a smartphone'-concept for on-site testing, e.g., in food safety, environment and health monitoring.

  5. Dye-Doped Silica Nanoparticle Labels/Protein Microarray for Detection of Protein Biomarkers

    OpenAIRE

    Wu, Hong; Huo, Qisheng; Varnum, Susan; Wang, Jun; Liu, Guodong; Nie, Zimin; Liu, Jun; Lin, Yuehe

    2008-01-01

    We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of a biomarker, Interleukin-6 (IL-6), on a microarray format. The tris (2,2’-bipyridyl)ruthenium (II)chloride hexahydrate (Rubpy) dye was incorporated into silica nanoparticles using a simple one-step microemulsion synthesis. In this synthesis process, Igepal CA520 was used as ...

  6. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  7. Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

    Science.gov (United States)

    Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

    2010-01-01

    The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

  8. Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

    Directory of Open Access Journals (Sweden)

    Sascha D Braun

    Full Text Available Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL and narrow spectrum (NSBL beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13, blaGIM (2/2, blaKPC (27/27, blaNDM (5/5, blaIMP-2/4/7/8/13/14/15/16/31 (10/10, blaOXA-23 (12/13, blaOXA-40-group (7/7, blaOXA-48-group (32/33, blaOXA-51 (1/1 and blaOXA-58 (1/1. Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16, blaOXA-2 (4/4, blaOXA-9 (33/33, OXA-10 (3/3, blaOXA-51 (25/25, blaOXA-58 (2/2, CTX-M1/M15 (17/17 and blaVIM (1/1]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4% isolates, including Acinetobacter baumannii (28/28, Enterobacter spec. (5/5, Escherichia coli (4/4, Klebsiella pneumoniae (62/63, Klebsiella oxytoca (0/2, Pseudomonas aeruginosa (12/12, Citrobacter freundii (1/1 and Citrobacter

  9. Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

    Directory of Open Access Journals (Sweden)

    Elena Serena

    Full Text Available INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin and functional properties. The spontaneous contraction frequency was (0.83±0.2 Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2O(2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2O(2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

  10. Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

    Directory of Open Access Journals (Sweden)

    Kreil David P

    2008-08-01

    Full Text Available Abstract Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer. To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes

  11. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

    Science.gov (United States)

    Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

    2007-01-01

    Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

  12. Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

    Science.gov (United States)

    Hu, Wenchao; Liu, Yuting; Yan, Jun

    2014-01-01

    Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

  13. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Yansheng Liu

    Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  14. Production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas.

    Science.gov (United States)

    Kampf, Caroline; Olsson, Ingmarie; Ryberg, Urban; Sjöstedt, Evelina; Pontén, Fredrik

    2012-05-31

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts (1 2). Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) (3 4). The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins

  15. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

    Science.gov (United States)

    El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

    2015-01-30

    In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  16. The Involvement of Thaumatin-Like Proteins in Plant Food Cross-Reactivity: A Multicenter Study Using a Specific Protein Microarray

    Science.gov (United States)

    Palacín, Arantxa; Rivas, Luis A.; Gómez-Casado, Cristina; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; Bonny, José A. Cumplido; Flores, Enrique; García-Alvarez-Eire, Mar G.; García-Nuñez, Ignacio; Fernández, Francisco J.; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Torres, Maria; Losada, Susana Varela; Villalba, Mayte; Vega, Francisco; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

    2012-01-01

    Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy. PMID:22970164

  17. The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

    Directory of Open Access Journals (Sweden)

    Arantxa Palacín

    Full Text Available Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG and another against pollens but tolerant to food-plant allergens (PAG, were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%, chestnut TLP (24% and plane pollen TLP (22% proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

  18. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Science.gov (United States)

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  19. Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

    Science.gov (United States)

    Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

    2010-01-01

    We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

  20. Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

    International Nuclear Information System (INIS)

    Liu Yingshuai; Li Xuelian; Bao Shujuan; Lu Zhisong; Li Changming; Li Qing

    2013-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml −1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors. (paper)

  1. Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

    Science.gov (United States)

    Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

    2013-05-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

  2. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2015-01-01

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  3. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  4. Candidate Genes for Testicular Cancer Evaluated by In Situ Protein Expression Analyses on Tissue Microarrays

    Directory of Open Access Journals (Sweden)

    Rolf I. Skotheim

    2003-09-01

    Full Text Available By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intratubular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT.

  5. Optimization of protein samples for NMR using thermal shift assays

    International Nuclear Information System (INIS)

    Kozak, Sandra; Lercher, Lukas; Karanth, Megha N.; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane

    2016-01-01

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor"® provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  6. Optimization of protein samples for NMR using thermal shift assays

    Energy Technology Data Exchange (ETDEWEB)

    Kozak, Sandra [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Lercher, Lukas; Karanth, Megha N. [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Meijers, Rob [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Carlomagno, Teresa, E-mail: teresa.carlomagno@oci.uni-hannover.de [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Boivin, Stephane, E-mail: sboivin77@hotmail.com, E-mail: s.boivin@embl-hamburg.de [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany)

    2016-04-15

    Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor{sup ®} provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

  7. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    Directory of Open Access Journals (Sweden)

    Rom William N

    2005-08-01

    Full Text Available Abstract Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD. Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

  8. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

    Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

    2011-09-01

    A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    International Nuclear Information System (INIS)

    Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

    2005-01-01

    Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

  10. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun; Peng, Chengbin; Li, Yue

    2016-01-01

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  11. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  12. NM23 protein expression in colorectal carcinoma using TMA (tissue microarray: association with metastases and survival

    Directory of Open Access Journals (Sweden)

    Levindo Alves de Oliveira

    2010-12-01

    Full Text Available CONTEXT: NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. OBJECTIVE: To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. METHODS: One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001. NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57, vascular invasion (P = 0.85, lymphatic invasion (P = 0.41, perineural infiltration (P = 0.46, staging (P = 0.19, lymph node metastases (P = 0.08, or liver metastases (P = 0.59. Disease-free survival showed significant association (P = 0.01 with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13. CONCLUSIONS: NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma

  13. The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling

    Directory of Open Access Journals (Sweden)

    Johana A. Luna Coronell

    2018-02-01

    Full Text Available Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology. Keywords: Autoantibody tumor biomarker, Cancer immunology, Colorectal cancer, Immunomics, Protein microarray

  14. High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

    Science.gov (United States)

    Landry, James P; Fei, Yiyan; Zhu, X D

    2011-12-01

    Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

  15. Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

    Science.gov (United States)

    Palacín, Arantxa; Gómez-Casado, Cristina; Rivas, Luis A.; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; de Frutos, Consolación; Álvarez-Eire, Genoveva García; Fernández, Francisco J.; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Sirvent, Sofía; Torres, María J.; Varela-Losada, Susana; Rodríguez, Rosalía; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

    2012-01-01

    The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens. PMID:23272072

  16. Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs using microarray in a multicenter study.

    Directory of Open Access Journals (Sweden)

    Arantxa Palacín

    Full Text Available The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

  17. Identification of proteomic biomarkers of preeclampsia using protein microarray and tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Karol Charkiewicz

    2015-05-01

    Full Text Available Preeclampsia (PE is the leading cause of death of the fetus and the mother. The exact pathomechanism has not so far been clarified. PE coexists with many other diseases, but it is often difficult to explain the association between them and find a clear reason for their occurrence. There are many predictive factors, but none are highly specific in preeclampsia. The diagnosis of preeclampsia seems to be very complex, which is another argument for the exploration of knowledge on this subject. Although many of the discoveries have hitherto been made in the field of proteomics, still no single specific biomarker of preeclampsia has been discovered. Research at the genome level is important because it can help us understand the genetic predisposition of patients affected by this disease. Nevertheless, researchers have recently become more interested in the pathophysiology of PE, and they are trying to answer the question: what is the real, direct cause of preeclampsia? Thus, the discovery of a protein that is a good predictor of preeclampsia development would significantly accelerate the medical care of pregnant women, and consequently reduce the risk of occurrence of HELLP syndrome and fetal death. Apart from the predictive and diagnostic function, such a discovery would help us to better understand the pathogenesis of preeclampsia and to find in the future a medical drug to suppress this disease. In order to make a breakthrough in this field, scientists need to use the most modern methods of proteomics, which allow for the analysis of small amounts of biological material in the shortest possible time, thereby giving a lot of information about existing proteins in the sample. Such optimization allows two methods, most commonly used by researchers: tandem mass spectrometry and protein microarray technique.

  18. Challenges in the Development of Functional Assays of Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Sophie Demarche

    2012-11-01

    Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  19. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    Energy Technology Data Exchange (ETDEWEB)

    Cary,; Bruce, R [Santa Fe, NM; Stubben, Christopher J [Los Alamos, NM

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  20. Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

    Science.gov (United States)

    Mendoza, A; Torrisi, D M; Sell, S; Cady, N C; Lawrence, D A

    2016-01-21

    Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

  1. A homogeneous fluorometric assay platform based on novel synthetic proteins

    International Nuclear Information System (INIS)

    Vardar-Schara, Goenuel; Krab, Ivo M.; Yi, Guohua; Su, Wei Wen

    2007-01-01

    Novel synthetic recombinant sensor proteins have been created to detect analytes in solution, in a rapid single-step 'mix and read' noncompetitive homogeneous assay process, based on modulating the Foerster resonance energy transfer (FRET) property of the sensor proteins upon binding to their targets. The sensor proteins comprise a protein scaffold that incorporates a specific target-capturing element, sandwiched by genetic fusion between two molecules that form a FRET pair. The utility of the sensor proteins was demonstrated via three examples, for detecting an anti-biotin Fab antibody, a His-tagged recombinant protein, and an anti-FLAG peptide antibody, respectively, all done directly in solution. The diversity of sensor-target interactions that we have demonstrated in this study points to a potentially universal applicability of the biosensing concept. The possibilities for integrating a variety of target-capturing elements with a common sensor scaffold predict a broad range of practical applications

  2. Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

    Science.gov (United States)

    Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

    2017-04-28

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

  3. A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

    Science.gov (United States)

    Cleton, N B; van Maanen, K; Bergervoet, S A; Bon, N; Beck, C; Godeke, G-J; Lecollinet, S; Bowen, R; Lelli, D; Nowotny, N; Koopmans, M P G; Reusken, C B E M

    2017-12-01

    The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public

  4. A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

    1998-01-01

    To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

  5. A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

    Science.gov (United States)

    Loch, Christian M; Strickler, James E

    2012-11-01

    Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Methodological Challenges in Protein Microarray and Immunohistochemistry for the Discovery of Novel Autoantibodies in Paediatric Acute Disseminated Encephalomyelitis

    Science.gov (United States)

    Peschl, Patrick; Ramberger, Melanie; Höftberger, Romana; Jöhrer, Karin; Baumann, Matthias; Rostásy, Kevin; Reindl, Markus

    2017-01-01

    Acute disseminated encephalomyelitis (ADEM) is a rare autoimmune-mediated demyelinating disease affecting mainly children and young adults. Differentiation to multiple sclerosis is not always possible, due to overlapping clinical symptoms and recurrent and multiphasic forms. Until now, immunoglobulins reactive to myelin oligodendrocyte glycoprotein (MOG antibodies) have been found in a subset of patients with ADEM. However, there are still patients lacking autoantibodies, necessitating the identification of new autoantibodies as biomarkers in those patients. Therefore, we aimed to identify novel autoantibody targets in ADEM patients. Sixteen ADEM patients (11 seronegative, 5 seropositive for MOG antibodies) were analysed for potential new biomarkers, using a protein microarray and immunohistochemistry on rat brain tissue to identify antibodies against intracellular and surface neuronal and glial antigens. Nine candidate antigens were identified in the protein microarray analysis in at least two patients per group. Immunohistochemistry on rat brain tissue did not reveal new target antigens. Although no new autoantibody targets could be found here, future studies should aim to identify new biomarkers for therapeutic and prognostic purposes. The microarray analysis and immunohistochemistry methods used here have several limitations, which should be considered in future searches for biomarkers. PMID:28327523

  7. Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

    Science.gov (United States)

    Groome, N P

    1981-10-01

    The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

  8. Carbohydrate microarrays

    DEFF Research Database (Denmark)

    Park, Sungjin; Gildersleeve, Jeffrey C; Blixt, Klas Ola

    2012-01-01

    In the last decade, carbohydrate microarrays have been core technologies for analyzing carbohydrate-mediated recognition events in a high-throughput fashion. A number of methods have been exploited for immobilizing glycans on the solid surface in a microarray format. This microarray...... of substrate specificities of glycosyltransferases. This review covers the construction of carbohydrate microarrays, detection methods of carbohydrate microarrays and their applications in biological and biomedical research....

  9. A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

    Science.gov (United States)

    2015-07-27

    total protein in each sample was quantified by Bradford assay (Bio-Rad). RAW264.7 cell lysate preparations were boiled for 10 min with NuPAGE LDS Sample...RPMA assays , cells were harvested, washed with PBS, and then lysed in a mixture of T-PER Reagent (Thermo Scientific) and 2X Tris-Glycine SDS sample... assay (RIPA) buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche), and phosphatase inhibitors (Roche). The amount of

  10. Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development

    DEFF Research Database (Denmark)

    Petersen, Jesper; Poulsen, Lena; Birgens, Henrik

    2009-01-01

    microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More...... specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can...

  11. Miniaturized Aptamer-Based Assays for Protein Detection

    Directory of Open Access Journals (Sweden)

    Alessandro Bosco

    2016-09-01

    Full Text Available The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range assay conditions.

  12. Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial.

    Science.gov (United States)

    Iresjö, Britt-Marie; Engström, Cecilia; Lundholm, Kent

    2016-06-01

    Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short-term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty-two patients (11/group) scheduled for upper GI surgery due to malignant or benign disease received a continuous peripheral all-in-one TPN infusion (30 kcal/kg/day, 0.16 gN/kg/day) or saline infusion for 12 h prior operation. Biopsies from the rectus abdominis muscle were taken at the start of operation for isolation of muscle RNA RNA expression microarray analyses were performed with Agilent Sureprint G3, 8 × 60K arrays using one-color labeling. 447 mRNAs were differently expressed between study and control patients (P nutrition; particularly anabolic signaling S6K1 (P parenteral nutrition is effective to promote muscle protein metabolism. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  13. Potency assay design for adjuvanted recombinant proteins as malaria vaccines.

    Science.gov (United States)

    Giersing, Birgitte K; Dubovsky, Filip; Saul, Allan; Denamur, Francoise; Minor, Philip; Meade, Bruce

    2006-05-15

    Many licensed vaccines are composed of live, attenuated or inactivated whole-cell microorganisms, or they comprise purified components from whole-cell extracts or culture supernatants. For some diseases, pathology is fairly well understood, and there may be known correlates of protection that provide obvious parameters for assessment of vaccine potency. However, this is not always the case, and some effective vaccines are routinely used even though the mechanisms or correlates of protection are unknown. Some more modern vaccine approaches employ purified recombinant proteins, based on molecules that appear on the surface of the pathogen. This is one of the strategies that has been adopted in the quest to develop a malaria vaccine. Use of these parasite antigens as vaccine candidates is supported by substantial epidemiological data, and some have demonstrated the ability to elicit protective responses in animal models of malaria infection. However, there is as yet no immunological correlate of protection and no functional assays or animal models that have demonstrated the ability to predict efficacy in humans. There is little precedence for the most appropriate and practical method for assessing potency of vaccines based on these recombinant molecules for malaria vaccines. This is likely because the majority of malaria vaccine candidates have only recently entered clinical evaluation. The PATH Malaria Vaccine Initiative (MVI) convened a panel with expertise in potency assay design from industry, governmental institutions, and regulatory bodies to discuss and review the rationale, available methods, and best approaches for assessing the potency of recombinant proteins, specifically for their use as malarial vaccines. The aim of this meeting was to produce a discussion document on the practical potency assessment of recombinant protein malaria vaccines, focusing on early phase potency assay development.

  14. Antibody Microarray Analyses of Signal Transduction Protein Expression and Phosphorylation during Porcine Oocyte Maturation

    Czech Academy of Sciences Publication Activity Database

    Pelech, S.; Jelínková, Lucie; Šušor, Andrej; Zhang, H.; Shi, X.; Pavlok, Antonín; Kubelka, Michal; Kovářová, Hana

    2008-01-01

    Roč. 7, č. 7 (2008), s. 2860-2871 ISSN 1535-3893 R&D Projects: GA ČR GA204/06/1297 Grant - others:GA AV ČR(CZ) 1QS500450568 Program:1Q Institutional research plan: CEZ:AV0Z50450515 Keywords : antibody microarray * pig * frog Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.684, year: 2008

  15. cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

    Science.gov (United States)

    Quinn, Patrick; Bowers, Robert M; Zhang, Xiaoyu; Wahlund, Thomas M; Fanelli, Michael A; Olszova, Daniela; Read, Betsy A

    2006-08-01

    Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

  16. Expression of the G protein-coupled estrogen receptor (GPER in endometriosis: a tissue microarray study

    Directory of Open Access Journals (Sweden)

    Samartzis Nicolas

    2012-04-01

    Full Text Available Abstract Background The G protein-coupled estrogen receptor (GPER is thought to be involved in non-genomic estrogen responses as well as processes such as cell proliferation and migration. In this study, we analyzed GPER expression patterns from endometriosis samples and normal endometrial tissue samples and compared these expression profiles to those of the classical sex hormone receptors. Methods A tissue microarray, which included 74 samples from different types of endometriosis (27 ovarian, 19 peritoneal and 28 deep-infiltrating and 30 samples from normal endometrial tissue, was used to compare the expression levels of the GPER, estrogen receptor (ER-alpha, ER-beta and progesterone receptor (PR. The immunoreactive score (IRS was calculated separately for epithelium and stroma as the product of the staining intensity and the percentage of positive cells. The expression levels of the hormonal receptors were dichotomized into low (IRS  =6 expression groups. Results The mean epithelial IRS (+/−standard deviation, range of cytoplasmic GPER expression was 1.2 (+/−1.7, 0–4 in normal endometrium and 5.1 (+/−3.5, 0–12 in endometriosis (p p = 0.71, of ER-alpha 10.6 (+/−2.4, 3–12 and 9.8 (+/−3.0, 2–12; p = 0.26, of ER-beta 2.4 (+/−2.2; 0–8 and 5.6 (+/−2.6; 0–10; p p p p = 0.001, of ER-beta 1.8 (+/−2.0; 0–8 and 5.4 (+/−2.5; 0–10; p p���= 0.044, respectively. Cytoplasmic GPER expression was not detectable in the stroma of endometrium and endometriosis. The observed frequency of high epithelial cytoplasmic GPER expression levels was 50% (n = 30/60 in the endometriosis and none (0/30 in the normal endometrium samples (p p = 0.01, as compared to peritoneal (9/18, 50% or deep-infiltrating endometriotic lesions (7/22, 31.8%. The frequency of high stromal nuclear GPER expression levels was 100% (n = 74/74 in endometriosis and 76.7% (n = 23/30 in normal endometrium (p

  17. Nanotechnology: moving from microarrays toward nanoarrays.

    Science.gov (United States)

    Chen, Hua; Li, Jun

    2007-01-01

    Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

  18. Photo-cross-linked small-molecule microarrays as chemical genomic tools for dissecting protein-ligand interactions.

    Science.gov (United States)

    Kanoh, Naoki; Asami, Aya; Kawatani, Makoto; Honda, Kaori; Kumashiro, Saori; Takayama, Hiroshi; Simizu, Siro; Amemiya, Tomoyuki; Kondoh, Yasumitsu; Hatakeyama, Satoru; Tsuganezawa, Keiko; Utata, Rei; Tanaka, Akiko; Yokoyama, Shigeyuki; Tashiro, Hideo; Osada, Hiroyuki

    2006-12-18

    We have developed a unique photo-cross-linking approach for immobilizing a variety of small molecules in a functional-group-independent manner. Our approach depends on the reactivity of the carbene species generated from trifluoromethylaryldiazirine upon UV irradiation. It was demonstrated in model experiments that the photogenerated carbenes were able to react with every small molecule tested, and they produced multiple conjugates in most cases. It was also found in on-array immobilization experiments that various small molecules were immobilized, and the immobilized small molecules retained their ability to interact with their binding proteins. With this approach, photo-cross-linked microarrays of about 2000 natural products and drugs were constructed. This photo-cross-linked microarray format was found to be useful not merely for ligand screening but also to study the structure-activity relationship, that is, the relationship between the structural motif (or pharmacophore) found in small molecules and its binding affinity toward a protein, by taking advantage of the nonselective nature of the photo-cross-linking process.

  19. Comparison of automated microarray detection with real-time PCR assays for detection of respiratory viruses in specimens obtained from children.

    Science.gov (United States)

    Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

    2009-03-01

    Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.

  20. Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children▿

    Science.gov (United States)

    Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

    2009-01-01

    Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions. PMID:19158263

  1. Evaluation of Colorimetric Assays for Analyzing Reductively Methylated Proteins: Biases and Mechanistic Insights

    OpenAIRE

    Brady, Pamlea N.; Macnaughtan, Megan A.

    2015-01-01

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated...

  2. Detection of proteins using a colorimetric bio-barcode assay.

    Science.gov (United States)

    Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

    2007-01-01

    The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

  3. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  4. Low cut-off values increase diagnostic performance of protein S assays

    NARCIS (Netherlands)

    Mulder, Rene; ten Kate, Min Ki; Kluin-Nelemans, Hanneke C.; Mulder, Andre B.

    Conflicting data have been reported on the accuracy of protein S (PS) assays for detection of hereditary PS deficiency. In this study we assessed the diagnostic performance of two total PS antigen assays, four free PS assays and three PS activity assays in a group of 28 heterozygous carriers of

  5. Current Knowledge on Microarray Technology - An Overview

    African Journals Online (AJOL)

    Erah

    This paper reviews basics and updates of each microarray technology and serves to .... through protein microarrays. Protein microarrays also known as protein chips are nothing but grids that ... conditioned media, patient sera, plasma and urine. Clontech ... based antibody arrays) is similar to membrane-based antibody ...

  6. Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

  7. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    Science.gov (United States)

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

    Science.gov (United States)

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-10-01

    Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

  9. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    Directory of Open Access Journals (Sweden)

    Chih-Yuan eChiang

    2015-07-01

    Full Text Available Burkholderia is a diverse genus of Gram-negative bacteria that cause high mortality rate in humans and cattle. The lack of effective therapeutic treatments poses serious public health threats. Insights toward host-Burkholderia spp. interaction are critical in understanding the pathogenesis of the infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray (RPMA technology was previously proven to characterize novel biomarkers and molecular signatures associated with infectious diseases and cancers. In the present study, this technology was utilized to interrogate changes in host protein expression and post-translational phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections and of which eight proteins were selected for further validation by immunoblotting. Kinetic expression patterns of phosphorylated AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune responses. Modulating inflammatory responses by perturbing AMPK-α1, Src, and GSK3β activities may provide novel therapeutic targets for future treatments.

  10. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  11. Cell-Based Microarrays for In Vitro Toxicology

    Science.gov (United States)

    Wegener, Joachim

    2015-07-01

    DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

  12. Verification of the harmonization of human epididymis protein 4 assays.

    Science.gov (United States)

    Ferraro, Simona; Borille, Simona; Carnevale, Assunta; Frusciante, Erika; Bassani, Niccolò; Panteghini, Mauro

    2016-10-01

    Serum human epididymis protein 4 (HE4) has gained relevance as an ovarian cancer (OC) biomarker and new automated methods have replaced the first released manual EIA by tracing results to it. We verified agreement and bias of automated methods vs. EIA as well as possible effects on patients' management. One hundred and fifteen serum samples were measured by Abbott Architect i2000, Fujirebio Lumipulse G1200, Roche Modular E170, and Fujirebio EIA. Passing-Bablok regression was used to compare automated assays to EIA and agreement between methods was estimated by Lin's concordance correlation coefficient (CCC). The bias vs. EIA was estimated and compared to specifications derived from HE4 biological variation. Median (25th-75th percentiles) HE4 concentrations (pmol/L) were 84.5 (60.1-148.8) for EIA, 82.7 (50.3-153.9) for Abbott, 89.1 (55.2-154.9) for Roche, and 112.2 (67.8-194.2) for Fujirebio. Estimated regressions and agreements (95% confidence interval) were: Abbott=1.01(0.98-1.03) EIA-4.8(-7.5/-2.6), CCC=0.99(0.99-1.00); Roche=0.91(0.89-0.93) EIA+5.7(4.2/8.0), CCC=0.98(0.98-0.99); Fujirebio=1.20(1.17-1.24) EIA+ 2.4(-0.6/4.9), CCC=0.97(0.96-0.98). The average bias vs. EIA resulted within the desirable goal for Abbott [-3.3% (-6.1/-0.5)] and Roche [-0.2% (-3.0/2.5)]. However, while for Abbott the bias was constant and acceptable along the measurement concentration range, Roche bias increased up to -28% for HE4 values >250 pmol/L. Lumipulse showed a markedly positive bias [25.3% (21.8/28.8)]. Abbott and Roche assays exhibited a good comparability in the range of HE4 values around the previously recommended 140 pmol/L cut-off. For patient monitoring, however, the assay used for determining serial HE4 must not be changed as results from different systems in lower and higher concentration ranges can markedly differ.

  13. Protein assay structured on paper by using lithography

    Science.gov (United States)

    Wilhelm, E.; Nargang, T. M.; Al Bitar, W.; Waterkotte, B.; Rapp, B. E.

    2015-03-01

    There are two main challenges in producing a robust, paper-based analytical device. The first one is to create a hydrophobic barrier which unlike the commonly used wax barriers does not break if the paper is bent. The second one is the creation of the (bio-)specific sensing layer. For this proteins have to be immobilized without diminishing their activity. We solve both problems using light-based fabrication methods that enable fast, efficient manufacturing of paper-based analytical devices. The first technique relies on silanization by which we create a flexible hydrophobic barrier made of dimethoxydimethylsilane. The second technique demonstrated within this paper uses photobleaching to immobilize proteins by means of maskless projection lithography. Both techniques have been tested on a classical lithography setup using printed toner masks and on a lithography system for maskless lithography. Using these setups we could demonstrate that the proposed manufacturing techniques can be carried out at low costs. The resolution of the paper-based analytical devices obtained with static masks was lower due to the lower mask resolution. Better results were obtained using advanced lithography equipment. By doing so we demonstrated, that our technique enables fabrication of effective hydrophobic boundary layers with a thickness of only 342 μm. Furthermore we showed that flourescine-5-biotin can be immobilized on the non-structured paper and be employed for the detection of streptavidinalkaline phosphatase. By carrying out this assay on a paper-based analytical device which had been structured using the silanization technique we proofed biological compatibility of the suggested patterning technique.

  14. A DNA Microarray Analysis of Chemokine and Receptor Genes in the Rat Dental Follicle – Role of Secreted Frizzled-Related Protein-1 in Osteoclastogenesis

    Science.gov (United States)

    Liu, Dawen; Wise, Gary E.

    2007-01-01

    The dental follicle, a loose connective tissue sac that surrounds the unerupted tooth, appears to regulate the osteoclastogenesis needed for eruption; i.e., bone resorption to form an eruption pathway. Thus, DNA microarray studies were conducted to determine which chemokines and their receptors were expressed chronologically in the dental follicle, chemokines that might attract osteoclast precursors. In the rat first mandibular molar, a major burst of osteoclastogenesis occurs at day 3 with a minor burst at day 10. The results of the microarray confirmed our previous studies showing the gene expression of molecules such as CSF-1 and MCP-1 in the dental follicle cells. Other new genes also were detected, including secreted frizzled-related protein-1 (SFRP-1), which was found to be down-regulated at days 3 and 9. Using rat bone marrow cultures to conduct in vitro osteoclastogenic assays, it was demonstrated that SFRP-1 inhibited osteoclast formation in a concentration-dependent fashion. However, with increasing concentrations of SFRP-1, the number of TRAP-positive mononuclear cells increased suggesting that SFRP-1 inhibits osteoclast formation by inhibiting the fusion of mononuclear cells (osteoclast precursors). Co-culturing bone marrow mononuclear cells and dental follicle cells demonstrated that the dental follicle cells were secreting a product(s) that inhibited osteoclastogenesis, as measured by counting of TRAP-positive osteoclasts. Adding an antibody either to SFRP-1 or OPG partially restored osteoclastogenesis. Adding both anti-SFRP-1 and anti-OPG fully negated the inhibitory effect of the follicle cells upon osteoclastogenesis. These results strongly suggest that SFRP-1 and OPG, both secreted by the dental follicle cells, use different pathways to exert their inhibitory effect on osteoclastogenesis. Based on these in vitro studies of osteoclastogenesis, it is likely that the down-regulation of SFRP-1 gene expression in the dental follicle at days 3 and 9 is

  15. Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection.

    Science.gov (United States)

    Gu, Xuefang; Yan, Yuerong; Jiang, Guoqing; Adkins, Jason; Shi, Jian; Jiang, Guomin; Tian, Shu

    2014-03-01

    A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle-protein-bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL(-1) for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.

  16. Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

    Directory of Open Access Journals (Sweden)

    Shuaizheng Jia

    Full Text Available The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

  17. Microarray and Proteomic Analysis of Brassinosteroid- and Gibberellin-Regulated Gene and Protein Expression in Rice

    OpenAIRE

    Yang, Guangxiao; Komatsu, Setsuko

    2016-01-01

    Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze exp...

  18. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination.

    Science.gov (United States)

    Field, Anjalie; Field, Jeffrey

    2010-08-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.

  19. Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

    Science.gov (United States)

    Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

    2017-06-20

    Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

  20. Biomarker Identification for Prostate Cancer and Lymph Node Metastasis from Microarray Data and Protein Interaction Network Using Gene Prioritization Method

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Arias

    2012-01-01

    Full Text Available Finding a genetic disease-related gene is not a trivial task. Therefore, computational methods are needed to present clues to the biomedical community to explore genes that are more likely to be related to a specific disease as biomarker. We present biomarker identification problem using gene prioritization method called gene prioritization from microarray data based on shortest paths, extended with structural and biological properties and edge flux using voting scheme (GP-MIDAS-VXEF. The method is based on finding relevant interactions on protein interaction networks, then scoring the genes using shortest paths and topological analysis, integrating the results using a voting scheme and a biological boosting. We applied two experiments, one is prostate primary and normal samples and the other is prostate primary tumor with and without lymph nodes metastasis. We used 137 truly prostate cancer genes as benchmark. In the first experiment, GP-MIDAS-VXEF outperforms all the other state-of-the-art methods in the benchmark by retrieving the truest related genes from the candidate set in the top 50 scores found. We applied the same technique to infer the significant biomarkers in prostate cancer with lymph nodes metastasis which is not established well.

  1. A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

    Science.gov (United States)

    Mitsios, Nick; Saka, Mohamad; Krupinski, Jerzy; Pennucci, Roberta; Sanfeliu, Coral; Wang, Qiuyu; Rubio, Francisco; Gaffney, John; Kumar, Pat; Kumar, Shant; Sullivan, Matthew; Slevin, Mark

    2007-01-01

    Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents. PMID:17997827

  2. Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

    Directory of Open Access Journals (Sweden)

    Yasser Baaj

    2009-01-01

    Full Text Available We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio for “homozygous” DNA from healthy individuals. “Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.

  3. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    Science.gov (United States)

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

    Science.gov (United States)

    Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

    2017-01-01

    The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

  5. A panel of prognostic protein markers for progression in non-muscle invasive bladder cancer - a multicenter tissue microarray validation study

    DEFF Research Database (Denmark)

    Fristrup, Niels; Birkenkamp-Demtröder, Karin; Ulhøi, Benedicte Parm

    2012-01-01

    cohort of 283 patients with long-term follow-up. For validation of the results we used three independent patient cohorts with long-term follow-up from Sweden, Spain, and Taiwan. In total 649 primary NMIBC tissue-microarray specimens from patients with long-term follow-up were used. Protein expression......Bladder cancer is the fifth most common cancer in the Western world. The histopathological parameters used in the clinic cannot precisely predict the individual disease course. Bladder cancer patients are therefore monitored thoroughly for disease recurrence and progression by urine and cystoscopy...... Ta and T1 urothelial carcinomas. Transcripts from the five genes encoding these proteins were previously included in gene expression signatures for outcome prediction for non-muscle invasive bladder cancer (NMIBC). As a training-set, we used primary NMIBC tissue-microarray specimens from a Danish...

  6. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    DEFF Research Database (Denmark)

    Batchelor, Miranda; Hopkins, Katie L; Liebana, Ernesto

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum ...

  7. Recombinant HT{sub m4} gene, protein and assays

    Science.gov (United States)

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  8. Design of a covalently bonded glycosphingolipid microarray

    DEFF Research Database (Denmark)

    Arigi, Emma; Blixt, Klas Ola; Buschard, Karsten

    2012-01-01

    , the major classes of plant and fungal GSLs. In this work, a prototype "universal" GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release...... of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-G(M1), with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2...

  9. Endotoxin testing of proteins for parenteral administration using the Mono Mac 6 assay

    DEFF Research Database (Denmark)

    Moesby, Lise; Hansen, E W; Christensen, J D

    2000-01-01

    Pharmaceutical products containing proteins cause problems in testing for endotoxin and pyrogens. Many proteins interfere with the LAL test and the proteins are immunogenic in rabbits. The monocytic cell line Mono Mac 6 is an alternative assay for detection of endotoxin and other pyrogens....

  10. An HTRF® Assay for the Protein Kinase ATM.

    Science.gov (United States)

    Adams, Phillip; Clark, Jonathan; Hawdon, Simon; Hill, Jennifer; Plater, Andrew

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase that plays a key role in the regulation of DNA damage pathways and checkpoint arrest. In recent years, there has been growing interest in ATM as a therapeutic target due to its association with cancer cell survival following genotoxic stress such as radio- and chemotherapy. Large-scale targeted drug screening campaigns have been hampered, however, by technical issues associated with the production of sufficient quantities of purified ATM and the availability of a suitable high-throughput assay. Using a purified, functionally active recombinant ATM and one of its physiological substrates, p53, we have developed an in vitro FRET-based activity assay that is suitable for high-throughput drug screening.

  11. Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

    Science.gov (United States)

    Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

    2015-07-01

    Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

  12. Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

    Science.gov (United States)

    Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

    2016-07-01

    The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

  13. Analysis of protein stability and ligand interactions by thermal shift assay.

    Science.gov (United States)

    Huynh, Kathy; Partch, Carrie L

    2015-02-02

    Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

  14. Protein Microarray Chips

    OpenAIRE

    Klenkar, Goran

    2007-01-01

    Livet tas för givet av de flesta. Det finns däremot många som ägnar stora delar av sitt liv för att försöka lösa dess mysterier. En del av lösningen ligger i att förstå hur alla molekyler är sammanlänkade i det gigantiska nätverk som definierar den levande organismen. Under det senaste seklet har en hel del forskning utförts för att kartlägga dessa nätverk. Resultatet av dessa mödor kan vi se i de läkemedel som vi har idag och som har utvecklats för att bota eller åtminstone lindra olika sjuk...

  15. Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

    OpenAIRE

    Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

    2010-01-01

    We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is...

  16. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    International Nuclear Information System (INIS)

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-01-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

  17. Cysteine residue is not essential for CPM protein thermal-stability assay.

    Science.gov (United States)

    Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

    2015-05-01

    A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

  18. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

    Directory of Open Access Journals (Sweden)

    Weatherford Wendy

    2005-05-01

    Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

  19. High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

    Science.gov (United States)

    Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

    2005-05-31

    High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

  20. Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays.

    Science.gov (United States)

    Seevaratnam, Rajini; Patel, Barkha P; Hamadeh, Mazen J

    2009-06-01

    The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

  1. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

    Science.gov (United States)

    Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung

    2013-03-01

    The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Development of an enzyme-linked immunosorbent assay method to detect mustard protein in mustard seed oil

    NARCIS (Netherlands)

    Koppelman, S.J.; Vlooswijk, R.; Bottger, G.; Duijn, G. van; Schaft, P. van der; Dekker, J.; Bemgen, H. van

    2007-01-01

    An enzyme-linked immunosorbent assay for the detection of mustard protein was developed. The assay is based on a polyclonal antiserum directed against a mixture of mustard proteins raised in rabbits. The assay has a detection limit of 1.5 ppm (milligrams per kilogram) and is suitable for the

  3. A two-hybrid assay to study protein interactions within the secretory pathway.

    Directory of Open Access Journals (Sweden)

    Danielle H Dube

    Full Text Available Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H, a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.

  4. A simple coated-tube assay for alpha-foeto protein for clinical use

    International Nuclear Information System (INIS)

    Dakubu, S.; Ahene, I.S.; Foli, A.K.

    1977-01-01

    A standard method for coating plastic tubes with antiserum has been applied to coat tubes with rabbit antiserum to human alpha-foeto protein. The coated plastic tubes have been used to set up a radioimmunoassay system which is sensitive and convenient for use on the occasional clinical sample. For a successful coated-tube assay, it was found necessary to modify the final incubation mixture from what was suitable in a standard double antibody assay system. (orig.) [de

  5. Lipid Microarray Biosensor for Biotoxin Detection.

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.; Edel, Joshua B.; Meyer, Grant D.; Craighead, Harold G.

    2006-05-01

    We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates by TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4

  6. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    International Nuclear Information System (INIS)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue; Roelofs, Dick; Chen, Fajun; Zhu-Salzman, Keyan; Liang, Yuyong; Sun, Yucheng; Ge, Feng

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues. -- Highlights: • We examined the effects of Bt proteins on gene expression of Folsomia candida. • Eleven transcripts were up-regulated by Bt proteins (Cry1Ab and Cry1Ac). • Only three of the eleven transcripts were annotated. • The responses of 11 transcripts were tested on both Cry1Ab and Cry1Ac. • These transcripts did not respond to the Bt proteins in Bt-rice residues. -- Eleven potential molecular biomarkers of Folsomia candida to Cry1Ab and Cry1Ac were screened by microarray and qPCR analysis

  8. Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

    2013-03-01

    Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

  9. Protein Analytical Assays for Diagnosing, Monitoring, and Choosing Treatment for Cancer Patients

    Directory of Open Access Journals (Sweden)

    Alicia D. Powers

    2012-01-01

    Full Text Available Cancer treatment is often hindered by inadequate methods for diagnosing the disease or insufficient predictive capacity regarding therapeutic efficacy. Targeted cancer treatments, including Bcr-Abl and EGFR kinase inhibitors, have increased survival for some cancer patients but are ineffective in other patients. In addition, many patients who initially respond to targeted inhibitor therapy develop resistance during the course of treatment. Molecular analysis of cancer cells has emerged as a means to tailor treatment to particular patients. While DNA analysis can provide important diagnostic information, protein analysis is particularly valuable because proteins are more direct mediators of normal and diseased cellular processes. In this review article, we discuss current and emerging protein assays for improving cancer treatment, including trends toward assay miniaturization and measurement of protein activity.

  10. The influence of hapten density on the assay of penicilloylated proteins in fluids

    International Nuclear Information System (INIS)

    Lee, D.; Dewdney, J.M.; Edwards, R.G.

    1985-01-01

    The use of inhibition radioimmunoassays for the measurement of penicilloylated proteins in biological fluids is compromised by the dominant influence of hapten density. Precise quantitation, and therefore assessment of antigenicity and immunogenicity, cannot be achieved in the absence of knowledge of the number and distribution of haptenic groups on the protein carrier. These assays may not, therefore, be appropriate for the measurement of potential allergenic residues in food products. (Auth.)

  11. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    Science.gov (United States)

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  12. Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

    Science.gov (United States)

    Ding, Baoqing; Yuan, Yao-Wu

    2016-04-01

    The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

  13. Microarray analysis of androgen-regulated gene expression in testis: the use of the androgen-binding protein (ABP-transgenic mouse as a model

    Directory of Open Access Journals (Sweden)

    Grossman Gail

    2005-12-01

    Full Text Available Abstract Background Spermatogenesis is an androgen-dependent process, yet the molecular mechanisms of androgens' actions in testis are poorly understood. Transgenic mice overexpressing rat androgen-binding protein (ABP in their testes have reduced levels of intratesticular androgens and, as a result, show a progressive impairment of spermatogenesis. We used this model to characterize changes in global gene expression in testis in response to reduced bioavailability of androgens. Methods Total RNA was extracted from testes of 30-day old transgenic and wild-type control mice, converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays. Microarray results were confirmed by real-time reverse transcription polymerase chain reaction. Results Three-hundred-eighty-one genes (3.05% of all transcripts represented on the chips were up-regulated and 198 genes (1.59% were down-regulated by at least a factor of 2 in the androgen-deficient animals compared to controls. Genes encoding membrane proteins, intracellular signaling molecules, enzymes, proteins participating in the immune response, and those involved in cytoskeleton organization were significantly overrepresented in the up-regulated group. Among the down-regulated transcripts, those coding for extracellular proteins were overrepresented most dramatically, followed by those related to proteolysis, cell adhesion, immune response, and growth factor, cytokine, and ion channel activities. Transcripts with the greatest potential impact on cellular activities included several transcription factors, intracellular signal transducers, secreted signaling molecules and enzymes, and various cell surface molecules. Major nodes in the up-regulated network were IL-6, AGT, MYC, and A2M, those in the down-regulated network were IL-2, -4, and -10, MAPK8, SOCS1, and CREB1. Conclusion Microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional

  14. Solid-phase immunoradiometric assay for C-reactive protein using magnetisable cellulose particles

    International Nuclear Information System (INIS)

    Beer, F.C. de; Pepys, M.B.

    1982-01-01

    An immunoradiometric assay (IRMA) for C-reactive protein (CRP) was developed using magnetisable cellulose particles as the solid-phase support for anti-CRP antibodies. 125 I-labelled immunopurified anti-CRP antibody was used to quantitate the amount of CRP taken up by the solid phase. Unbound label was easily and rapidly removed by decantation after sedimenting the particles on a magnet. The assay could detect 1 μg CRP/l and had a range of up to 10 mg/l with the portion of the standard curve between 10 μg/l and 2-3 mg/l being linear. Fifty samples per hour could be processed manually from serum to CRP result with an intra-assay CV of 5.2% and an inter-assay CV of 10.0%, based on 5 replicates of 5 samples with CRP levels between 2 mg/l and 180 mg/l run in 5 separate assays. Fifty clinical samples were assayed in parallel with a standard electroimmunoassay and yielded a linear correlation coefficient (r) of 0.975 and a slope of 0.98. With its single, brief incubation step including all reagents and its simple phase separation procedure the present method may be the assay of choice when precise measurement of CRP concentrations is required rapidly. (Auth.)

  15. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    International Nuclear Information System (INIS)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-01-01

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

  16. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  17. Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

    Science.gov (United States)

    Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

    2017-01-05

    BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Emerging putative associations between non-coding RNAs and protein-coding genes in Neuropathic Pain. Added value from re-using microarray data.

    Directory of Open Access Journals (Sweden)

    Enrico Capobianco

    2016-10-01

    Full Text Available Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs. This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve injury, and studied in a rat model, using two neuronal tissues, namely dorsal root ganglion (DRG and sciatic nerve (SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes, and re-purposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parent genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to neuropathic pain. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN, and 8 in DRG, antisense RNA (31 asRNA in SN, and 12 in DRG and pseudogenes (456 in SN, 56 in DRG. In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly

  19. Lectin binding assays for in-process monitoring of sialylation in protein production.

    Science.gov (United States)

    Xu, Weiduan; Chen, Jianmin; Yamasaki, Glenn; Murphy, John E; Mei, Baisong

    2010-07-01

    Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galbeta1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(beta1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galbeta1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galbeta1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.

  20. Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

    DEFF Research Database (Denmark)

    Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

    2011-01-01

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

  1. Analysis of Nanobody-Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.

    Science.gov (United States)

    Früholz, Simone; Pimpl, Peter

    2017-01-01

    Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.

  2. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

    2013-01-01

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.

    Science.gov (United States)

    Carmichael, W W; An, J

    1999-01-01

    Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

  5. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac)

    DEFF Research Database (Denmark)

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida....... A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were...... tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues....

  6. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

    Science.gov (United States)

    Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

    2016-02-01

    To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

  7. Identification and prognostic value of anterior gradient protein 2 expression in breast cancer based on tissue microarray.

    Science.gov (United States)

    Guo, Jilong; Gong, Guohua; Zhang, Bin

    2017-07-01

    Breast cancer has attracted substantial attention as one of the major cancers causing death in women. It is crucial to find potential biomarkers of prognostic value in breast cancer. In this study, the expression pattern of anterior gradient protein 2 in breast cancer was identified based on the main molecular subgroups. Through analysis of 69 samples from the Gene Expression Omnibus database, we found that anterior gradient protein 2 expression was significantly higher in non-triple-negative breast cancer tissues compared with normal tissues and triple-negative breast cancer tissues (p gradient protein 2 expression pattern. Furthermore, we performed immunohistochemical analysis. The quantification results revealed that anterior gradient protein 2 is highly expressed in non-triple-negative breast cancer (grade 3 excluded) and grade 1 + 2 (triple-negative breast cancer excluded) tumours compared with normal tissues. Anterior gradient protein 2 was significantly highly expressed in non-triple-negative breast cancer (grade 3 excluded) and non-triple-negative breast cancer tissues compared with triple-negative breast cancer tissues (p gradient protein 2 was significantly highly expressed in grade 1 + 2 (triple-negative breast cancer excluded) and grade 1 + 2 tissues compared with grade 3 tissues (p gradient protein 2 expression was significantly associated with histologic type, histological grade, oestrogen status and progesterone status. Univariate analysis of clinicopathological variables showed that anterior gradient protein 2 expression, tumour size and lymph node status were significantly correlated with overall survival in patients with grade 1 and 2 tumours. Cox multivariate analysis revealed anterior gradient protein 2 as a putative independent indicator of unfavourable outcomes (p = 0.031). All these data clearly showed that anterior gradient protein 2 is highly expressed in breast cancer and can be regarded as a putative biomarker for

  8. No evidence of chromosome damage in children and adolescents with differentiated thyroid carcinoma after receiving {sup 131}I radiometabolic therapy, as evaluated by micronucleus assay and microarray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Federico, Giovanni; Fiore, Lisa; Massart, Francesco; Saggese, Giuseppe [Azienda Ospedaliero-Universitaria Pisana, Department of Pediatrics, Unit of Pediatric Endocrinology and Diabetes, Pisa (Italy); Boni, Giuseppe; Lazzeri, Patrizia; Mariani, Giuliano [Azienda Ospedaliero-Universitaria Pisana, Unit of Nuclear Medicine, Pisa (Italy); Fabiani, Barbara; Verola, Carmela; Scarpato, Roberto [University of Pisa, Department of Biology, Unit of Genetics, Mutagenesis and Environmental Epidemiology, Pisa (Italy); Traino, Claudio [Azienda Ospedaliero-Universitaria Pisana, Health Physics Service, Pisa (Italy)

    2008-11-15

    As {sup 131}I therapy, used to achieve ablation of thyroid gland remnant, can cause chromosome damage in cultured peripheral lymphocytes especially, we investigated whether administration of radioiodine may induce early genome damage in peripheral T lymphocytes of adolescents with differentiated thyroid carcinoma (DTC). We studied 11 patients, aged 14.8 {+-} 3.1 years, who assumed {sup 131}I (range: 1.11-4.44 GBq) to ablate thyroid remnant. A blood sample for micronucleus assay and for evaluating expression of some genes involved in the DNA repair or the apoptosis pathways was obtained from each patient 1 h before (T{sub 0}) and 24 (T{sub 1}) and 48 h (T{sub 2}) post-radioiodine administration. Compared to T{sub 0}, we did not find any difference in the number of micronucleated cells at both T{sub 1} and T{sub 2} in any subject. Nine out of 11 patients had altered expression levels in a majority of the DNA repair and apoptosis genes at T{sub 1}, which decreased at T{sub 2}. We demonstrated for the first time that peripheral cells of DTC children and adolescents who received {sup 131}I at a mean dosage of 3.50 {+-} 0.37 GBq did not show chromosome damage within 48 h from the end of radiometabolic therapy. This may be due to a prompt activation of the cell machinery that maintains the integrity of the genome to prevent harmful double-strand breaks from progressing to chromosome mutations, either by repairing the lesions or by eliminating the most seriously damaged cells via apoptosis. (orig.)

  9. A systems biology approach to the pathogenesis of obesity-related nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis.

    Science.gov (United States)

    Calvert, Valerie S; Collantes, Rochelle; Elariny, Hazem; Afendy, Arian; Baranova, Ancha; Mendoza, Michael; Goodman, Zachary; Liotta, Lance A; Petricoin, Emanuel F; Younossi, Zobair M

    2007-07-01

    Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse-phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor-mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non-progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD.

  10. Performance of scintillation proximity assay (SPA) to measure the level of VEGFR 1 protein

    International Nuclear Information System (INIS)

    LEE, So-Young; LIM, Jae-Cheong; KIM, Jin-Joo

    2014-01-01

    Scintillation proximity assay is a one of radioimmunoassay and can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Due to its simplicity and high-throughput protocol, it is broadly applicable to immunology, receptor binding, monitoring receptor-ligand interactions and enzyme reactions. Briefly, an antibody or receptor is coated on SPA beads. When a radiolabeled antigen or ligand binds to the beads, the SPA beads stimulate to emit short range electrons. The 3 H and 125 I are commonly used for radiolabeling and the produced photons are detectable with a liquid scintillation counter (LSC). A binding affinity of unlabeled ligands can be determined by competitive reaction of the radiolabeled ligands. Bevacizumab is a recombinant humanized monoclonal antibody, and can stop or delay growth of tumors by inhibiting vascular endothelial growth factor (VEGF). Bevacizumab was approved by FDA for metastatic cancer such as colorectal cancers, ovarian cancers, breast cancers and glioblastoma multiform of the brain. Recently, Dan G. duda et al. was reported that the concentration of vascular endothelial growth factor receptor-1 (VEGFR-1) in plasma may potentially be used as a negative selection biomarker. In this study, we describe a method using scintillation proximity assay to detect the amounts of VEGFR-1 protein. This method is successfully used to measure the concentration of VEGFR-1 protein in human cell extracts. In summary, a simple and sensitive assay is developed for measuring the amount of VEGFR 1 protein in cancer cell lysate using SPA beads. The antibody coating on the beads and antigen binding are achieved in one mixing step

  11. Performance of scintillation proximity assay (SPA) to measure the level of VEGFR 1 protein

    Energy Technology Data Exchange (ETDEWEB)

    LEE, So-Young; LIM, Jae-Cheong; KIM, Jin-Joo [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2014-10-15

    Scintillation proximity assay is a one of radioimmunoassay and can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Due to its simplicity and high-throughput protocol, it is broadly applicable to immunology, receptor binding, monitoring receptor-ligand interactions and enzyme reactions. Briefly, an antibody or receptor is coated on SPA beads. When a radiolabeled antigen or ligand binds to the beads, the SPA beads stimulate to emit short range electrons. The {sup 3}H and {sup 125}I are commonly used for radiolabeling and the produced photons are detectable with a liquid scintillation counter (LSC). A binding affinity of unlabeled ligands can be determined by competitive reaction of the radiolabeled ligands. Bevacizumab is a recombinant humanized monoclonal antibody, and can stop or delay growth of tumors by inhibiting vascular endothelial growth factor (VEGF). Bevacizumab was approved by FDA for metastatic cancer such as colorectal cancers, ovarian cancers, breast cancers and glioblastoma multiform of the brain. Recently, Dan G. duda et al. was reported that the concentration of vascular endothelial growth factor receptor-1 (VEGFR-1) in plasma may potentially be used as a negative selection biomarker. In this study, we describe a method using scintillation proximity assay to detect the amounts of VEGFR-1 protein. This method is successfully used to measure the concentration of VEGFR-1 protein in human cell extracts. In summary, a simple and sensitive assay is developed for measuring the amount of VEGFR 1 protein in cancer cell lysate using SPA beads. The antibody coating on the beads and antigen binding are achieved in one mixing step.

  12. Probing intracellular motor protein activity using an inducible cargo trafficking assay.

    Science.gov (United States)

    Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

    2010-10-06

    Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. A novel protease activity assay using a protease-responsive chaperone protein

    International Nuclear Information System (INIS)

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-01-01

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  14. A novel protease activity assay using a protease-responsive chaperone protein

    Energy Technology Data Exchange (ETDEWEB)

    Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  15. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Science.gov (United States)

    Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

    2014-01-01

    Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  16. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Katja Limmer

    Full Text Available Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA, parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  17. Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

    Science.gov (United States)

    Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

    2013-01-01

    Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

  18. A protein chip membrane-capture assay for botulinum neurotoxin activity

    International Nuclear Information System (INIS)

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-01-01

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC 50 s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC 50 of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays

  19. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo

    2012-10-01

    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  20. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    Milk contains many different proteins of which the larger constituents like the caseins and major whey constituents are well characterized. We have for some time been studying the structure and function of proteins associated with the milk fat globule membrane like lactadherin, MUC1/15, xanthine...... oxidoreductase along with minor whey constituents like osteopontin, EPV20 etc. The enterocyte migration rate is a key parameter in maintaining intestinal homeostasis and intestinal repair when recovering from infection or intestinal diseases like Crohns and ulcerative colitis. We developed a novel in vitro wound...... healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  1. G protein-coupled receptor internalization assays in the high-content screening format.

    Science.gov (United States)

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  2. A histidine-rich protein 2-based malaria drug sensitivity assay for field use.

    Science.gov (United States)

    Noedl, Harald; Attlmayr, Bernhard; Wernsdorfer, Walther H; Kollaritsch, Herwig; Miller, Robert S

    2004-12-01

    With the spread of antimalarial drug resistance, simple and reliable tools for the assessment of antimalarial drug resistance, particularly in endemic regions and under field conditions, have become more important than ever before. We therefore developed a histidine-rich protein 2 (HRP2)-based drug sensitivity assay for testing of fresh isolates of Plasmodium falciparum in the field. In contrast to the HRP2 laboratory assay, the field assay uses a procedure that further simplifies the handling and culturing of malaria parasites by omitting centrifugation, washing, the use of serum, and dilution with uninfected red blood cells. A total of 40 fresh Plasmodium falciparum isolates were successfully tested for their susceptibility to dihydroartemisinin, mefloquine, quinine, and chloroquine (50% inhibitory concentration [IC50] = 3.43, 61.89, 326.75, and 185.31 nM, respectively). Results very closely matched those obtained with a modified World Health Organization schizont maturation assay (R2 = 0.96, P < 0.001; mean log difference at IC50 = 0.054).

  3. Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Parida, S; Clavijo, A

    2007-05-14

    Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.

  4. Manual evaluation of tissue microarrays in a high-throughput research project: The contribution of Indian surgical pathology to the Human Protein Atlas (HPA) project.

    Science.gov (United States)

    Navani, Sanjay

    2016-04-01

    The Human Protein Atlas (HPA) program (www.proteinatlas.org) is an international program that has been set up to allow for a systematic exploration of the human proteome using antibody-based proteomics. This is accomplished by combining high-throughput generation of affinity-purified (mono-specific) antibodies with protein profiling in a multitude of tissues/cell types assembled in tissue microarrays. Twenty-six surgical pathologists over a seven-and-half year period have annotated and curated approximately sixteen million tissue images derived from immunostaining of normal and cancer tissues by approximately 23 000 antibodies. Web-based annotation software that allows for a basic and rapid evaluation of immunoreactivity in tissues has been utilized. Intensity, fraction of immunoreactive cells and subcellular localization were recorded for each given cell population. A text comment summarizing the characteristics for each antibody was added. The methods used and the challenges encountered for this exercise, the largest effort ever by a single group of surgical pathologists, are discussed. Manual annotation of digital images is an important tool that may be successfully utilized in high-throughput research projects. This is the first time an Indian private pathology laboratory has been associated with cutting-edge research internationally providing a classic example of developed and emerging nation collaboration. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

    Science.gov (United States)

    Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

    2012-09-18

    The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics

  6. A simple purification and activity assay of the coagulant protein from Moringa oleifera seed.

    Science.gov (United States)

    Ghebremichael, Kebreab A; Gunaratna, K R; Henriksson, Hongbin; Brumer, Harry; Dalhammar, Gunnel

    2005-06-01

    Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1--4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications.

  7. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins

    Science.gov (United States)

    2016-04-01

    responses. The kit also contains buffer solution , beads conjugated to antibodies, wash buffers, a positive control sample, and a standard agent...the standard solution provided with 2 of the kits (Rat Liver Injury Panel and Rat Cytokine–Chemokine) to generate the standard curve that allows for...are possibly the result of human error. Through a miscommunication , the initial sample concentration was below the recommended protein concentration

  8. Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

    Science.gov (United States)

    Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John

    2006-01-01

    The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.

  9. Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay

    International Nuclear Information System (INIS)

    Blaszczak, L.C.; Halligan, N.G.; Seitz, D.E.

    1989-01-01

    Radioiodination of p-(trimethylstannyl)penicillin V with [ 125 I]Na using a modification of the chloramine-T method is simple, high yielding, and site-specific. The structure and penicillin binding protein (PBP) affinity of p-[ 125 I]-penicillin V (IPV) are similar to penicillin G and the product can be used directly without purification in the PBP assay. Because of the high degree of stability toward autoradiolysis and equivalent PBP binding affinity, IPV can be used in place of [ 3 H]-penicillin G or [ 14 C]-penicillin G for these experiments. (author)

  10. Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

    Science.gov (United States)

    Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

    2011-10-01

    The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

  11. A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

    Science.gov (United States)

    Shibahara, Yusuke; Uesaka, Yoshihiko; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2013-01-15

    Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 μg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

  13. Comparison of Colorimetric Assays with Quantitative Amino Acid Analysis for Protein Quantification of Generalized Modules for Membrane Antigens (GMMA)

    OpenAIRE

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A.; Saul, Allan; Gerke, Christiane

    2014-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Int...

  14. New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

    OpenAIRE

    Siitari, H; Turunen, P; Schrimsher, J; Nunn, M

    1990-01-01

    A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-am...

  15. Solid-phase immunoradiometric assay for serum amyloid A protein using magnetisable cellulose particles

    International Nuclear Information System (INIS)

    De Beer, F.C.; Dyck, R.F.; Pepys, M.B.

    1982-01-01

    An immunoradiometric assay for human serum amyloid A protein (SAA) was developed using magnetisable cellulose particles as the solid phase. Rabbit antiserum to SAA was raised by immunization with SAA isolated from acute-phase serum by gel filtration in formic acid. The antiserum was rendered monospecific for SAA by solid-phase immunoabsorption with normal human serum, which contains only traces of SAA, and some was coupled covalently to the cellulose particles. Immunopurified anti-SAA antibodies were isolated from the monospecific anti-SAA serum by binding to, and elution from insolubilized acute-phase serum and were radiolabelled with 125 I. The assay was calibrated with an acute phase serum which contained 6000 times more SAA than normal sera with the lowest detectable level of SAA, and an arbitrary value of 6000 U/l was assigned to this standard. Sera were tested in the native, undenatured state and there was no increase in SAA immunoreactivity following alkali treatment or heating. The assay range was from 1-2000 U/l so that all SAA levels above 6 U/l could be measured on a single (1:6) dilution of serum. The intra- and interassay coefficients of variation were 11.7 and 15.0% respectively. Among 100 healthy normal subjects (50 male, 50 female) the median SAA level was 9 U/l, range <1-100, with 93% below 20 U/l and only 2% below the lower limit of sensitivity of the assay (1 U/l). (Auth.)

  16. An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in Escherichia coli.

    Science.gov (United States)

    Natarajan, Aravind; Haitjema, Charles H; Lee, Robert; Boock, Jason T; DeLisa, Matthew P

    2017-05-19

    The extracellular expression of recombinant proteins using laboratory strains of Escherichia coli is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (i.e., 10 10-12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of E. coli single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanisms-enabling the exploration of protein secretion in pathogenesis as well as the creation of designer E. coli strains with greatly expanded secretomes-all without the need for expensive exogenous reagents, assay instruments, or robotic automation.

  17. A practical method for extending the biuret assay to protein determination of corn-based products.

    Science.gov (United States)

    Liu, Zelong; Pan, Junhui

    2017-06-01

    A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    Science.gov (United States)

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

  19. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    Science.gov (United States)

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

  1. Magnetic levitation as a platform for competitive protein-ligand binding assays.

    Science.gov (United States)

    Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

    2012-07-17

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

  2. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts

    OpenAIRE

    Redmile-Gordon, M.A.; Armenise, E.; White, R.P.; Hirsch, P.R.; Goulding, K.W.T.

    2013-01-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colori...

  3. Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.

    Science.gov (United States)

    Mačinković, Igor S; Abughren, Mohamed; Mrkic, Ivan; Grozdanović, Milica M; Prodanović, Radivoje; Gavrović-Jankulović, Marija

    2013-12-01

    High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

    Science.gov (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka

    2013-12-13

    Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

  5. Palmtop spectrophotometer for DNA and protein measurement in micro-nanoliter assays

    International Nuclear Information System (INIS)

    Qiu Tian; Huang Guoliang; Yang Xiaoyong; Ma Li; Yang Xu

    2011-01-01

    Spectrophotometer, an important tool in life science, medicine, and analytical fields, usually uses an optical path of 10 mm or more for absorbance measurement of UV light. This corresponds to a sample consumption of ≥ 50 μL in volume and a narrow measuring range of 0.5-50 ng/μL for nucleic acid samples and 0.05-2 mg/mL for protein samples. Higher concentrations must be diluted for measurement. In this paper, we developed an advanced palmtop spectrophotometer for the measurement of both DNA and protein concentrations in micro-nanoliter assays. We constructed a fiber transmission and a fiber reflection absorbance detection scheme illuminated by either UV-LED or deuterium lamp. The sensitivity of 0.5 ng/μL and a wide measuring range of 0.5-2000 ng/μL in concentrations were obtained for DNA, and the sensitivity of 0.05 mg/mL and a wide measuring range of 0.05-100 mg/mL were also obtained for protein. However, sample consumption is only 1 μL in volume for fiber transmission detection scheme and 500 nL for fiber reflection detection scheme. The linear correlation coefficient of measured concentrations to theoretical concentrations is greater than 0.99. With the profit of this work, a miniaturized spectrophotometer with better sensitivity and wider measuring range can be produced for analytical applications.

  6. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

    Science.gov (United States)

    Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

    2013-12-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

  7. Assay of mouse-cell clones for retrovirus p30 protein by use of an automated solid-state radioimmunoassay

    International Nuclear Information System (INIS)

    Kennel, S.J.; Tnnant, R.W.

    1979-01-01

    A solid-state radioimmunoassay system has been developed that is useful for automated analysis of samples in microtiter plates. Assays for interspecies and type-specific antigenic determinants of the C-type retrovirus protein, p30, have been used to identify clones of cells producing this protein. This method allows testing of at least 1000 clones a day, making it useful for studies of frequencies of virus protein induction, defective virus production, and formation of recombinant viruses

  8. Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay.

    Science.gov (United States)

    Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C J; Wallace, John C; Polyak, Steven W

    2008-11-15

    Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme's properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.

  9. Protein-ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI).

    Science.gov (United States)

    Grøftehauge, Morten K; Hajizadeh, Nelly R; Swann, Marcus J; Pohl, Ehmke

    2015-01-01

    Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.

  10. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    International Nuclear Information System (INIS)

    Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

    2015-01-01

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs

  11. Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yi; Keegan, Gemma L., E-mail: gemmakeegan@gmail.com [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland); Stranik, Ondrej [Leibniz Institute of Photonic Technology, Department of NanoBiophotonics (Germany); Brennan-Fournet, Margaret E. [CMP-EMSE, MOC, Department of Bioelectronics, Ecole Nationale Superieure des Mines (France); McDonagh, Colette [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland)

    2015-07-15

    Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs.

  12. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA)

    International Nuclear Information System (INIS)

    Nicol, Alcina F; Pirmez, Claude; Pires, Andréa Rodrigues Cordovil; Souza, Simone R de; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Silva, José R Lapa e

    2008-01-01

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm 2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm 2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development

  13. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA).

    Science.gov (United States)

    Nicol, Alcina F; Pires, Andréa Rodrigues Cordovil; de Souza, Simone R; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Lapa e Silva, José R; Pirmez, Claude

    2008-10-07

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development.

  14. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  15. Synthesis of an 125I analog of MK-0591 and characterization of a 5-lipoxygenase activating protein binding assay

    International Nuclear Information System (INIS)

    Eggler, J.F.; Cheng, J.B.; Cooper, K.; Hanak, L.M.; Pillar, J.S.

    1994-01-01

    The 125 I analog of MK-0591,1, has been prepared for use as a radioligand for developing a 5-lipoxygenase activating protein (FLAP) binding assay. The radiosynthesis involves a two step oxidative iododestannylation-saponification procedure. A FLAP binding assay has been developed in human neutrophil membranes. The binding of 1 to human neutrophil FLAP is rapid, reversible, of high affinity, saturable and selective for FLAP inhibitors. (author)

  16. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

    Science.gov (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J

    2015-11-01

    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  17. Microarrays for Universal Detection and Identification of Phytoplasmas

    DEFF Research Database (Denmark)

    Nicolaisen, Mogens; Nyskjold, Henriette; Bertaccini, Assunta

    2013-01-01

    Detection and identification of phytoplasmas is a laborious process often involving nested PCR followed by restriction enzyme analysis and fine-resolution gel electrophoresis. To improve throughput, other methods are needed. Microarray technology offers a generic assay that can potentially detect...... and differentiate all types of phytoplasmas in one assay. The present protocol describes a microarray-based method for identification of phytoplasmas to 16Sr group level....

  18. Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

    DEFF Research Database (Denmark)

    Jensen, A T; Gaafar, A; Ismail, A

    1996-01-01

    An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...... samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter...

  19. Ligand-protein conjugated quantification assay by UV spectrophotometry in 99mTc indirect labeling

    International Nuclear Information System (INIS)

    Basualdo, Daniel A.; Rabiller, Graciela; Poch, Carolina; El Tamer, Elias A.

    2009-01-01

    Objective: Quantify IgG-HYNIC conjugated for obtaining substitution ratio and as a chemical quality control for 99m Tc labeling of this immunoglobulin. Introduction: The Operational Guidance on Hospital Radiopharmacy by IAEA states that the procedures performed in a Radiopharmacy Laboratory fall into three operational levels. At present, Nuclear Medicine Centre of 'Hospital de Clinicas' has an operational level 2b which requires the preparation of radiopharmaceuticals from approved reagent kits and radionuclide generators, and labeling of autologous blood cells. Centre's goal is to reach an operational level 3a, which allows us to compounding radiopharmaceuticals from drugs and radionuclides for diagnosis; modification to existing commercial kits; related research and development. In approach of that goal, we addressed the optimization of conjugation of proteins and peptides with S-HYNIC so as to bring about the procedures involved. In this work, was conjugated nonspecific polyclonal immunoglobulin G (IgG) with S-HYNIC. Our interest was focused in calculate how many HYNIC groups were incorporated per IgG molecule so that in later stages can be obtained a correlate with labeling efficiency. Materials and methods: A sample of IgG-HYNIC conjugate of 0.2 ml was diluted in 4 ml of benzaldehyde o-sulfonic acid (1 mg / ml, 0.1 M NaAc, pH 4.7). The reaction was incubated at room temperature overnight in darkness. As a negative control took 0.2 ml of IgG-HYNIC conjugate in 4 ml of NaAc buffer 0.1 M. 3 ml of benzaldehyde o-sulfonic acid (1 mg / ml 0.1 M NaAc, pH 4.7) was used as blank. The absorption of the hydrazone was read at 343 nm. The hydrazine concentration was calculated using a molar extinction coefficient ε (343 nm) 17000 M-1cm-1. Results: Molar substitution ratio (MSR) was calculated. The MSR indicates the number of HYNIC groups incorporated in the IgG-HYNIC conjugate determined by the spectrophotometric assay. Conclusions: In labeling with a bifunctional

  20. Impact of protein supplementation and exercise in preventing changes in gene expression profiling in woman muscles after long-term bedrest as revealed by microarray analysis.

    Science.gov (United States)

    Chopard, Angele; Lecunff, Martine; Danger, Richard; Teusan, Raluca; Jasmin, Bernard J.; Marini, Jean-Francois; Leger, Jean

    Long duration space flights have a dramatic impact on human physiology and under such a condition, skeletal muscles are known to be one of the most affected systems. A thorough understanding of the basic mechanisms leading to muscle impairment under microgravity, which causes significant loss of muscle mass as well as structural disorders, is necessary for the development of efficient space flight countermeasures. This study was conducted under the aegis of the European Space Agency (ESA), the National Aeronautics and Space Administration of the USA (NASA), the Canadian Space Agency (CSA), and the French "Centre National d'Etudes Spatiales" (CNES). It gave us the opportunity to investigate for the first time the effects of prolonged disuse (long-term bedrest, LTBR) on the transcriptome of different muscle types in healthy women (control, n=8), as well as the potential beneficial impact of protein supplementation (nutrition, n=8) and a combined resistance and aerobic exercise training program (exercise, n=8). Pre- (LTBR -8) and post- (LTBR +59) biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles from each subject. Skeletal muscle gene expression profiles were obtained using a custom made microarray containing 6681 muscle-relevant genes. 555 differentiallyexpressed and statistically-significant genes were identified in control group following 60 days of LTBR, including 348 specific for SOL, 83 specific for VL, and 124 common for the two types of muscle (p<0.05). After LTBR, both muscle types exhibited a consistent decrease in pathways involved in fatty acid oxidation, ATP synthesis, and oxidative phosphorylation (p<0.05). However, the postural SOL muscle exhibited a higher level of changes with mRNA encoding proteins involved in protein synthesis and activation of protein degradation (mainly ubiquitinproteasome components) (p<0.05). Major changes in muscle function, such as those involved in calcium signaling and muscle structure including

  1. Enzyme-linked immunosorbent assay characterization of Basal variation and heritability of systemic microfibrillar-associated protein 4

    DEFF Research Database (Denmark)

    Sækmose, Susanne Gjørup; Schlosser, Anders; Holst, René

    2013-01-01

    Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation...... in systemic MFAP4 (sMFAP4) has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing...

  2. Detection of NT-pro BNP using fluorescent protein modified by streptavidin as a label in immunochromatographic assay

    Directory of Open Access Journals (Sweden)

    Haixia Li

    2016-12-01

    Full Text Available A novel fluorescent immunochromatographic assay for the detection of NT-proBNP in human serum has been developed. Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody labeled with fluorescent protein and “sandwiched” by another monoclonal antibody immobilized on the nitrocellulose membrane, the fluorescence and concentration of analytes were measured and then calculated by fluoroanalyzer. The fluorescent protein is a fusion protein and was prepared through the application of Streptavidin gene SA, β subunit cpcB of Phycocyanin, lyase alr0617, and phycoerythrobilin synthetase gene ho1, pebA, pebB for covalent binding. It is characterized with higher stability, good solubility in water and it is not easy to quench fluorescence. Take the advantages of fluorescent protein, the immunochromatographic assay exhibited a wide linear range for NT-proBNP from 200 pg ml−1 to 26,000 pg ml−1, with a detection limit of 47 pg ml−1 under optimal conditions. Compared with chemiluminescence immunoassay (CLIA, 131 human serum samples were analyzed and the correlation coefficient of the developed immunoassay was 0.978. These results demonstrated that fluorescent immunochromatographic assay is a more rapid, sensitive, specific method and could be developed into a platform for more biomarkers determination in clinical practice. Keywords: NT-pro BNP, Fluorescent protein, Immunochromatographic assay

  3. Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

    Science.gov (United States)

    Wang, Xiao-Peng; Zhao, Xin-Huai

    2017-06-01

    Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  4. Fibre optic microarrays.

    Science.gov (United States)

    Walt, David R

    2010-01-01

    This tutorial review describes how fibre optic microarrays can be used to create a variety of sensing and measurement systems. This review covers the basics of optical fibres and arrays, the different microarray architectures, and describes a multitude of applications. Such arrays enable multiplexed sensing for a variety of analytes including nucleic acids, vapours, and biomolecules. Polymer-coated fibre arrays can be used for measuring microscopic chemical phenomena, such as corrosion and localized release of biochemicals from cells. In addition, these microarrays can serve as a substrate for fundamental studies of single molecules and single cells. The review covers topics of interest to chemists, biologists, materials scientists, and engineers.

  5. Transcriptome-Wide Identification of Differentially Expressed Genes in Solanum lycopersicon L. in Response to an Alfalfa-Protein Hydrolysate Using Microarrays

    Directory of Open Access Journals (Sweden)

    Andrea Ertani

    2017-07-01

    Full Text Available An alfalfa-based protein hydrolysate (EM has been tested in tomato (Solanum lycopersicon L. plants at two different concentrations (0.1 and 1 mL L-1 to get insight on its efficacy as biostimulant in this species and to unravel possible metabolic targets and molecular mechanisms that may shed light on its mode of action. EM was efficient in promoting the fresh biomass and content in chlorophyll and soluble sugars of tomato plants, especially when it was applied at the concentration of 1 mL L-1. This effect on plant productivity was likely related to the EM-dependent up-regulation of genes identified via microarray and involved in primary carbon and nitrogen metabolism, photosynthesis, nutrient uptake and developmental processes. EM also up-regulated a number of genes implied in the secondary metabolism that leads to the synthesis of compounds (phenols and terpenes functioning in plant development and interaction with the environment. Concomitantly, phenol content was enhanced in EM-treated plants. Several new genes have been identified in tomato as potential targets of EM action, like those involved in detoxification processes from reactive oxygen species and xenobiotic (particularly glutathione/ascorbate cycle-related and ABC transporters, and defense against abiotic and biotic stress. The model hypothesized is that elicitors present in the EM formulation like auxins, phenolics, and amino acids, may trigger a signal transduction pathway via modulation of the intracellular levels of the hormones ethylene, jasmonic acid and abscissic acid, which then further prompt the activation of a cascade events requiring the presence and activity of many kinases and transcription factors to activate stress-related genes. The genes identified suggest these kinases and transcription factors as players involved in a complex crosstalk between biotic and abiotic stress signaling pathways. We conclude that EM acts as a biostimulant in tomato due to its capacity to

  6. Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein

    DEFF Research Database (Denmark)

    Rasmussen, M; Dahl, M; Buus, S

    2014-01-01

    . We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide...... as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed....../ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer...

  7. The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

    Science.gov (United States)

    Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

    2015-10-01

    Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    Science.gov (United States)

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

  9. Enzymeaticial analysis and soluble proteins assays on radioprotective effects of cordyceps militaris

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Beong Gyu [Wonkwang Health Science College, Iksan (Korea, Republic of); Park, Joon Chul [Ansan 1 College, Ansan (Korea, Republic of)

    2001-06-01

    Effect of single pre-administration of Cordyceps militaries (Cm) extract on the survival ratio, body weight and organ weight changes and blood cell counts after whole-body {gamma}-irradiation were investigated. The single pre-administration of Cm extract at 24 hrs before {gamma}-irradiation increased the 40-day survival ration of irradiated mice from 60.1% to 71/4%. The administration of Cm extract completely prevented weight reductions of spleen and thymus produced by {gamma}-irradiation (P<0.01, P<0.05). Similar but somewhat less radioprotective effect was also found in the testis of the Cm treated mice. The administration of Cm extract retarded the reduction of both leukocyte and lymphocyte counts occured during the first 7 days and accelerated the recovery of the counts thereafter. The extract also accelerated the recovery of the erythrocyte counts occurred after the day 21th. SDS-polyacrylamide gel electrophoresis of the soluble proteins extracted from various organs did not reveal differences to any extent in all groups except in the levers of the irradiated and extract treated groups, in which some proteins were missing or less present. Also, the result of general intra and extra mycelial enzyme assays with Cm, extramycelial enzyme activity was relatively higher than the intramycelial enzyme. Cm appeared to indicate that {alpha}-amylase was the highest among the enzymes and gluosidase and chitinase were followed. Since the spleen, thymus and testis have been well known as radiosensitive organs, the protective action of Cm extract on irradiated mice may be responsible for its enhancing recovery of these organs. Although the exact mechanism in protective effect of Cm extract on irradiated mice is not clear yet, the present study is the first report regarding the Cm which was tested and found to be a potential radioprotective agent.

  10. Enzymeaticial analysis and soluble proteins assays on radioprotective effects of cordyceps militaris

    International Nuclear Information System (INIS)

    Yoo, Beong Gyu; Park, Joon Chul

    2001-01-01

    Effect of single pre-administration of Cordyceps militaries (Cm) extract on the survival ratio, body weight and organ weight changes and blood cell counts after whole-body γ-irradiation were investigated. The single pre-administration of Cm extract at 24 hrs before γ-irradiation increased the 40-day survival ration of irradiated mice from 60.1% to 71/4%. The administration of Cm extract completely prevented weight reductions of spleen and thymus produced by γ-irradiation (P<0.01, P<0.05). Similar but somewhat less radioprotective effect was also found in the testis of the Cm treated mice. The administration of Cm extract retarded the reduction of both leukocyte and lymphocyte counts occured during the first 7 days and accelerated the recovery of the counts thereafter. The extract also accelerated the recovery of the erythrocyte counts occurred after the day 21th. SDS-polyacrylamide gel electrophoresis of the soluble proteins extracted from various organs did not reveal differences to any extent in all groups except in the levers of the irradiated and extract treated groups, in which some proteins were missing or less present. Also, the result of general intra and extra mycelial enzyme assays with Cm, extramycelial enzyme activity was relatively higher than the intramycelial enzyme. Cm appeared to indicate that α-amylase was the highest among the enzymes and gluosidase and chitinase were followed. Since the spleen, thymus and testis have been well known as radiosensitive organs, the protective action of Cm extract on irradiated mice may be responsible for its enhancing recovery of these organs. Although the exact mechanism in protective effect of Cm extract on irradiated mice is not clear yet, the present study is the first report regarding the Cm which was tested and found to be a potential radioprotective agent

  11. Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

    Science.gov (United States)

    Zhang, Xu; Zhu, Qing; Tian, Tian; Zhao, Changlong; Zang, Jianye; Xue, Ting; Sun, Baolin

    2015-05-15

    It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII. This study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.

  12. DNA Microarray Technology

    Science.gov (United States)

    Skip to main content DNA Microarray Technology Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions Funding ...

  13. Clinical applications of HPLC-competitive protein binding assay for serum 25-hydroxyvitamin D

    International Nuclear Information System (INIS)

    Yang Shouli

    1989-01-01

    This report describes the clinical applications of HPLC-competitive protein binding assay for serum 25(OH) Vit D in 156 cases. Serum 25(OH) Vit D of normal human was 33.1 +- 14.8 nmol/L (13.2 +- 5.9 ng/ml), while for various diseases are as follows: rickets, 18.0 +- 9.0 nmol/L (7.2 +- 3.6 ng/ml, n = 36, P 0.1); pneumonia of children, 33.8 +- 14.8 nmol/L (13.5 +- 5.9 ng/ml, n = 10, P > 0.5); cirrhosis, 13.8 +- 11.3 nmol/L (5.5 +- 4.5 ng/ml, n = 9, P 0.2); administration of anticonvulsant (1 to 15 years), 19.0 +- 6.5 nmol/L (7.6 +- 2.6 ng/ml, n = 19, P 6 months), 15.3 +- 5.0 nmol/L (6.1 +- 2.0 ng/ml, n = 6, P 0.5); pregnant women, 39.8 +- 16.5 nmol/L (15.9 +- 6.6 ng/ml, n = 7, P > 0.1). We found it is a useful reference value for most of the above diseased state especially for differentiation between rickets and hypervitaminosis

  14. Human chorionic gonadotropin (HCG) and alphafeto protein (AFP) in sudanese pregnant women using immunoradiometric assay

    International Nuclear Information System (INIS)

    Abdalla, O. M.; Khalid, M. M.; Hassan, A.; Ali, N. I.; Khalid, A. SH.; Abdelhadi, H. A.; Khair, L. A. M.; Almahi, W. A.; Gaafar, A.; Abdalla, H.; Basheer, H.

    2004-12-01

    In this study 672 pregnant Sudanese women were involved in order to determine the reference values of human chorionic gonadotropin (HCG) and alpha feto protein (AFP). Blood samples were collected from different maternity centers in Khartoum and Omdurman maternity. Sensitive immunoradiometric assay (IRMA), method was used for measuring HCG and AFP in maternal serum. The data collected reveals that, the behavior of both AFP and HCG resemble that of the international one, where the peak concentrations of HCG are reached at 7-9 weeks of pregnancy then decrease, then staying relatively constant during the second trimester and increasing slightly towards term. The maternal serum concentration of AFP increases during pregnancy, reaching its peak during the last trimester. The concentration of AFP and HCG in maternal serum with relative couples was also compared to that of ir relative couples. Relative couples showed significant increase in maternal AFP level in the first and third trimesters (p=0.001and 0.000) respectively. The HCG concentration in both groups was not significantly different throughout the pregnancy (p>0.15). It is recommended that each laboratory establishes its own normal values. Since sudanese obstetrician depends previously on values from abroad, this study may help them to handle their patients depending on our own reference values.(Author)

  15. Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

    Science.gov (United States)

    Reid, Michael S; Le, X Chris; Zhang, Hongquan

    2018-04-27

    Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Human chorionic gonadotropin (HCG) and alphafeto protein (AFP) in Sudanese pregnant women using immunoradiometric assay

    International Nuclear Information System (INIS)

    Abdalla, O. M.; Khalid, M. M.; Hassan, A.; Ali, N. I.; Khalid, A. SH.; Abdelhadi, H. A.; Khair, L. A. M.; Almahi, W. A.; Gaafar, A.; Abdalla, H.; Basheer, H.

    2004-01-01

    In this study 672 pregnant Sudanese women were involved in order to determine the reference values of Human Chorionic Gonadotropin (HCG) and alpha feto protein (AFP). Blood samples were collected from different maternity centers in Khartoum and Omdurman maternity. Sensitive immunoradiometric assay (IRMA), method was used for measuring HCG and AFP in maternal serum. The data collected reveals that, the behavior of both AFP and HCG resemble that of the international one, where the peak concentrations of HCG are reached at 7-9 weeks of pregnancy then decrease, then staying relatively constant during the second trimester and increasing slightly towards term. The maternal serum concentration of AFP increases during pregnancy, reaching its peak during the last trimester. The concentration of AFP and HCG in maternal serum with relative couples was also compared to that of ir relative couples. Relative couples showed significant increase in maternal AFP level in the first and third trimesters (p= 0.001and 0.000) respectively. The HCG concentration in both groups was not significantly different throughout the pregnancy (p> 0.15). It is recommended that each laboratory establishes its own normal values. Since Sudanese obstetrician depends previously on values from abroad, this study may help them to handle their patients depending on our own reference values. (Authors)

  17. DNA Microarray Technology; TOPICAL

    International Nuclear Information System (INIS)

    WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

    2002-01-01

    Collaboration between Sandia National Laboratories and the University of New Mexico Biology Department resulted in the capability to train students in microarray techniques and the interpretation of data from microarray experiments. These studies provide for a better understanding of the role of stationary phase and the gene regulation involved in exit from stationary phase, which may eventually have important clinical implications. Importantly, this research trained numerous students and is the basis for three new Ph.D. projects

  18. Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

    Science.gov (United States)

    Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

    2017-05-29

    Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2018-06-01

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  20. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  1. IgG immune responses to different proteins of Helicobacter Pylori as defined by immunoblot assay

    Directory of Open Access Journals (Sweden)

    Raeiszadeh M

    2000-09-01

    Full Text Available Helicobacter pylori (H.pylori is an etiologic factor for chronic gastritis and peptic ulcers. Serological testing of H.pylori infection is common in Iran, as other parts of the world. There are geographical variations in the humoral immune response to various H. pylori strains in different parts of the worl. We studied the immunogenic proteins of H.pylori by means of an Immunoblot assay with antigens of H.pylori strains isolated in Iran. Sera of 64 patients suffering from dyspepsia were analyzed to determine antibodlies which were good marker of infection and the antibody patterns associated with peptic ulcer.54 out of 64 dyspeptic patients were infected by H. pylori based on positive culture or positive results of both rapid urease test and direct examination. 14 out of fity-four had peptic ulcers and the rest were catagoriied as patients with non-ulcer dyspepsia. Some of them had multiple erosions in the gut or deodenum. Tweny –two major bands were identified by immunoblot. Of these, IgG antibodies against 10 protients, and they produced immunoreative bands at 14, 16, 22, 26, 32 , 32, 44, 87, 92, 120 Kda. Antibody patterns were not identical in the patients. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection(sensitivity, 90% and specificity, 80% Major serological cross reactions were found at moderate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of at least one band at 14, 16, 22, 26, 32, 35Kda was the best marker of infection (sensitivity, 90% and specificity, 80%. Major serological crossreactions were found at moderate molerate molecular weight bands (50, 52, 54, 60, 66 KDa. The presence of antibodies to 120 Kda protein (Cag A and 87 Kda Protein (Vac A were not associated with the presence of peptic ulcers. These were in contradiction to results obtained across Europe and U.S but in agreement with Asian studies. However the presence of at least one band at either 32 or 35 Kda was

  2. A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

    Science.gov (United States)

    Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

    2012-01-01

    Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

  3. Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

    OpenAIRE

    Feng, Sunlian; Kendall, Lon V.; Hodzic, Emir; Wong, Scott; Lorenzana, Edward; Freet, Kimberly; Ku, Karin S.; Luciw, Paul A.; Barthold, Stephen W.; Khan, Imran H.

    2004-01-01

    Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated t...

  4. Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins

    International Nuclear Information System (INIS)

    Huang Yong; Shi Ruina; Zhong Xuefei; Wang Dan; Zhao Meiping; Li Yuanzong

    2007-01-01

    The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins

  5. Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

    Science.gov (United States)

    Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

    2016-11-19

    The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

  6. Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

    Directory of Open Access Journals (Sweden)

    Kei Kanie

    2016-11-01

    Full Text Available The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV, an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I, and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

  7. An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

    Science.gov (United States)

    Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

    2018-03-12

    In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

  8. Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

    Science.gov (United States)

    Kottom, Theodore J; Limper, Andrew H

    2011-10-01

    Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

  9. Fabrication of Biomolecule Microarrays for Cell Immobilization Using Automated Microcontact Printing.

    Science.gov (United States)

    Foncy, Julie; Estève, Aurore; Degache, Amélie; Colin, Camille; Cau, Jean Christophe; Malaquin, Laurent; Vieu, Christophe; Trévisiol, Emmanuelle

    2018-01-01

    Biomolecule microarrays are generally produced by conventional microarrayer, i.e., by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method.

  10. Clinical applications of HPLC-competitive protein binding assay for serum 25-hydroxyvitamin D

    Energy Technology Data Exchange (ETDEWEB)

    Shouli, Yang [China-Japan Friendship Hospital, Beijing, BJ (China). Inst. of Clinical Medical Science; and others

    1989-08-01

    This report describes the clinical applications of HPLC-competitive protein binding assay for serum 25(OH) Vit D in 156 cases. Serum 25(OH) Vit D of normal human was 33.1 +- 14.8 nmol/L (13.2 +- 5.9 ng/ml), while for various diseases are as follows: rickets, 18.0 +- 9.0 nmol/L (7.2 +- 3.6 ng/ml, n = 36, P < 0.005); hypervitaminosis D, 116.8 +- 31.3 nmol/L (46.7 +- 12.3 ng/ml, n = 6, P < 0.001); renal diseases of children, 25.3 +- 7.3 nmol/L (10.1 +- 2.9 ng/ml, n = 9. P > 0.1); pneumonia of children, 33.8 +- 14.8 nmol/L (13.5 +- 5.9 ng/ml, n = 10, P > 0.5); cirrhosis, 13.8 +- 11.3 nmol/L (5.5 +- 4.5 ng/ml, n = 9, P < 0.001); vasculitis, 25.5 +- 15.3 nmol/L (10.2 +- 6.1 ng/ml, n = 5, P > 0.2); administration of anticonvulsant (1 to 15 years), 19.0 +- 6.5 nmol/L (7.6 +- 2.6 ng/ml, n = 19, P < 0.001); administration of glococorticoids (> 6 months), 15.3 +- 5.0 nmol/L (6.1 +- 2.0 ng/ml, n = 6, P < 0.005); hyperthyroidism, 34.0 +- 23.5 nmol/L (13.6 +- 9.4 ng/ml, n = 13, P > 0.5); pregnant women, 39.8 +- 16.5 nmol/L (15.9 +- 6.6 ng/ml, n = 7, P > 0.1). We found it is a useful reference value for most of the above diseased state especially for differentiation between rickets and hypervitaminosis.

  11. Definition of purified enzyme-linked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis.

    Science.gov (United States)

    Cho, Yun Sang; Lee, Sang-Eun; Ko, Young Joon; Cho, Donghee; Lee, Hyang Shim; Hwang, Inyeong; Nam, Hyangmi; Heo, Eunjung; Kim, Jong Man; Jung, Sukchan

    2009-01-01

    Enzyme-linked immunosorbent assay (ELISA) has been developed as the ancillary diagnosis of bovine tuberculosis at ante-mortem to overcome the disadvantages of intradermal skin test. In this study, the antigenic proteins were purified, applied to bTB ELISA, and identified through proteomic analysis. Culture filtrate protein of Mycobacterium bovis was fractionated by MonoQ column chromatography, and examined the antigenicity by immunoblotting. The antigenic 20 kDa protein was in-gel digested and identified the antigenome by LTQ mass spectrometer and peptide match fingerprinting, which were MPB64, MPB70, MPB83, Fas, Smc, Nrp, RpoC, Transposase, LeuA, and MtbE. The 20 kDa protein exhibited the highest antigenicity to bTB positive cattle in ELISA and would be useful for bTB serological diagnosis.

  12. Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

    Science.gov (United States)

    Ashok, Yashwanth; Jaakola, Veli-Pekka

    2016-01-01

    Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

  13. Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

    Science.gov (United States)

    Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

    2016-01-01

    Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies. © 2016 Elsevier Inc. All rights reserved.

  14. Setting up a Bioluminescence Resonance Energy Transfer high throughput screening assay to search for protein/protein interaction inhibitors in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Cyril eCouturier

    2012-09-01

    Full Text Available Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed interactome. Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET technique was primarily developed to allow the dynamic monitoring of protein-protein interactions in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of protein-protein interactions and here is described why and how to set up and optimize a High Throughput Screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence substrate concentration, number of cells and medium composition used on the Z’ factor, and expected interferences for colored or fluorescent compounds.

  15. Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

    Directory of Open Access Journals (Sweden)

    Pace Umberto

    2006-05-01

    Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate

  16. A gene-protein assay for human epidermal growth factor receptor 2 (HER2: brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17 in formalin-fixed, paraffin-embedded breast cancer tissue sections

    Directory of Open Access Journals (Sweden)

    Nitta Hiroaki

    2012-05-01

    Full Text Available Abstract Background The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH or immunohistochemistry (IHC, respectively. Our objective was to combine the US Food and Drug Administration (FDA-approved HER2 & chromosome 17 centromere (CEN17 brightfield ISH (BISH and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. Methods The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative] and Calu-3 [HER2 positive (amplified gene, protein positive]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5 and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. Results HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein

  17. Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

    Science.gov (United States)

    Rasmussen, M; Dahl, M; Buus, S; Djurisic, S; Ohlsson, J; Hviid, T V F

    2014-08-01

    The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use. © 2014 John Wiley & Sons A/S. Published by John Wiley

  18. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

    Science.gov (United States)

    Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

    2015-05-01

    Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

  19. Protein A磁珠检测朊病毒方法的建立%The foundation of Protein A magnetism to detect Prion assay

    Institute of Scientific and Technical Information of China (English)

    李奇; 杨阳; 吕鹏; 宋博翠; 白宗瑞; 陈志宝

    2009-01-01

    建立Protein A磁珠法检测朊病毒,将朊病毒抗体IH2包被后的Protein A磁珠与蛋白酶K消化过的攻毒小鼠脑组织匀浆液混合,经免疫沉淀缓冲液温和洗脱杂蛋白,再经0.2 M甘氨酸将目的蛋白洗脱并且利用聚丙酰胺凝胶电泳检测.通过聚丙酰胺凝胶电泳检测出25 KD的目的蛋白带,经与正常未消化的朊蛋白条带相比较,证实检测出朊病毒.Protein A磁珠具有高效特异的与抗体IgG结合的特点,与多克隆抗体混合后可有效结合IgG.该方法较免疫组织化学和Western-blot技术简便快速,且磁珠可以回收再使用,使成本低廉.%Objective The country need the fast and effective detection for Prion asthe high risk country where Scrapie and BSE may bresk out. The common assay of Western-Blot and Immunohistochemistry consume a long time and the associated experience is needed. Protein A magnetism has the feature of binding IgG differential.h could bind the IgG adequately,when mixing with polyclonal antibody.After intermixing with brain tissue protein digested by protein kinase, Prion could easily elute from Protein A magnetism.Interest protein band is 25KD through SDS-PAGE. When definiting the molecular weight of Priori,the result of detection is educed by SDS-PAGE electrophoresis. The assay is more handier and faster than common assay. The magnetism can reclaim and reuse,which reduce the cost.The assay of detection is a innovation and many branches could generally applicated it.

  20. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

    Science.gov (United States)

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

    2014-12-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

  1. Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes.

    Science.gov (United States)

    Debaize, Lydie; Jakobczyk, Hélène; Rio, Anne-Gaëlle; Gandemer, Virginie; Troadec, Marie-Bérengère

    2017-01-01

    Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

  2. Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens.

    Science.gov (United States)

    Rose, Alexandra; Funk, Deborah; Neiger, Reto

    2016-04-01

    To compare total protein (TP) concentrations in canine pleural and abdominal fluid specimens as measured by refractometry and biuret assay. Diagnostic test evaluation. Data regarding 92 pleural and 148 abdominal fluid specimens from dogs with various diseases. TP concentrations in fluid specimens as measured by refractometry and biuret assay were recorded. Strength of association between sets of measurements was assessed by Spearman rank correlations and Bland-Altman plots. Optimal concentration cutoff for diagnostic discrimination between exudate and nonexudate was identified by construction of receiver operating characteristic curves. Median TP concentration in pleural fluid specimens was 2.7 g/dL (range, 0.3 to 4.8 g/dL) for refractometry and 2.9 g/dL (range, 0.7 to 5.8 g/dL) for biuret assay. Median TP concentration in abdominal fluid specimens was 3.5 g/dL (range, 0.1 to 6.0 g/dL) for refractometry and 3.5 g/dL (range, 0.6 to 5.7 g/dL) for biuret assay. Correlation was significant between refractometric and biuret results for pleural (ρ = 0.921) and abdominal (ρ = 0.908) fluid. Bland-Altman plots revealed bias of -0.18 g/dL for pleural fluid and -0.03 g/dL for abdominal fluid for refractometry versus biuret assay. With a TP concentration of ≥ 3 g/dL used to distinguish exudate from nonexudate, sensitivity of refractometry was 77% for pleural fluid and 80% for abdominal fluid. Specificity was 100% and 94%, respectively. Refractometry yielded acceptable results for measurement of TP concentration in canine pleural and abdominal fluid specimens, providing a more rapid and convenient method than biuret assay.

  3. Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe

    DEFF Research Database (Denmark)

    Alava, M.A.; Gonzalez-Ramon, N.; Heegaard, Peter M. H.

    1997-01-01

    The pattern of plasma proteins changes greatly following infection, inflammation or tissue injury. The concentration of some proteins referred to as acute phase proteins (APPs) significantly increases within hours or days after the onset of these processes. In contrast, the concentration of other...... during the inflammation. In addition to CRP and Hp, a serum alpha(2)-globulin was observed to be the major acute phase (MAP) protein in pigs. Pig-MAP is a new mammalian plasma protein, which is the pig counterpart of a recently cloned human serum protein denominated PK-120 or MRP. Pig-MAP shows promise...... for the presence of infectious, inflammatory and pathological lesions in animals. The ability to monitor the APP concentration in serum of pigs will improve the quality and safety of the meat produced as well as provide important diagnostic information for animal health and welfare. The serum concentration of APP...

  4. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration

    NARCIS (Netherlands)

    Heijboer, Annemieke C.; Blankenstein, Marinus A.; Kema, Ido P.; Buijs, Madelon M.

    2012-01-01

    Recent recognition of its broad pathophysiological importance has triggered an increased interest in 25-hydroxyvitamin D [25(OH)D]. By consequence, throughput in 25(OH)D testing has become an issue for clinical laboratories, and several automated assays for measurement of 25(OH)D are now available.

  5. Accuracy of 6 Routine 25-Hydroxyvitamin D Assays: Influence of Vitamin D Binding Protein Concentration

    NARCIS (Netherlands)

    Heijboer, A.C.; Blankenstein, M.A.; Kema, I.P.; Buijs, M.M.

    2012-01-01

    BACKGROUND: Recent recognition of its broad pathophysiological importance has triggered an increased interest in 25-hydroxyvitamin D [25(OH)D]. By consequence, throughput in 25(OH)D testing has become an issue for clinical laboratories, and several automated assays for measurement of 25(OH)D are now

  6. A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

    DEFF Research Database (Denmark)

    Robey, R W; Honjo, Y; van de Laar, A

    2001-01-01

    The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compo...

  7. LINEARIZATION OF THE BRADFORD PROTEIN ASSAY TO APPLICATION IN COW MILK PROTEINS QUANTIFICATION BY UV-Vis SPECTROPHOTOMETRY METHOD

    Directory of Open Access Journals (Sweden)

    Alessa Siqueira de Oliveira dos Santos

    2015-01-01

    Full Text Available Reliable methods for determination and quantification of total protein in food are essential information to ensure quality and safety of food trade. The objective of this study was to evaluate the linearity of calibration curves obtained from different proteins (blood serum albumin-BSA, α-LA, β-LG, caseins (CN: αs, β and κ-CAS with the reagent of Bradford. Comercial UHT skimmed bovine milk was analyzed for the determination of total protein using the Bradford method by reading at 595 nm. The determination of the concentrations of total milk protein was achieved by linear regression. The Bradford method showed a high sensitivity for the determination of total proteins in bovine milk dilution 1:25 to values closer to those obtained by the Kjeldahl method. The results showed that the calibration curve of standard proteins β-CN and BSA obtained better linearity with less variation in the absorbance measurements for the determination of total protein of milk.

  8. Linearization of the Bradford protein assay to application in cow milk proteins quantification by UV-Vis spectrophotometry method.

    OpenAIRE

    SANTOS, A. S. de O. dos; COSTA, F. F.; ESTEVES, W. T.; BRITO, M. A. V. P. e; FURTADO, M. A. M.; MARTINS, M. F.

    2015-01-01

    Reliable methods for determination and quantification of total protein in food are essential information to ensure quality and safety of food trade. The objective of this study was to evaluate the linearity of calibration curves obtained from different proteins (blood serum albumin-BSA, α-LA, β-LG, αs, β and κ-CAS) with the reagent of Bradford. Comercial UHT skimmed bovine milk was analyzed for the determination of total protein using the Bradford method by reading at 595 nm. The determinatio...

  9. A Rational Approach for Discovering and Validating Cancer Markers in Very Small Samples Using Mass Spectrometry and ELISA Microarrays

    Directory of Open Access Journals (Sweden)

    Richard C. Zangar

    2004-01-01

    Full Text Available Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF or early-stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result, a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.

  10. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  11. Carbohydrate Microarrays in Plant Science

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik; Pedersen, H.L.; Vidal-Melgosa, S.

    2012-01-01

    Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important...... industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high...... for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities....

  12. Probing intracellular motor protein activity using an inducible cargo trafficking assay

    NARCIS (Netherlands)

    L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

    2010-01-01

    textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

  13. Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Welner, Simon; Trier, Nicole Hartwig; Morten Frisch, Morten

    2013-01-01

    Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation...

  14. Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

    Science.gov (United States)

    Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

    2016-01-14

    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

  15. Evaluation of five commercially available assays and measurement of serum total protein concentration via refractometry for the diagnosis of failure of passive transfer of immunity in foals.

    Science.gov (United States)

    Davis, Rachel; Giguère, Steeve

    2005-11-15

    To determine and compare sensitivity, specificity, accuracy, and predictive values of measurement of serum total protein concentration by refractometry as well as 5 commercially available kits for the diagnosis of failure of passive transfer (FPT) of immunity in foals. Prospective study. 65 foals with various medical problems and 35 clinically normal foals. IgG concentration in serum was assessed by use of zinc sulfate turbidity (assay C), glutaraldehyde coagulation (assay D), 2 semiquantitative immunoassays (assays F and G), and a quantitative immunoassay (assay H). Serum total protein concentration was assessed by refractometry. Radial immunodiffusion (assays A and B) was used as the reference method. For detection of IgG or = 6.0 g/dL indicated adequate IgG concentrations. Most assays were adequate as initial screening tests. However, their use as a definitive test would result in unnecessary treatment of foals with adequate IgG concentrations.

  16. Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

    Science.gov (United States)

    Akimoto, Masaru; Hokazono, Eisaku; Ota, Eri; Tateishi, Takiko; Kayamori, Yuzo

    2016-01-01

    Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein. © The Author(s) 2015.

  17. Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers.

    Science.gov (United States)

    Kasari, Marje; Padrik, Peeter; Vaasa, Angela; Saar, Kristi; Leppik, Krista; Soplepmann, Jaan; Uri, Asko

    2012-03-15

    A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Expression of a hantavirus N protein and its efficacy as antigen in immune assays

    Directory of Open Access Journals (Sweden)

    L.T.M. Figueiredo

    2008-07-01

    Full Text Available Hantavirus cardiopulmonary syndrome (HCPS has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

  20. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    Science.gov (United States)

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay

    Directory of Open Access Journals (Sweden)

    Tommy Baumann

    2013-10-01

    Full Text Available We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90 also accumulate in the T-cell uropod. Using the in situ proximity ligation assay (PLA we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET. We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the uropod upon cell stimulation.

  2. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...... for malaria but free of leishmaniasis was negative in both assays....

  3. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.

    Science.gov (United States)

    Hill, Ryan C; Oman, Trent J; Shan, Guomin; Schafer, Barry; Eble, Julie; Chen, Cynthia

    2015-08-26

    Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.

  4. Laser-Induced Breakdown Spectroscopy Based Protein Assay for Cereal Samples.

    Science.gov (United States)

    Sezer, Banu; Bilge, Gonca; Boyaci, Ismail Hakki

    2016-12-14

    Protein content is an important quality parameter in terms of price, nutritional value, and labeling of various cereal samples. However, conventional analysis methods, namely, Kjeldahl and Dumas, have major drawbacks such as long analysis time, titration mistakes, and carrier gas dependence with high purity. For this reason, there is an urgent need for rapid, reliable, and environmentally friendly technologies for protein analysis. The present study aims to develop a new method for protein analysis in wheat flour and whole meal by using laser-induced breakdown spectroscopy (LIBS), which is a multielemental, fast, and simple spectroscopic method. Unlike the Kjeldahl and Dumas methods, it has potential to analyze a high number of samples in considerably short time. In the study, nitrogen peaks in LIBS spectra of wheat flour and whole meal samples with different protein contents were correlated with results of the standard Dumas method with the aid of chemometric methods. A calibration graph showed good linearity with the protein content between 7.9 and 20.9% and a 0.992 coefficient of determination (R 2 ). The limit of detection was calculated as 0.26%. The results indicated that LIBS is a promising and reliable method with its high sensitivity for routine protein analysis in wheat flour and whole meal samples.

  5. Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

    Science.gov (United States)

    Dinca, V; Ranella, A; Farsari, M; Kafetzopoulos, D; Dinescu, M; Popescu, A; Fotakis, C

    2008-10-01

    The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

  6. Detection of Antibodies to U.S. Isolates of Avian Pneumovirus by a Recombinant Nucleocapsid Protein-Based Sandwich Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Gulati, Baldev R.; Munir, Shirin; Patnayak, Devi P.; Goyal, Sagar M.; Kapur, Vivek

    2001-01-01

    The nucleocapsid (N) protein of subgroup C (United States-specific) avian pneumovirus (APV/US) was expressed in Escherichia coli, and antibodies to the recombinant N protein were shown to specifically recognize the ≈47-kDa N protein of APV/US by Western immunoblot analysis. The recombinant APV/US N protein was used in a sandwich-capture enzyme-linked immunosorbent assay (ELISA), and the resulting assay was found to be more sensitive and specific than the routine indirect ELISA for the detecti...

  7. Detection of anti-spermatozoal antibodies by a 125I-protein-A radioimmunological assay

    International Nuclear Information System (INIS)

    Czuppon, A.B.

    1985-01-01

    A newly developed solid-phase radioimmunobinding assay (RIBA) using detergent-solubilized sperm antigen has been used to evaluate anti-sperm antibodies. Results showed that none of the fertile females and males were positive in the RIBA, whereas approximately 6% of the females and 3% of the males with unexplained infertility were positive. These results are similar to those obtained using a chemically synthesized spermatozoal decapeptide antigen. The RIBA is simple to perform, requires no vital or intact spermatozoa, and large numbers of sera (up to 400) can be processed in one day with a total incubation time of 90 min. (Auth.)

  8. Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay.

    Directory of Open Access Journals (Sweden)

    Kazunori Sano

    Full Text Available INTRODUCTION: The definitive diagnosis of genetic prion diseases (gPrD requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau protein in the cerebrospinal fluid (CSF, but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS. We recently developed a new in vitro amplification technology, designated "real-time quaking-induced conversion (RT-QUIC", to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined. METHOD/PRINCIPAL FINDINGS: 56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N, and 24 cases of genetic CJD (gCJD, comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%, FFI (100%, gCJD E200K (87%, and gCJD V203I (100%. On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%, FFI (0%, gCJD E200K (73%, and gCJD V203I (67%. Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI. CONCLUSION/SIGNIFICANCE: RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

  9. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro CardTM

    OpenAIRE

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro cardTM has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro cardTM using the sensitive proximity extension assay (PEA). Among 92 proteins in ...

  10. Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

    Science.gov (United States)

    Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

    2006-01-01

    Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

  11. Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

    Directory of Open Access Journals (Sweden)

    Manish Biyani

    2015-07-01

    Full Text Available Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density, ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.

  12. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    OpenAIRE

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Brad...

  13. Proximity probing assays for simultaneous visualization of protein complexes in situ

    DEFF Research Database (Denmark)

    Moreira, José; Thorsen, Stine Buch; Brünner, Nils

    2013-01-01

    EVALUATION OF: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print). Techniques for in situ detection and quantification of proteins...... in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer...

  14. 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

    Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory...... disease. To investigate ECP release from in vitro activated human eosinophils and to study the involvement of eosinophils in health and disease, we have developed a sensitive and specific enzyme immunoassay. ECP was purified from normal human peripheral blood eosinophils and polyclonal antibodies to ECP...

  15. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  16. Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

    Science.gov (United States)

    Hodgson, Derek J; Aubin, Yves

    2017-05-10

    A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  17. Identification of Acute Phase Proteins and Assays Applicable in Nondomesticated Mammals

    DEFF Research Database (Denmark)

    Bertelsen, M. F.; Kjelgaard-Hansen, M.; Grøndahl, C.

    2009-01-01

    potential APPs-serum amyloid A (SAA), C-reactive protein (CRP), and haptoglobin (Hp)-was evaluated in eight species. For SAA, a turbidimetric immunoassay (TIA) demonstrated significant detective abilities ill the Asian elephant (Elaphas maximus), impala (Aepyceros melampus), musk ox (Ovibos moschatus...

  18. Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations

    Energy Technology Data Exchange (ETDEWEB)

    Nielson, Carrie M.; Jones, Kerry S.; Chun, Rene F.; Jacobs, Jon M.; Wang, Ying; Hewison, Martin; Adams, John S.; Swanson, Christine M.; Lee, Christine G.; Vanderschueren, Dirk; Pauwels, Steven; Prentice, Ann; Smith, Richard D.; Shi, Tujin; Gao, Yuqian; Schepmoes, Athena A.; Zmuda, Joseph M.; Lapidus, Jodi; Cauley, Jane A.; Bouillon, Roger; Schoenmakers, Inez; Orwoll, Eric S.

    2016-05-01

    Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitaminDbinding protein (DBP) used to calculate f25OHD.

  19. Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

    Science.gov (United States)

    Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

    2016-07-01

    Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. © 2016 Institute of Food Technologists®

  20. Evaluation of a microarray-based genotyping assay for the rapid detection of cytochrome P450 2C19 *2 and *3 polymorphisms from whole blood using nanoparticle probes.

    Science.gov (United States)

    Buchan, Blake W; Peterson, Jess F; Cogbill, Christopher H; Anderson, Dennis K; Ledford, Joellen S; White, Mary N; Quigley, Neil B; Jannetto, Paul J; Ledeboer, Nathan A

    2011-10-01

    Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

  1. Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

    DEFF Research Database (Denmark)

    Dufva, Martin; Petersen, Jesper; Poulsen, Lena

    2009-01-01

    DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to f...

  2. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    Science.gov (United States)

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  3. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  4. On-chip mitochondrial assay microfluidic devices and protein nanopore/nanotube hybrid transistor

    Science.gov (United States)

    Lim, Taesun

    Tremendous efforts to understand the cause, mechanism of development and the way to treat various diseases as well as an early diagnosis have been made so far and people are still working hardly on these researches. Even now, countless people are suffering from diseases such as Alzhemer's disease, Parkinson's disease, diabetes and cancer without knowing clues to cure their diseases completely. Generally speaking, we still have a long way to go through to comprehensively figure out these our long-lasting homeworks. One of possible solutions is to merge current advanced technology and science together to find a powerful synergetic effect for a specific purpose that can be tailored depending on user's need. Here this research tried to put nanotechnology and biological science together to find a way to resolve current challenges by developing a new generation of the analytical sensing device. Mitochondrial functions and biological roles in regulating life and death control will be discussed indicating mitochondrion is a crucial organism to monitor to obtain important information regarding degenerative diseases and aging process. On-chip mitochondrial functional assay microsensor that could facilitate the mitochondrial evaluation will be extensively demonstrated and discussed in both technical and biological perspectives. The novel fusion technological approach will be demonstrated by combining artificial cell membrane with carbon nanotube electronics to interrogate interactions between biomolecules and electronic circuitries. In addition, molecular dynamics at the cell membrane could be investigated closely which can help understand the cell-cell communication and the regulation of ion transport.

  5. A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

    DEFF Research Database (Denmark)

    Zhang, Zhao; Hejesen, Christian; Kjelstrup, Michael Brøndum

    2014-01-01

    . The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies......Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers...

  6. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    Science.gov (United States)

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  7. Exploring physical and chemical factors influencing the properties of recombinant prion protein and the real-time quaking-induced conversion (RT-QuIC) assay.

    Science.gov (United States)

    Cheng, Keding; Sloan, Angela; Avery, Kristen M; Coulthart, Michael; Carpenter, Michael; Knox, J David

    2014-01-01

    Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.

  8. Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor.

    Science.gov (United States)

    Lv, Xiaoyi; Chen, Liangliang; Zhang, Hongyan; Mo, Jiaqing; Zhong, Furu; Lv, Changwu; Ma, Ji; Jia, Zhenhong

    2013-01-15

    A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. ELISA-BASE: an integrated bioinformatics tool for analyzing and tracking ELISA microarray data

    OpenAIRE

    White, Amanda M.; Collett, James R.; Seurynck-Servoss, Shannon L.; Daly, Don S.; Zangar, Richard C.

    2009-01-01

    Summary:ELISA-BASE is an open source database for capturing, organizing and analyzing enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Software Environment (BASE) database system.

  10. AKT1, LKB1, and YAP1 revealed as MYC interactors with NanoLuc-based protein-fragment complementation assay. | Office of Cancer Genomics

    Science.gov (United States)

    The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. Here we report the development of a NanoLuc®-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells.

  11. Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

    Science.gov (United States)

    Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J Stephen

    2012-09-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

  12. An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

    DEFF Research Database (Denmark)

    Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard

    2008-01-01

    characterized and validated for use in sandwich enzyme-linked immunosorbent assay (ELISA). Based on two of these, we established an ELISA that allows for measurements of mouse SP-D in various body fluids. The final ELISA was optimized and calibrated with a standard of purified recombinant mouse SP-D, which......Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of other...... innate immune cells, such as macrophages and neutrophils. It also modulates the adaptive immune response by interacting with antigen-presenting cells and T cells. Monoclonal anti-mouse-SP-D antibodies were raised from SP-D deficient mice using recombinant SP-D as antigen. Ten monoclonal antibodies were...

  13. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

    Directory of Open Access Journals (Sweden)

    Caroline E. Moore

    2016-03-01

    Full Text Available Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1. A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

  14. Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

    Directory of Open Access Journals (Sweden)

    Sorette M

    2004-12-01

    Full Text Available Abstract Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

  15. Enzyme-linked immunosorbent assay characterization of basal variation and heritability of systemic microfibrillar-associated protein 4.

    Directory of Open Access Journals (Sweden)

    Susanne Gjørup Sækmose

    Full Text Available BACKGROUND: Microfibrillar-associated protein 4 (MFAP4 is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM, and variation in systemic MFAP4 (sMFAP4 has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation. METHODS: The sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years by mixed-effect linear regression modeling. RESULTS: The practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml. The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h(2 = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors. CONCLUSIONS: The described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively

  16. Simple assay method for proteins carrying sexual hormones (PTHS); values in men, women, and during pregnancy

    International Nuclear Information System (INIS)

    Tafurt, C.A.; Estrada, R. de.

    1977-01-01

    Starting from the fact that the binding forces between steroid hormones and their carrier proteins are similar to those between antigens and antibodies, the paper describes PTHS determination by a dilution method analogous to antiserum labelling for radioimmunoassay. The method consists of the following steps: 1) Plasma dilution, 2) incubation of the solutions with 20,000 dpm 1,2 3 H testosterone, 3) separation of the tracer fraction bound to PTHS by precipitation with ammonium sulfate, 4) centrifugation and measurement of the supernatant, 5) presentation of the findings in a graphical system with the bound steroid fraction, referred to the free steroid (U/L) as the ordinate and the plasma dilutions as the abscissa. The values represent the label in 50% of the sites. The method offers the highest sensitivity, i.e. the steepest parts of the dilution curves where 50% of the binding sites are located. The method also dispenses with tedious processes such as dialysis. The following PTHS values were obtained: 1/5 in men, 1/93 in women, and 1/360 in pregnant women. There were no cross-reactions. (AJ) [de

  17. DNA microarrays : a molecular cloning manual

    National Research Council Canada - National Science Library

    Sambrook, Joseph; Bowtell, David

    2002-01-01

    .... DNA Microarrays provides authoritative, detailed instruction on the design, construction, and applications of microarrays, as well as comprehensive descriptions of the software tools and strategies...

  18. Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology.

    Science.gov (United States)

    Wu, Yun; Ma, Jie; Wang, Yipeng; Zhang, Yonghong; Hou, Yongjin; Zhang, Chunming; Sun, Huanqin; Sun, Jianping; Wang, Zikang; Li, Ning

    2018-04-06

    Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood. Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 μg/l and GP73 0.482 μg/l. The functional sensitivity was AFP 0.010 μg/l and GP73 0.640 μg/l. The range of detection was AFP 0.01-50 μg/l and GP73 0.64-100 μg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) μg/l and GP73 was 42.60 (39.10, 57.40) μg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) μg/l and GP73 was 136.10 (92.10, 261.70) μg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) μg/l and GP73 was 186.25 (96.73, 262.03) μg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001). The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B

  19. A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

    Science.gov (United States)

    Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

    2017-07-01

    Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

    Science.gov (United States)

    Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

    2015-05-01

    In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  1. Characterization of equine vitamin D-binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease.

    Science.gov (United States)

    Pihl, Tina H; Jacobsen, Stine; Olsen, Dorthe T; Højrup, Peter; Grosche, Astrid; Freeman, David E; Andersen, Pia H; Houen, Gunnar

    2017-06-01

    OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases. RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.

  2. Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and surface-enhanced Raman scattering.

    Science.gov (United States)

    Kong, Xianming; Yu, Qian; Lv, Zhongpeng; Du, Xuezhong

    2013-10-11

    Tandem assays of protein and glucose in combination with mannose-functionalized Fe3 O4 @SiO2 and Ag@SiO2 tag particles have promising potential in effective magnetic separation and highly sensitive and selective SERS assays of biomaterials. It is for the first time that tandem assay of glucose is developed using SERS based on the Con A-sandwiched microstructures between the functionalized magnetic and tag particles. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

    Directory of Open Access Journals (Sweden)

    Zong-Xiu Wang

    Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

  4. Geiger mode avalanche photodiodes for microarray systems

    Science.gov (United States)

    Phelan, Don; Jackson, Carl; Redfern, R. Michael; Morrison, Alan P.; Mathewson, Alan

    2002-06-01

    New Geiger Mode Avalanche Photodiodes (GM-APD) have been designed and characterized specifically for use in microarray systems. Critical parameters such as excess reverse bias voltage, hold-off time and optimum operating temperature have been experimentally determined for these photon-counting devices. The photon detection probability, dark count rate and afterpulsing probability have been measured under different operating conditions. An active- quench circuit (AQC) is presented for operating these GM- APDs. This circuit is relatively simple, robust and has such benefits as reducing average power dissipation and afterpulsing. Arrays of these GM-APDs have already been designed and together with AQCs open up the possibility of having a solid-state microarray detector that enables parallel analysis on a single chip. Another advantage of these GM-APDs over current technology is their low voltage CMOS compatibility which could allow for the fabrication of an AQC on the same device. Small are detectors have already been employed in the time-resolved detection of fluorescence from labeled proteins. It is envisaged that operating these new GM-APDs with this active-quench circuit will have numerous applications for the detection of fluorescence in microarray systems.

  5. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  6. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-09-10

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  7. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    International Nuclear Information System (INIS)

    Bloch, Donald B.; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-01-01

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: → A two-hybrid assay was developed to study interactions in macromolecular complexes. → The assay was applied to interactions between components of mRNA P-bodies. → The assay effectively and efficiently identified protein interaction domains. → P-body assembly in mammalian cells differs from that in other species.

  8. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

  9. HER2/neu (c-erbB-2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen

    2009-01-01

    intraepithelial neoplasia (CIN)1 (n = 262), CIN2 (n = 230), CIN3 (n = 186) and invasive carcinoma (n = 136), for HER2/neu protein expression by immunohistochemistry (IHC) and for HER2/neu gene amplification by chromogenic in situ hybridization (CISH). We found moderate or strong immunohistochemical positivity...... and invasive cervical carcinoma specimens. When present, Her-2/neu positivity is more commonly seen in higher grades of cervical dysplasia and in carcinoma. However, this large TMA study shows that HER2/neu oncoprotein expression and HER2/neu gene amplification overall are uncommon events in cervical neoplasia....... This provides compelling evidence that HER2/neu plays no major role in the development and progression of cervical neoplasia....

  10. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection. PMID:28936257

  11. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Directory of Open Access Journals (Sweden)

    Malin Berggrund

    2016-01-01

    Full Text Available The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA. Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  12. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™.

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  13. Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

    Directory of Open Access Journals (Sweden)

    Peng Huiru

    2011-04-01

    Full Text Available Abstract Background Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat. Results In this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress. Conclusion Our results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future.

  14. Plant-pathogen interactions: what microarray tells about it?

    Science.gov (United States)

    Lodha, T D; Basak, J

    2012-01-01

    Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

  15. Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

    Directory of Open Access Journals (Sweden)

    Andrzej Chruscinski

    Full Text Available Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen. Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient

  16. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

    Science.gov (United States)

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

  17. Microarray Expression Analyses of Arabidopsis Guard Cells and Isolation of a Recessive Abscisic Acid Hypersensitive Protein Phosphatase 2C MutantW⃞

    Science.gov (United States)

    Leonhardt, Nathalie; Kwak, June M.; Robert, Nadia; Waner, David; Leonhardt, Guillaume; Schroeder, Julian I.

    2004-01-01

    Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell–expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription–PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)–regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type–specific genomic scale analyses of gene function. PMID:14973164

  18. Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

    Science.gov (United States)

    Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

    2013-01-01

    Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

  19. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  20. A cell spot microarray method for production of high density siRNA transfection microarrays

    Directory of Open Access Journals (Sweden)

    Mpindi John-Patrick

    2011-03-01

    Full Text Available Abstract Background High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. Results Here, we describe the optimization of a miniaturized cell spot microarray (CSMA method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. Conclusions The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.

  1. Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.

    Directory of Open Access Journals (Sweden)

    Casey R Richardson

    2010-05-01

    Full Text Available MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery.We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes.Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other

  2. The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5 protein as an antigen for vaccinating against Grass carp reovirus

    Directory of Open Access Journals (Sweden)

    Xu Dan

    2011-03-01

    Full Text Available Abstract Background Grass carp reovirus (GCRV is the causative pathogen of grass carp hemorrhagic disease, one of the major diseases damaging grass carp Ctenopharyngon idellus breeding industry in China. Prevention and control of the disease is impeded largely due to the lack of research in economic subunit vaccine development. This study aimed to evaluate the potential of viral outer shell protein VP5 as subunit vaccine. Methods The vp5 gene was isolated from the viral genome through RT-PCR and genetically engineered to express the recombinant VP5 protein in E coli. The viral origin of the recombinant protein was confirmed by Western blot analysis with a monoclonal antibody against viral VP5 protein. Polyclonal antibody against the recombinant VP5 protein was prepared from mice. A microneutralization assay was developed to test its neutralizing ability against GCRV infection in cell culture. Results The GST-VP5 fusion protein (rVP5 was produced from E. Coli with expected molecular weight of 90 kDa. The protein was purified and employed to prepare anti-VP5 polyclonal antibody from mice. The anti-VP5 antibody was found to neutralize GCRV through in vitro microneutralization assay and viral progeny quantification analysis. Conclusions The present study showed that the viral VP5 protein was involved in viral infection and bacterially-expressed VP5 could be suitable for developing subunit vaccine for the control of GCRV infection.

  3. Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I

    2001-01-01

    We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...... used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...

  4. Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA regions of the host genome.

    Science.gov (United States)

    Benferhat, Rima; Josse, Thibaut; Albaud, Benoit; Gentien, David; Mansuroglu, Zeyni; Marcato, Vasco; Souès, Sylvie; Le Bonniec, Bernard; Bouloy, Michèle; Bonnefoy, Eliette

    2012-10-01

    Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways, such as cell adhesion, axonal guidance, development, and coagulation were closely related to RVFV-induced disorders. In particular, we show in this work that NSs targeted and modified the expression of genes coding for coagulation factors, demonstrating for the first time that this hemorrhagic virus impairs the host coagulation cascade at the transcriptional level.

  5. Factorial microarray analysis of zebra mussel (Dreissena polymorpha: Dreissenidae, Bivalvia adhesion

    Directory of Open Access Journals (Sweden)

    Faisal Mohamed

    2010-05-01

    Full Text Available Abstract Background The zebra mussel (Dreissena polymorpha has been well known for its expertise in attaching to substances under the water. Studies in past decades on this underwater adhesion focused on the adhesive protein isolated from the byssogenesis apparatus of the zebra mussel. However, the mechanism of the initiation, maintenance, and determination of the attachment process remains largely unknown. Results In this study, we used a zebra mussel cDNA microarray previously developed in our lab and a factorial analysis to identify the genes that were involved in response to the changes of four factors: temperature (Factor A, current velocity (Factor B, dissolved oxygen (Factor C, and byssogenesis status (Factor D. Twenty probes in the microarray were found to be modified by one of the factors. The transcription products of four selected genes, DPFP-BG20_A01, EGP-BG97/192_B06, EGP-BG13_G05, and NH-BG17_C09 were unique to the zebra mussel foot based on the results of quantitative reverse transcription PCR (qRT-PCR. The expression profiles of these four genes under the attachment and non-attachment were also confirmed by qRT-PCR and the result is accordant to that from microarray assay. The in situ hybridization with the RNA probes of two identified genes DPFP-BG20_A01 and EGP-BG97/192_B06 indicated that both of them were expressed by a type of exocrine gland cell located in the middle part of the zebra mussel foot. Conclusions The results of this study suggested that the changes of D. polymorpha byssogenesis status and the environmental factors can dramatically affect the expression profiles of the genes unique to the foot. It turns out that the factorial design and analysis of the microarray experiment is a reliable method to identify the influence of multiple factors on the expression profiles of the probesets in the microarray; therein it provides a powerful tool to reveal the mechanism of zebra mussel underwater attachment.

  6. Factorial microarray analysis of zebra mussel (Dreissena polymorpha: Dreissenidae, Bivalvia) adhesion.

    Science.gov (United States)

    Xu, Wei; Faisal, Mohamed

    2010-05-28

    The zebra mussel (Dreissena polymorpha) has been well known for its expertise in attaching to substances under the water. Studies in past decades on this underwater adhesion focused on the adhesive protein isolated from the byssogenesis apparatus of the zebra mussel. However, the mechanism of the initiation, maintenance, and determination of the attachment process remains largely unknown. In this study, we used a zebra mussel cDNA microarray previously developed in our lab and a factorial analysis to identify the genes that were involved in response to the changes of four factors: temperature (Factor A), current velocity (Factor B), dissolved oxygen (Factor C), and byssogenesis status (Factor D). Twenty probes in the microarray were found to be modified by one of the factors. The transcription products of four selected genes, DPFP-BG20_A01, EGP-BG97/192_B06, EGP-BG13_G05, and NH-BG17_C09 were unique to the zebra mussel foot based on the results of quantitative reverse transcription PCR (qRT-PCR). The expression profiles of these four genes under the attachment and non-attachment were also confirmed by qRT-PCR and the result is accordant to that from microarray assay. The in situ hybridization with the RNA probes of two identified genes DPFP-BG20_A01 and EGP-BG97/192_B06 indicated that both of them were expressed by a type of exocrine gland cell located in the middle part of the zebra mussel foot. The results of this study suggested that the changes of D. polymorpha byssogenesis status and the environmental factors can dramatically affect the expression profiles of the genes unique to the foot. It turns out that the factorial design and analysis of the microarray experiment is a reliable method to identify the influence of multiple factors on the expression profiles of the probesets in the microarray; therein it provides a powerful tool to reveal the mechanism of zebra mussel underwater attachment.

  7. The impact of pre-analytical variables on the stability of neurofilament proteins in CSF, determined by a novel validated SinglePlex Luminex assay and ELISA.

    Science.gov (United States)

    Koel-Simmelink, Marleen J A; Vennegoor, Anke; Killestein, Joep; Blankenstein, Marinus A; Norgren, Niklas; Korth, Carsten; Teunissen, Charlotte E

    2014-01-15

    Neurofilament (Nf) proteins have been shown to be promising biomarkers for monitoring and predicting disease progression for various neurological diseases. The aim of this study was to evaluate the effects of pre-analytical variables on the concentration of neurofilament heavy (NfH) and neurofilament light (NfL) proteins. For NfH an in-house newly-developed and validated SinglePlex Luminex assay was used; ELISA was used to analyze NfL. For the NfL ELISA assay, the intra- and inter-assay variation was respectively, 1.5% and 16.7%. Analytical performance of the NfH SinglePlex Luminex assay in terms of sensitivity (6.6pg/mL), recovery in cerebrospinal fluid (CSF) (between 90 and 104%), linearity (from 6.6-1250pg/mL), and inter- and intra-assay variation (<8%) were good. Concentrations of both NfL and NfH appeared not negatively affected by blood contamination, repeated freeze-thaw cycles (up to 4), delayed processing (up to 24hours) and during long-term storage at -20°C, 4°C, and room temperature. A decrease in concentration was observed during storage of both neurofilament proteins up to 21days at 37°C, which was significant by day 5. The newly developed NfH SinglePlex Luminex assay has a good sensitivity and is robust. Moreover, both NfH and NfL are stable under the most prevalent pre-analytical variations. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    Science.gov (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  9. Sensitive diagnosis of bovine tuberculosis in a farmed cervid herd with use of an MPB70 protein fluorescence polarization assay.

    Science.gov (United States)

    Surujballi, Om; Lutze-Wallace, Cyril; Turcotte, Claude; Savic, Mirjana; Stevenson, Dan; Romanowska, Anna; Monagle, Wendy; Berlie-Surujballi, Gloria; Tangorra, Erin

    2009-07-01

    After histopathological examination of a lesion found in a herd member returned a diagnosis of mycobacteriosis, a farmed herd (n = 47) of elk (Cervus elaphus nelsoni) and red deer (C. elaphus elaphus) was investigated for bovine tuberculosis with a battery of antemortem and postmortem diagnostic tests. Every animal was tested with the mid-cervical tuberculin skin test; all 47 had negative results. All of the 16 adult animals and 15 of the 31 calves (approximately 2-years-old) were blood-tested with a lymphocyte stimulation test (LST) and a fluorescence polarization assay (FPA), which detects antibody to the MPB70 protein antigen. At necropsy of the 31 blood-tested animals, tissues were harvested for histopathological examination and culture of mycobacteria. Mycobacterium bovis was isolated from 16 of the 31 animals, and a scotochromogen was also isolated from 1 of the 16 whose tissues yielded M. bovis. Each of these 16 animals, 15 of which were calves, also received a histopathological diagnosis of mycobacteriosis. Other species of mycobacteria, including those belonging to the M. avium and M. terrae complexes, were isolated from an additional 7 animals. The FPA was scored "positive" or "suspect" for 16 animals, 13 (81%) of which were culture-positive for M. bovis. The other 3 animals that were culture-positive for M. bovis had negative FPA results. Of the 3 FPA-positive or FPA-suspect animals that were culture-negative, 2 were suspected to have mycobacteriosis on the basis of the histopathological examination. The 7 animals from which Mycobacterium species other than M. bovis were cultured were all FPA-negative. The only animal with positive LST results was also FPA-positive and culture-positive for M. bovis. The M. bovis isolates had an identical spoligotype pattern, with an octal code of 664073777777600. This is the first report of the isolation and identification of this strain type in Canada.

  10. A novel TaqMan® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70.

    Science.gov (United States)

    Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall'Olio, Raffaele; Nanetti, Antonio

    2018-04-01

    Nosema ceranae is now a widespread honey bee pathogen with high incidence in apiculture. Rapid and reliable detection and quantification methods are a matter of concern for research community, nowadays mainly relying on the use of biomolecular techniques such as PCR, RT-PCR or HRMA. The aim of this technical paper is to provide a new qPCR assay, based on the highly-conserved protein coding gene Hsp70, to detect and quantify the microsporidian Nosema ceranae affecting the western honey bee Apis mellifera. The validation steps to assess efficiency, sensitivity, specificity and robustness of the assay are described also. Copyright © 2018 Elsevier GmbH. All rights reserved.

  11. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  12. Polyadenylation state microarray (PASTA) analysis.

    Science.gov (United States)

    Beilharz, Traude H; Preiss, Thomas

    2011-01-01

    Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

  13. Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

    Science.gov (United States)

    Fournier-Wirth, C; Coste, J

    2007-01-01

    Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.

  14. A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

    Directory of Open Access Journals (Sweden)

    Andrew Gehring

    2014-06-01

    Full Text Available Shiga toxins 1 and 2 (Stx1 and Stx2 from Shiga toxin-producing E. coli (STEC bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies and pooled horseradish peroxidase (HRP-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB, the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic and/or B-PER (a cell-disrupting, protein extraction reagent. Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

  15. Microarray-based IgE detection in tears of patients with vernal keratoconjunctivitis.

    Science.gov (United States)

    Leonardi, Andrea; Borghesan, Franco; Faggian, Diego; Plebani, Mario

    2015-11-01

    A specific allergen sensitization can be demonstrated in approximately half of the vernal keratoconjunctivitis (VKC) patients by conventional allergic tests. The measurement of specific IgE in tears using a multiplex allergen microarray may offer advantages to identify local sensitization to a specific allergen. In spring-summer 2011, serum and tears samples were collected from 10 active VKC patients (three females, seven males) and 10 age-matched normal subjects. Skin prick test, symptoms score and full ophthalmological examination were performed. Specific serum and tear IgE were assayed using ImmunoCAP ISAC, a microarray containing 103 components derived from 47 allergens. Normal subjects resulted negative for the presence of specific IgE both in serum and in tears. Of the 10 VKC patients, six resulted positive to specific IgE in serum and/or tears. In three of these six patients, specific IgE was found positive only in tears. Cross-reactivity between specific markers was found in three patients. Grass, tree, mites, animal but also food allergen-specific IgE were found in tears. Conjunctival provocation test performed out of season confirmed the specific local conjunctival reactivity. Multiple specific IgE measurements with single protein allergens using a microarray technique in tear samples are a useful, simple and non-invasive diagnostic tool. ImmunoCAP ISAC detects allergen sensitization at component level and adds important information by defining both cross- and co-sensitization to a large variety of allergen molecules. The presence of specific IgE only in tears of VKC patients reinforces the concept of possible local sensitization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

    Science.gov (United States)

    Rao, Archana N; Grainger, David W

    2014-04-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

  17. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

    Science.gov (United States)

    Rao, Archana N.; Grainger, David W.

    2014-01-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

  18. DNA microarray-based PCR ribotyping of Clostridium difficile.

    Science.gov (United States)

    Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2015-02-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

    OpenAIRE

    Rao, Archana N.; Grainger, David W.

    2014-01-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surface...

  20. Contributions of procoagulants and anticoagulants to the international normalized ratio and thrombin generation assay in patients treated with warfarin: potential role of protein Z as a powerful determinant of coagulation assays.

    Science.gov (United States)

    Choi, Qute; Kim, Ji-Eun; Hyun, Jungwon; Han, Kyou-Sup; Kim, Hyun Kyung

    2013-07-01

    The effects of warfarin are measured with the international normalized ratio (INR). However, the thrombin generation assay (TGA) may offer more information about global coagulation. We analyzed the monitoring performance of the TGA and INR and investigated the impact of procoagulants (fibrinogen, factor (F)II, FVII, FIX, and FX) and anticoagulants (proteins C, S, and Z) on them. The TGA was performed on a calibrated automated thrombogram, producing lag time, endogenous thrombin potential (ETP), and peak thrombin in 239 patients treated with warfarin. Pro- and anticoagulant levels were also measured. The INR was significantly and inversely correlated with ETP. The therapeutic range of ETP comparable to an INR range of 2.0-3.0 was 290.1-494.6. ETP showed comparable performance to the INR as a warfarin-monitoring parameter with respect to clinical complication rate. The median levels of FII, FVII, FIX, and FX and proteins C and Z tended to decrease gradually with increasing anticoagulation intensity according to the INR or ETP. Of note, protein Z levels decreased dramatically with increasing anticoagulation status. INRs were significantly determined by FII, FVII, and protein Z. ETP was significantly dependent on FVII, and proteins C and Z concentration. Protein Z significantly reduced the total amount of thrombin generation and prolonged PT value in vitro. The INR and ETP exhibit similar efficacy for warfarin monitoring according to the clinical complication rate. Protein Z is considered to be a significant determinant of INR and ETP in patients on warfarin therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Seeded Bayesian Networks: Constructing genetic networks from microarray data

    Directory of Open Access Journals (Sweden)

    Quackenbush John

    2008-07-01

    Full Text Available Abstract Background DNA microarrays and other genomics-inspired technologies provide large datasets that often include hidden patterns of correlation between genes reflecting the complex processes that underlie cellular metabolism and physiology. The challenge in analyzing large-scale expression data has been to extract biologically meaningful inferences regarding these processes – often represented as networks – in an environment where the datasets are often imperfect and biological noise can obscure the actual signal. Although many techniques have been developed in an attempt to address these issues, to date their ability to extract meaningful and predictive network relationships has been limited. Here we describe a method that draws on prior information about gene-gene interactions to infer biologically relevant pathways from microarray data. Our approach consists of using preliminary networks derived from the literature and/or protein-protein interaction data as seeds for a Bayesian network analysis of microarray results. Results Through a bootstrap analysis of gene expression data derived from a number of leukemia studies, we demonstrate that seeded Bayesian Networks have the ability to identify high-confidence gene-gene interactions which can then be validated by comparison to other sources of pathway data. Conclusion The use of network seeds greatly improves the ability of Bayesian Network analysis to learn gene interaction networks from gene expression data. We demonstrate that the use of seeds derived from the biomedical literature or high-throughput protein-protein interaction data, or the combination, provides improvement over a standard Bayesian Network analysis, allowing networks involving dynamic processes to be deduced from the static snapshots of biological systems that represent the most common source of microarray data. Software implementing these methods has been included in the widely used TM4 microarray analysis package.

  2. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    Science.gov (United States)

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  3. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

    Science.gov (United States)

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

  4. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Directory of Open Access Journals (Sweden)

    Anna Grazia Recchia

    Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  5. Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity.

    Science.gov (United States)

    Sommer, Jurg M; Buyue, Yang; Bardan, Sara; Peters, Robert T; Jiang, Haiyan; Kamphaus, George D; Gray, Elaine; Pierce, Glenn F

    2014-11-01

    Due to variability in the one-stage clotting assay, the performance of new factor IX (FIX) products should be assessed in this assay. The objective of this field study was to evaluate the accuracy of measuring recombinant FIX Fc fusion protein (rFIXFc) activity in clinical haemostasis laboratories using the one-stage clotting assay. Human haemophilic donor plasma was spiked with rFIXFc or BeneFIX® at 0.80, 0.20, or 0.05 IU/ml based on label potency. Laboratories tested blinded samples using their routine one-stage assay and in-house FIX plasma standard. The mean spike recoveries for BeneFIX (n=30 laboratories) were 121 %, 144 %, and 168 % of expected at nominal 0.80, 0.20, and 0.05 IU/ml concentrations, respectively. Corresponding rFIXFc spike recoveries were 88 %, 107 %, and 132 % of expected, respectively. All BeneFIX concentrations were consistently overestimated by most laboratories. rFIXFc activity was reagent-dependent; ellagic acid and silica gave higher values than kaolin, which underestimated rFIXFc. BeneFIX demonstrated significantly reduced chromogenic assay activity relative to one-stage assay results and nominal activity, while rFIXFc activity was close to nominal activity at three concentrations with better dilution linearity than the typical one-stage assay. In conclusion, laboratory- and reagent-specific assay variabilities were revealed, with progressively higher variability at lower FIX concentrations. Non-parallelism against the FIX plasma standard was observed in all one-stage assays with rFIXFc and BeneFIX, leading to significant overestimation of FIX activity at lower levels and generally high inter-laboratory variability. Compared to the accuracy currently achieved in clinical laboratories when measuring other rFIX products, most laboratories measured rFIXFc activity with acceptable accuracy and reliability using routine one-stage assay methods and commercially available plasma standards.

  6. Direct calibration of PICKY-designed microarrays

    Directory of Open Access Journals (Sweden)

    Ronald Pamela C

    2009-10-01

    Full Text Available Abstract Background Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples. Results Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu under the PICKY Download area. Conclusion PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.

  7. Kernel Based Nonlinear Dimensionality Reduction and Classification for Genomic Microarray

    Directory of Open Access Journals (Sweden)

    Lan Shu

    2008-07-01

    Full Text Available Genomic microarrays are powerful research tools in bioinformatics and modern medicinal research because they enable massively-parallel assays and simultaneous monitoring of thousands of gene expression of biological samples. However, a simple microarray experiment often leads to very high-dimensional data and a huge amount of information, the vast amount of data challenges researchers into extracting the important features and reducing the high dimensionality. In this paper, a nonlinear dimensionality reduction kernel method based locally linear embedding(LLE is proposed, and fuzzy K-nearest neighbors algorithm which denoises datasets will be introduced as a replacement to the classical LLE’s KNN algorithm. In addition, kernel method based support vector machine (SVM will be used to classify genomic microarray data sets in this paper. We demonstrate the application of the techniques to two published DNA microarray data sets. The experimental results confirm the superiority and high success rates of the presented method.

  8. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

  9. Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

    Science.gov (United States)

    Lung, O; Furukawa-Stoffer, T; Burton Hughes, K; Pasick, J; King, D P; Hodko, D

    2017-12-01

    Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site. © 2016 Her Majesty the Queen in Right of Canada.

  10. The ability of in vitro antioxidant assays to predict the efficiency of a cod protein hydrolysate and brown seaweed extract to prevent oxidation in marine food model systems.

    Science.gov (United States)

    Jónsdóttir, Rósa; Geirsdóttir, Margrét; Hamaguchi, Patricia Y; Jamnik, Polona; Kristinsson, Hordur G; Undeland, Ingrid

    2016-04-01

    The ability of different in vitro antioxidant assays to predict the efficiency of cod protein hydrolysate (CPH) and Fucus vesiculosus ethyl acetate extract (EA) towards lipid oxidation in haemoglobin-fortified washed cod mince and iron-containing cod liver oil emulsion was evaluated. The progression of oxidation was followed by sensory analysis, lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) in both systems, as well as loss of redness and protein carbonyls in the cod system. The in vitro tests revealed high reducing capacity, high DPPH radical scavenging properties and a high oxygen radical absorbance capacity (ORAC) value of the EA which also inhibited lipid and protein oxidation in the cod model system. The CPH had a high metal chelating capacity and was efficient against oxidation in the cod liver oil emulsion. The results indicate that the F. vesiculosus extract has a potential as an excellent natural antioxidant against lipid oxidation in fish muscle foods while protein hydrolysates are more promising for fish oil emulsions. The usefulness of in vitro assays to predict the antioxidative properties of new natural ingredients in foods thus depends on the knowledge about the food systems, particularly the main pro-oxidants present. © 2015 Society of Chemical Industry.

  11. Evaluation of a novel Dot-ELISA assay utilizing a recombinant protein for the effective diagnosis of Taenia pisiformis larval infections.

    Science.gov (United States)

    Chen, Lin; Yang, Deying; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-08-29

    Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in domestic breeds of the rabbit Oryctolagus cuniculus that results in economic losses. At present, there is no convenient and effective method for the rapid detection of T. pisiformis larvae. Here, we developed and tested the efficacy of a Dot-ELISA assay for the diagnosis of T. pisiformis larval infections in rabbits, based on the expression of the recombinant fusion protein (rTp1) from the Tp1 gene. Rapid amplification of cDNA ends (RACE) was used to amplify the 3' ends of the Tp1 gene, based on the unigene similar to Ts1 gene (EU009656.1) which comes from transcriptome sequencing of T. pisiformis. The Tp1 gene was successfully amplified, cloned and expressed in BL21 (DE3). Western blot analysis revealed that the recombinant Tp1 protein is specifically recognized by rabbit T. pisiformis cysticercosis antisera. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with T. pisiformis larvae and with other parasitic infections. Results showed that this Dot-ELISA assay had both high sensitivity (92.9-97.6%) and specificity (95.2-98.4%) to detect T. pisiformis larval infections. We also found very low levels of cross-reaction with other parasitic infections. This study has revealed that our novel Dot-ELISA assay utilizing the recombinant fusion protein, rTp1, has a strong potential for the effective diagnosis of T. pisiformis infections in rabbits. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    OpenAIRE

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactiv...

  13. Evaluation of apoptosis and apoptosis proteins as possible markers of radiation at doses 0.1-2 Gy, in comparison to the micronucleus assay in three cell lines

    International Nuclear Information System (INIS)

    Jaworska, A.; Angelis, P. de

    1997-01-01

    In recent years the interest in apoptosis as possible indicator of radiation damage has increased. Studies have been done to examine the induction of apoptosis after ionizing radiation using morphological criteria, characteristic DNA damage pattern(ladders), early DNA damage using flow cytometry and/or expression of the proteins involved in apoptosis. But the picture which emerges from these investigations is unclear. Some researchers suggest that apoptosis studies can be used as potential assays of biological dosimetry, others doubt if apoptosis can be used as a marker of irradiation at all. Most studies have been done using relatively high doses of radiation. In this study we focus on apoptosis induction after relatively small doses (0,1-2 Gy). We detected apoptosis with the in situ terminal deoxynucleotidyl transferase assay by flow cytometry, and measured the expression of proteins that regulate apoptosis (Bcl-2, Bax, P53) with Western blotting. As comparison we used the cytokinesis-block micronucleus assay as a reference. The studies were carried out in three lymphoid cell lines: the mouse lymphoma L5178Y resistant and sensitive cell lines widely used in radiobiological studies, and the human pre-B cell leukemia Reh cells. Our results indicate that we can not consider the examined parameters of apoptosis as markers of radiation in these cell lines. (author)

  14. Homogeneous competitive assay of ligand affinities based on quenching fluorescence of tyrosine/tryptophan residues in a protein via Főrster-resonance-energy-transfer

    Science.gov (United States)

    Xie, Yanling; Yang, Xiaolan; Pu, Jun; Zhao, Yunsheng; Zhang, Ying; Xie, Guoming; Zheng, Jun; Yuan, Huidong; Liao, Fei

    2010-11-01

    A new homogeneous competitive assay of ligand affinities was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Főrster-resonance-energy-transfer using a fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a protein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate ligand. Chemometrics for deriving the binding ratio of the acceptor with either fluorescence signal was discussed; the dissociation constant ( Kd) of a nonfluorescent candidate ligand was calculated from its concentration to displace 50% binding of the acceptor. N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) and N-biotinyl-N'-dansyl-ethylenediamine (BDEDA) were used as the reference ligands and acceptors to streptavidin to test this new homogeneous competitive assay. Upon binding of an acceptor to streptavidin, there were the quench of streptavidin fluorescence at 340 nm and the characteristic fluorescence at 430 nm for BNEDA or at 525 nm for BDEDA. Kd of BNEDA and BDEDA was obtained via competitive binding against biotin. By quantifying BNEDA fluorescence, Kd of each tested nonfluorescent biotin derivative was consistent with that by quantifying streptavidin fluorescence using BNEDA or BDEDA as the acceptor. The overall coefficients of variation were about 10%. Therefore, this homogeneous competitive assay was effective and promising to high-throughput-screening.

  15. Development and application of an indirect enzyme-linked immunosorbent assay using recombinant truncated Cap protein for the diagnosis of porcine circovirus-like virus P1.

    Science.gov (United States)

    Wen, Li-bin; Wen, Shi-fu; He, Kong-wang

    2016-01-19

    Porcine circovirus-like virus P1 is a newly discovered virus. To date, there has been no specific serological assay for use in the diagnosis of P1 infection. Because P1 has high homology to porcine circovirus type 2 (PCV2) at the nucleotide level, the C-terminal portion of the capsid protein (amino acids 73-114), a discriminative antigen, was expressed in a prokaryotic expression system. The recombinant product (rctCap), composed of three identical repeated domains, was shown to be strongly immunoreactive to P1-specific serum. This assay was validated by comparison with an indirect immunofluorescence assay (IFA). The diagnostic sensitivity and specificity of the rctCap enzyme-linked immunosorbent assay (ELISA) developed in this study are 93.6% and 98.3%, respectively, compared with the results from IFAs on 450 sera samples from pigs. The indirect ELISA that we developed with rctCap, the recombinant capsid fragment containing the 217-342 nt repeat domain, was sensitive, specific, and suitable for the large-scale detection of P1 infections in swine.

  16. Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

    Science.gov (United States)

    Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

    2003-01-01

    The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

  17. Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

    Science.gov (United States)

    Asai, Akira; Takakuma, Kazuyuki

    2017-01-01

    When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

  18. Gene protein detection platform--a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

    Science.gov (United States)

    Stålhammar, Gustav; Farrajota, Pedro; Olsson, Ann; Silva, Cristina; Hartman, Johan; Elmberger, Göran

    2015-08-01

    Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  20. DNA microarray technology in nutraceutical and food safety.

    Science.gov (United States)

    Liu-Stratton, Yiwen; Roy, Sashwati; Sen, Chandan K

    2004-04-15

    The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

  1. Antioxidant Activity of Fish Protein Hydrolysates in in vitro Assays and in Oil-in-Water Emulsions

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Andersen, Lisa Lystbæk; Jacobsen, Charlotte

    The aim of this study was to screen different protein hydrolysates with respect to their antioxidative properties in order to select the most promising extracts for further evaluation in oil-in-water emulsions. Three fractions of protein hydrolysates (Crude, >5kDa and 5kDa, 3-5kDa and...

  2. Mass spectrometry based biomarker discovery, verification, and validation--quality assurance and control of protein biomarker assays.

    Science.gov (United States)

    Parker, Carol E; Borchers, Christoph H

    2014-06-01

    In its early years, mass spectrometry (MS)-based proteomics focused on the cataloging of proteins found in different species or different tissues. By 2005, proteomics was being used for protein quantitation, typically based on "proteotypic" peptides which act as surrogates for the parent proteins. Biomarker discovery is usually done by non-targeted "shotgun" proteomics, using relative quantitation methods to determine protein expression changes that correlate with disease (output given as "up-or-down regulation" or "fold-increases"). MS-based techniques can also perform "absolute" quantitation which is required for clinical applications (output given as protein concentrations). Here we describe the differences between these methods, factors that affect the precision and accuracy of the results, and some examples of recent studies using MS-based proteomics to verify cancer-related biomarkers. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. DNA microarray technique for detecting food-borne pathogens

    Directory of Open Access Journals (Sweden)

    Xing GAO

    2012-08-01

    Full Text Available Objective To study the application of DNA microarray technique for screening and identifying multiple food-borne pathogens. Methods The oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogens, and then were validated by bioinformatic analyses. The 5' end of each probe was modified by amino-group and 10 Poly-T, and the optimized probes were synthesized and spotted on aldehyde-coated slides. The bacteria DNA template incubated with Klenow enzyme was amplified by arbitrarily primed PCR, and PCR products incorporated into Aminoallyl-dUTP were coupled with fluorescent dye. After hybridization of the purified PCR products with DNA microarray, the hybridization image and fluorescence intensity analysis was acquired by ScanArray and GenePix Pro 5.1 software. A series of detection conditions such as arbitrarily primed PCR and microarray hybridization were optimized. The specificity of this approach was evaluated by 16 different bacteria DNA, and the sensitivity and reproducibility were verified by 4 food-borne pathogens DNA. The samples of multiple bacteria DNA and simulated water samples of Shigella dysenteriae were detected. Results Nine different food-borne bacteria were successfully discriminated under the same condition. The sensitivity of genomic DNA was 102 -103pg/ μl, and the coefficient of variation (CV of the reproducibility of assay was less than 15%. The corresponding specific hybridization maps of the multiple bacteria DNA samples were obtained, and the detection limit of simulated water sample of Shigella dysenteriae was 3.54×105cfu/ml. Conclusions The DNA microarray detection system based on arbitrarily primed PCR can be employed for effective detection of multiple food-borne pathogens, and this assay may offer a new method for high-throughput platform for detecting bacteria.

  4. Testing a Microarray to Detect and Monitor Toxic Microalgae in Arcachon Bay in France

    Directory of Open Access Journals (Sweden)

    Linda K. Medlin

    2013-03-01

    Full Text Available Harmful algal blooms (HABs occur worldwide, causing health problems and economic damages to fisheries and tourism. Monitoring agencies are therefore essential, yet monitoring is based only on time-consuming light microscopy, a level at which a correct identification can be limited by insufficient morphological characters. The project MIDTAL (Microarray Detection of Toxic Algae—an FP7-funded EU project—used rRNA genes (SSU and LSU as a target on microarrays to identify toxic species. Furthermore, toxins were detected with a newly developed multiplex optical Surface Plasmon Resonance biosensor (Multi SPR and compared with an enzyme-linked immunosorbent assay (ELISA. In this study, we demonstrate the latest generation of MIDTAL microarrays (version 3 and show the correlation between cell counts, detected toxin and microarray signals from field samples taken in Arcachon Bay in France in 2011. The MIDTAL microarray always detected more potentially toxic species than those detected by microscopic counts. The toxin detection was even more sensitive than both methods. Because of the universal nature of both toxin and species microarrays, they can be used to detect invasive species. Nevertheless, the MIDTAL microarray is not completely universal: first, because not all toxic species are on the chip, and second, because invasive species, such as Ostreopsis, already influence European coasts.

  5. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  6. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    Directory of Open Access Journals (Sweden)

    Michael S Rogers

    Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

  7. Microarrays: Molecular allergology and nanotechnology for personalised medicine (II).

    Science.gov (United States)

    Lucas, J M

    2010-01-01

    Progress in nanotechnology and DNA recombination techniques have produced tools for the diagnosis and investigation of allergy at molecular level. The most advanced examples of such progress are the microarray techniques, which have been expanded not only in research in the field of proteomics but also in application to the clinical setting. Microarrays of allergic components offer results relating to hundreds of allergenic components in a single test, and using a small amount of serum which can be obtained from capillary blood. The availability of new molecules will allow the development of panels including new allergenic components and sources, which will require evaluation for clinical use. Their application opens the door to component-based diagnosis, to the holistic perception of sensitisation as represented by molecular allergy, and to patient-centred medical practice by allowing great diagnostic accuracy and the definition of individualised immunotherapy for each patient. The present article reviews the application of allergenic component microarrays to allergology for diagnosis, management in the form of specific immunotherapy, and epidemiological studies. A review is also made of the use of protein and gene microarray techniques in basic research and in allergological diseases. Lastly, an evaluation is made of the challenges we face in introducing such techniques to clinical practice, and of the future perspectives of this new technology. Copyright 2010 SEICAP. Published by Elsevier Espana. All rights reserved.

  8. Microarray expression profiling of human dental pulp from single subject.

    Science.gov (United States)

    Tete, Stefano; Mastrangelo, Filiberto; Scioletti, Anna Paola; Tranasi, Michelangelo; Raicu, Florina; Paolantonio, Michele; Stuppia, Liborio; Vinci, Raffaele; Gherlone, Enrico; Ciampoli, Cristian; Sberna, Maria Teresa; Conti, Pio

    2008-01-01

    Microarray is a recently developed simultaneous analysis of expression patterns of thousand of genes. The aim of this research was to evaluate the expression profile of human healthy dental pulp in order to find the presence of genes activated and encoding for proteins involved in the physiological process of human dental pulp. We report data obtained by analyzing expression profiles of human tooth pulp from single subjects, using an approach based on the amplification of the total RNA. Experiments were performed on a high-density array able to analyse about 21,000 oligonucleotide sequences of about 70 bases in duplicate, using an approach based on the amplification of the total RNA from the pulp of a single tooth. Obtained data were analyzed using the S.A.M. system (Significance Analysis of Microarray) and genes were merged according to their molecular functions and biological process by the Onto-Express software. The microarray analysis revealed 362 genes with specific pulp expression. Genes showing significant high expression were classified in genes involved in tooth development, protoncogenes, genes of collagen, DNAse, Metallopeptidases and Growth factors. We report a microarray analysis, carried out by extraction of total RNA from specimens of healthy human dental pulp tissue. This approach represents a powerful tool in the study of human normal and pathological pulp, allowing minimization of the genetic variability due to the pooling of samples from different individuals.

  9. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    Science.gov (United States)

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  10. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    Science.gov (United States)

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Enzyme-Linked Immunosorbent Assay Using a Virus Type-Specific Peptide Based on a Subdomain of Envelope Protein Erns for Serologic Diagnosis of Pestivirus Infections in Swine

    Science.gov (United States)

    Langedijk, J. P. M.; Middel, W. G. J.; Meloen, R. H.; Kramps, J. A.; de Smit, J. A.

    2001-01-01

    Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the Erns protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the Erns protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used. PMID:11230402

  12. External Quality Control for Dried Blood Spot Based C-reactive Protein Assay: Experience from the Indonesia Family Life Survey and the Longitudinal Aging Study in India

    Science.gov (United States)

    Hu, Peifeng; Herningtyas, Elizabeth H.; Kale, Varsha; Crimmins, Eileen M.; Risbud, Arun R.; McCreath, Heather; Lee, Jinkook; Strauss, John; O’Brien, Jennifer C.; Bloom, David E.; Seeman, Teresa E.

    2015-01-01

    Measurement of C-reactive protein, a marker of inflammation, in dried blood spots has been increasingly incorporated in community-based social surveys internationally. Although the dried blood spot based CRP assay protocol has been validated in the United States, it remains unclear whether laboratories in other less developed countries can generate C-reactive protein results of similar quality. We therefore conducted external quality monitoring for dried blood spot based C-reactive protein measurement for the Indonesia Family Life Survey and the Longitudinal Aging Study in India. Our results show that dried blood spot based C-reactive protein results in these two countries have excellent and consistent correlations with serum-based values and dried blood spot based results from the reference laboratory in the United States. Even though the results from duplicate samples may have fluctuations in absolute values over time, the relative order of C-reactive protein levels remains similar and the estimates are reasonably precise for population-based studies that investigate the association between socioeconomic factors and health. PMID:25879265

  13. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  14. Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors

    Science.gov (United States)

    Villa, Stefania; Legnani, Laura; Colombo, Diego; Gelain, Arianna; Lammi, Carmen; Bongiorno, Daniele; Ilboudo, Denise P.; McGee, Kellen E.; Bosch, Jürgen; Grazioso, Giovanni

    2018-03-01

    The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

  15. Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

    Science.gov (United States)

    Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

    2014-01-01

    The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

  16. A method of radiocompetitive assay of total thyroxine in the serum by means of enzymatic release of thyroxine from the transporting proteins

    International Nuclear Information System (INIS)

    Snarski, A.; Wyrwinski, J.

    1978-01-01

    Pepsin causes denaturation of the transporting proteins and liberates thyroxine which can be assayed by the radiocompetitive method. Change of the pH of the medium from acid to alkaline inactivates irreveribly pepsin. The enzymatic release of thyroxine is much simpler that the method of ethanol extraction and thermal denaturation of the transporting proteins applied up to now. The new technique of thyroxine release has been introduced for radiocompetitive determination of thyroxine using dextran coated charcoal for adsorption of the free hormone. A new method has been elaborated for preparation of working standards of thyroxine in a mixture of pepsin solution with hormone-free serum. The method is efficient and rapid. The normal range is from 50 to 130 nanomol/l. Over 7 000 determinations were done as yet in patients with suspected thyroid function disturbances. (author)

  17. FULL-LENGTH PEPTIDE ASSAY OF ANTIGENIC PROFILE OF ENVELOPE PROTEINS FROM SIBERIAN ISOLATES OF HEPATITIS C VIRUS

    Directory of Open Access Journals (Sweden)

    A. A. Grazhdantseva

    2010-01-01

    Full Text Available Antigenic profiles of envelope glycoproteins of hepatitis C virus presented by three genotypes 1b, 2a/2c and 3a, which are most widespread in the territory of Russia and, in particular, in Novosibirsk, were studied using a panel of overlapping synthetic peptides. It was shown that highly immunogenic peptide epitopes of Е1 and Е2 proteins common for all HCV genotypes, are located in amino acid positions 250-260, 315-325 (Е1 protein, 390-400 (hypervariable region 1, 430-440, and 680-690 (Е2 protein. The greatest inter-genotypic differences were recorded in positions 280-290, 410-430 and 520-540. A novel antigenic determinant was detected in the region of aa 280-290 of the Е1 protein which was typical only for HCV 2a/2c genotype. A broad variation in the boundaries for the most epitopes suggests a high variability of the Е1 and Е2 viral proteins; however, a similar repertoire of antibodies induced by different HCV genotypes indicates to an opportunity of designing a new generation of cross-reactive HCV vaccines based on mapping of the E1 and E2 antigenic regions.

  18. Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.

    Directory of Open Access Journals (Sweden)

    Thierry Gauthier

    Full Text Available Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous in vivo protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.

  19. Smart protein biogate as a mediator to regulate competitive host-guest interaction for sensitive ratiometric electrochemical assay of prion

    Science.gov (United States)

    Yu, Peng; Zhang, Xiaohua; Zhou, Jiawan; Xiong, Erhu; Li, Xiaoyu; Chen, Jinhua

    2015-11-01

    A novel competitive host-guest strategy regulated by protein biogate was developed for sensitive and selective analysis of prion protein. The methylene blue (MB)-tagged prion aptamer (MB-Apt) was introduced to the multiwalled carbon nanotubes-β-cyclodextrins (MWCNTs-β-CD) composites-modified glassy carbon (GC) electrode through the host-guest interaction between β-CD and MB. In the absence of prion, MB-Apt could be displaced by ferrocenecarboxylic acid (FCA) due to its stronger binding affinity to β-CD, resulting in a large oxidation peak of FCA. However, in the presence of prion, the specific prion-aptamer interaction drove the formation of protein biogate to seal the cavity of β-CD, which hindered the guest displacement of MB by FCA and resulted in the oxidation peak current of MB (IMB) increased and that of FCA (IFCA) decreased. The developed aptasensor showed good response towards the target (prion protein) with a low detection limit of 160 fM. By changing the specific aptamers, this strategy could be easily extended to detect other proteins, showing promising potential for extensive applications in bioanalysis.

  20. Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

    Science.gov (United States)

    Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

    2016-12-27

    The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

  1. Immunoradiometric assay for the determination of E. coli proteins in recombinant dna derived human growth hormone produced at IPEN-CNEN/SP

    International Nuclear Information System (INIS)

    Soares, Carlos R.J.

    1995-01-01

    An immunoradiometric assay (IRMA) for the determination of multiple antigens was set up in order to quantify E. coli (ECP) in lots of purified recombinant human growth hormone (rec-hGH). SDS-PAGE and Western Blotting techniques were carried out, in parallel, to confirm the results obtained by IRMA and to provide more information about the contaminants. Anti-ECP antibodies were obtained by rabbit immunization with ECP, which were submitted to the same purification process utilized for rec-hGH with the exception of the last step. A strain-process-specific assay was thus set up. The antiserum obtained was purified through an affinity column prepared with the same ECP used for immunization, this provided an highly sensitive assay (0,03 ng ECP/mL). This IRMA was shown to be specific, not presenting any cross reaction with hGH and studies carried out on precision, accuracy and linearity of response with dilution confirmed its validity as one of the fundamental purity tests for rec-hGH produced at IPEN-CNEN/SP, whose principles can be easily extended to the analysis of other similar products. These studies have also shown that the utilization of an affinity column, prepared with the described anti-ECP antiserum was very effective, providing rec-hGH lots with less then 10 parts per million (0,001%) of contaminating proteins. (author). 45 refs., 15 figs., 11 tabs

  2. An Evaluation Study of Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Protein Pap31 for Detection of Antibody against Bartonella bacilliformis Infection among the Peruvian Population

    Science.gov (United States)

    Angkasekwinai, Nasikarn; Atkins, Erin H.; Romero, Sofia; Grieco, John; Chao, Chien Chung; Ching, Wei Mei

    2014-01-01

    Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability. PMID:24515944

  3. Biocompatible Hydrogels for Microarray Cell Printing and Encapsulation

    Directory of Open Access Journals (Sweden)

    Akshata Datar

    2015-10-01

    Full Text Available Conventional drug screening processes are a time-consuming and expensive endeavor, but highly rewarding when they are successful. To identify promising lead compounds, millions of compounds are traditionally screened against therapeutic targets on human cells grown on the surface of 96-wells. These two-dimensional (2D cell monolayers are physiologically irrelevant, thus, often providing false-positive or false-negative results, when compared to cells grown in three-dimensional (3D structures such as hydrogel droplets. However, 3D cell culture systems are not easily amenable to high-throughput screening (HTS, thus inherently low throughput, and requiring relatively large volume for cell-based assays. In addition, it is difficult to control cellular microenvironments and hard to obtain reliable cell images due to focus position and transparency issues. To overcome these problems, miniaturized 3D cell cultures in hydrogels were developed via cell printing techniques where cell spots in hydrogels can be arrayed on the surface of glass slides or plastic chips by microarray spotters and cultured in growth media to form cells encapsulated 3D droplets for various cell-based assays. These approaches can dramatically reduce assay volume, provide accurate control over cellular microenvironments, and allow us to obtain clear 3D cell images for high-content imaging (HCI. In this review, several hydrogels that are compatible to microarray printing robots are discussed for miniaturized 3D cell cultures.

  4. Tissue Microarray TechnologyA Brief Review

    Directory of Open Access Journals (Sweden)

    Ramya S Vokuda

    2018-01-01

    Full Text Available In this era of modern revolutionisation in the field of medical laboratory technology, everyone is aiming at taking the innovations from laboratory to bed side. One such technique that is most relevant to the pathologic community is Tissue Microarray (TMA technology. This is becoming quite popular amongst all the members of this family, right from laboratory scientists to clinicians and residents to technologists. The reason for this technique to gain popularity is attributed to its cost effectiveness and time saving protocols. Though, every technique is accompanied by disadvantages, the benefits out number them. This technique is very versatile as many downstream molecular assays such as immunohistochemistry, cytogenetic studies, Fluorescent In situ-Hybridisation (FISH etc., can be carried out on a single slide with multiple numbers of samples. It is a very practical approach that aids effectively to identify novel biomarkers in cancer diagnostics and therapeutics. It helps in assessing the molecular markers on a large scale very quickly. Also, the quality assurance protocols in pathological laboratory has exploited TMA to a great extent. However, the application of TMA technology is beyond oncology. This review shall focus on the different aspects of this technology such as construction of TMA, instrumentation, types, advantages and disadvantages and utilisation of the technique in various disease conditions.

  5. The mitogenic activities of bean proteins determined by assay of the incorporation of sup(3)H - thymidine by human lymphocytes

    International Nuclear Information System (INIS)

    Derbyshire, E.; Carvalho, M.T.V.; Vitti, D.M.S.; Costa, C.P. da

    1988-01-01

    The proteins in a saline extract from cotyledons of the bean cultivar Goiano precoce included a protein with electrophoretic mobility equal to that of a commercial preparation of bean mitogen. The crude extract stimulated the incorporation of sup(3)H-tymidine by cultures of human lymphocytes at concentrations of extracted protein from 30 mu g - 300 mu g/culture, and the existence of an optimal concentration in the vicinity of 175 mu g/culture was indicated by the data. The range of active concentrations and the optimal concentration of the heterogeneous extract were 12-15 times greater than the corresponding values obtained when the commercial mitogen was employed. Microscopic examinations showed the presence of blast cells and mitotic figures only in cultures which included seed extract or commercial mitogen. (author)

  6. Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

    Science.gov (United States)

    Pegels, N; González, I; Fernández, S; García, T; Martín, R

    2012-01-01

    A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

  7. "Harshlighting" small blemishes on microarrays

    Directory of Open Access Journals (Sweden)

    Wittkowski Knut M

    2005-03-01

    Full Text Available Abstract Background Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs. Results We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Conclusion Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization.

  8. Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation.

    Science.gov (United States)

    Schabacker, Daniel S; Stefanovska, Ivana; Gavin, Igor; Pedrak, Casandra; Chandler, Darrell P

    2006-12-01

    Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition.

  9. High throughput production of mouse monoclonal antibodies using antigen microarrays

    DEFF Research Database (Denmark)

    De Masi, Federico; Chiarella, P.; Wilhelm, H.

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  10. A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

    Science.gov (United States)

    Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

    2015-01-01

    Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

  11. A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate acceptor

    Directory of Open Access Journals (Sweden)

    Angélique eLEVOYE

    2015-11-01

    Full Text Available Although G protein-coupled receptor (GPCR internalization has long been considered a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z’-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS of compounds that may modulate GPCRs internalization.

  12. Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

    Directory of Open Access Journals (Sweden)

    Francis M. F. Nunes

    2013-01-01

    Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  13. Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

    Science.gov (United States)

    Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

    2013-01-04

    RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

  14. High-Yield Production in Escherichia coli of Fungal Immunomodulatory Protein Isolated from Flammulina velutipes and Its Bioactivity Assay in Vivo

    Directory of Open Access Journals (Sweden)

    Shenkui Liu

    2013-01-01

    Full Text Available A fungal immunomodulatory protein isolated from Flammulina velutipes (FIP-fve has structural similarity to the variable region of the immunoglobulin heavy chain. In the present study, the recombinant bioactive FIP-fve protein with a His-tag in N-terminal of recombinant protein was expressed in transetta (DE3 at a high level under the optimized culturing conditions of 0.2 mM IPTG and 28 °C. The efficiency of the purification was improved with additional ultrasonication to the process of lysozyme lysis. The yield of the bioactive FIP-fve protein with 97.1% purity reached 29.1 mg/L with a large quantity for industrial applications. Enzyme-linked immunosorbent assay showed a maximum increase in interleukin-2 (IL-2 and gamma interferon (IFN-γ for the mice serum group of 5 mg/kg body mass (p < 0.01 with three doses of His-FIP-fve. However, the production of IL-4 had no apparent difference compared to the control.

  15. Discovery of a quorum sensing modulator pharmacophore by 3D small-molecule microarray screening

    DEFF Research Database (Denmark)

    Marsden, David M; Nicholson, Rebecca L; Skindersoe, Mette E

    2010-01-01

    ligand-binding domains of the LuxR homolog CarR from Erwinia carotovora subsp. carotovora. The 3D microarray platform was used to discover the biologically active chloro-pyridine pharmacophore, which was validated using a fluorometric ligand binding assay and ITC. Analogs containing the chloro...

  16. Characterization of equine vitamin D–binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease

    DEFF Research Database (Denmark)

    Pihl, Tina H.; Jacobsen, Stine; Olsen, Dorthe T.

    2017-01-01

    horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass...... spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed...

  17. Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay

    OpenAIRE

    Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J. Stephen

    2012-01-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interactio...

  18. Application of TMA (Tissue micro-array) in the observation of apoptotic cascade in postradiation damage in avian medicine

    International Nuclear Information System (INIS)

    Fridman, E.; Skarda, J.; Skardova, I.

    2006-01-01

    The study of apoptotic cascade by the use of relatively new technique in avian medicine: TMA may help in early detection and prevention of acquired immunodeficiency caused by the influence of a variety of pathogenic and non-pathogenic environmental factors, which may result in severe economical losses in conditions of intensive poultry farming. There has not been any report of applying this method in veterinary medicine. Tissue micro-array (TMA) technology allows rapid visualization of molecular targets in thousands of tissue specimens at a time, either at the DNA, RNA or protein level. The technique facilitates rapid translation of molecular discoveries to clinical applications. This technology has a number of advantages compared with conventional techniques: speed and high throughput, standardization and experimental uniformity, ease of use, all histochemical and molecular detection techniques can be used, decreased assay volume, preservation of original block, and conservation of valuable tissue etc. The aim of the present work were the study of immunosuppression and apoptotic cascade and possibilities of application of tissue micro-array in chicken in experimental condition and diagnostics in avian medicine in general. The selection of samples from avian primary immune organs: thymus and Bursa Fabric was done after gamma irradiation and infectious bursal virus infection (IBDV). (authors)

  19. Advanced microarray technologies for clinical diagnostics

    NARCIS (Netherlands)

    Pierik, Anke

    2011-01-01

    DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains

  20. In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

    Directory of Open Access Journals (Sweden)

    Andreas Weinhäusel

    2012-06-01

    Full Text Available The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses. In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively. The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics.

  1. The EADGENE Microarray Data Analysis Workshop

    DEFF Research Database (Denmark)

    de Koning, Dirk-Jan; Jaffrézic, Florence; Lund, Mogens Sandø

    2007-01-01

    Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from...... 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays...... statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful...

  2. The Manchester Acute Coronary Syndromes (MACS) decision rule: validation with a new automated assay for heart-type fatty acid binding protein.

    Science.gov (United States)

    Body, Richard; Burrows, Gillian; Carley, Simon; Lewis, Philip S

    2015-10-01

    The Manchester Acute Coronary Syndromes (MACS) decision rule may enable acute coronary syndromes to be immediately 'ruled in' or 'ruled out' in the emergency department. The rule incorporates heart-type fatty acid binding protein (h-FABP) and high sensitivity troponin T levels. The rule was previously validated using a semiautomated h-FABP assay that was not practical for clinical implementation. We aimed to validate the rule with an automated h-FABP assay that could be used clinically. In this prospective diagnostic cohort study we included patients presenting to the emergency department with suspected cardiac chest pain. Serum drawn on arrival was tested for h-FABP using an automated immunoturbidimetric assay (Randox) and high sensitivity troponin T (Roche). The primary outcome, a diagnosis of acute myocardial infarction (AMI), was adjudicated based on 12 h troponin testing. A secondary outcome, major adverse cardiac events (MACE; death, AMI, revascularisation or new coronary stenosis), was determined at 30 days. Of the 456 patients included, 78 (17.1%) had AMI and 97 (21.3%) developed MACE. Using the automated h-FABP assay, the MACS rule had the same C-statistic for MACE as the original rule (0.91; 95% CI 0.88 to 0.92). 18.9% of patients were identified as 'very low risk' and thus eligible for immediate discharge with no missed AMIs and a 2.3% incidence of MACE (n=2, both coronary stenoses). 11.1% of patients were classed as 'high-risk' and had a 92.0% incidence of MACE. Our findings validate the performance of a refined MACS rule incorporating an automated h-FABP assay, facilitating use in clinical settings. The effectiveness of this refined rule should be verified in an interventional trial prior to implementation. UK CRN 8376. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  3. AN EVALUATION STUDY OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA USING RECOMBINANT PROTEIN GRA1 FOR DETECTION OF IGG ANTIBODIES AGAINTS TOXOPLASMA GONDII INFECTIONS

    Directory of Open Access Journals (Sweden)

    Nina Difla Muflikhah

    2017-08-01

    Full Text Available Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, an intracellular protozoan parasite that live inside the cells of the reticulo endothelial and parenchymal cells of human and animals (mammals and birds. Some cases of toxoplasmosis usually have no symptoms, but in any cases caused severe symptoms, such as hydrocephalus, microcephalus, intracranial calcification, retinal damage, brain abscess, mental retardation, lymphadenopathy, and others. Its severe symptoms usually showed a long time after first exposure, except symptoms showed by congenital transmission caused by infected mother. Early diagnosis is important to prevent the illness but methods for toxoplasmosis screening are still too expensive for developing country. Enzyme-linked immunosorbent assay (ELISA allow the testing of a large number samples within short time frame and based on antibody or antigen detection. This study aimed to know the sensitivity and specificity of recombinat protein GRA1 as antigen using ELISA methods. We tested the sensitivity and spesificity of GRA1 protein as antigen in ELISA methods to diagnose toxoplasmosis and compared with ELISA Kit Commercial. Reliable laboratory testing is important to detect Toxoplasma gondii infection, and focused to improving the low cost and easy-to-use diagnostic instrument. Seventy sera collected and tested using both indirect ELISA, commercial ELISA kit and GRA1 protein coated as antigen. Fourty eight and fifty one samples showed positive IgG antibody result of ELISA-GRA1 and ELISA kit. Negative sample tested by ELISA-GRA1 was 22 samples and 19 sample tested by ELISA Kit. The sensitivity and specificity of GRA1-based on ELISA were 100% and 86.36%, positive prediction value (ppv was 94.11%. These data indicate that the recombinant protein GRA1 is a highly immunogenic protein in human toxoplasmosis and become a promising marker for the screening of toxoplasmosis.

  4. Canine models of inherited bleeding disorders in the development of coagulation assays, novel protein replacement and gene therapies.

    Science.gov (United States)

    Nichols, T C; Hough, C; Agersø, H; Ezban, M; Lillicrap, D

    2016-05-01

    Animal models of inherited bleeding disorders are important for understanding disease pathophysiology and are required for preclinical assessment of safety prior to testing of novel therapeutics in human and veterinary medicine. Experiments in these animals represent important translational research aimed at developing safer and better treatments, such as plasma-derived and recombinant protein replacement therapies, gene therapies and immune tolerance protocols for antidrug inhibitory antibodies. Ideally, testing is done in animals with the analogous human disease to provide essential safety information, estimates of the correct starting dose and dose response (pharmacokinetics) and measures of efficacy (pharmacodynamics) that guide the design of human trials. For nearly seven decades, canine models of hemophilia, von Willebrand disease and other inherited bleeding disorders have not only informed our understanding of the natural history and pathophysiology of these disorders but also guided the development of novel therapeutics for use in humans and dogs. This has been especially important for the development of gene therapy, in which unique toxicities such as insertional mutagenesis, germ line gene transfer and viral toxicities must be assessed. There are several issues regarding comparative medicine in these species that have a bearing on these studies, including immune reactions to xenoproteins, varied metabolism or clearance of wild-type and modified proteins, and unique tissue tropism of viral vectors. This review focuses on the results of studies that have been performed in dogs with inherited bleeding disorders that closely mirror the human condition to develop safe and effective protein and gene-based therapies that benefit both species. © 2016 International Society on Thrombosis and Haemostasis.

  5. An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

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    Laurenzi Ian J

    2009-12-01

    Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

  6. Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

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    Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

    2015-01-01

    Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze

  7. Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay.

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    Lv, Chao; Hong, Yang; Fu, Zhiqiang; Lu, Ke; Cao, Xiaodan; Wang, Tao; Zhu, Chuangang; Li, Hao; Xu, Rui; Jia, Bingguang; Han, Qian; Dou, Xuefeng; Shen, Yuanxi; Zhang, Zuhang; Zai, Jinli; Feng, Jintao; Lin, Jiaojiao

    2016-03-09

    Schistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed. Epitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats. ELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively. The application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single

  8. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

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    Paniego Norma

    2008-01-01

    Full Text Available Abstract Background Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion

  9. Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

    Science.gov (United States)

    Samolski, Ilanit; de Luis, Alberto; Vizcaíno, Juan Antonio; Monte, Enrique; Suárez, M Belén

    2009-10-13

    It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as

  10. Chemical composition, true nutrient digestibility, and true metabolizable energy of novel pet food protein sources using the precision-fed cecectomized rooster assay.

    Science.gov (United States)

    Deng, P; Utterback, P L; Parsons, C M; Hancock, L; Swanson, K S

    2016-08-01

    A wide variety of animal protein-based ingredients is commonly used in the pet food products. The raw ingredients and processing procedures used may greatly affect protein quality. Testing the quality of alternative protein sources is necessary and contributes to the sustainability of pet foods. The objective of this study was to test the chemical composition of 8 protein sources intended for use in dog and cat foods (calamari meal, pork peptone, alligator meal, lamb meal, venison meal, chicken meal, and 2 duck meals), and evaluate their true nutrient digestibility and nitrogen-corrected true ME (TMEn) using the precision-fed cecectomized rooster assay. Calamari meal and pork peptone had lower ash (4.4 and 3.6% of DM, respectively) but greater CP (88.1 and 80.5% of DM, respectively) and either greater or similar GE (5.6 and 5.3 kcal/g of DM, respectively) compared with alligator, lamb, venison, chicken, and duck meals (11.8 to 24.5% ash, 58.7 to 65.9% CP, and 4.6 to 5.3 kcal GE/g). Acid-hydrolyzed fat (AHF) was lower in calamari meal (8.7% of DM) compared with the other proteins tested (15.5-22.1% of DM). True nutrient digestibility was variable among the protein sources (52 to 79% of DM, 60 to 83% of OM, 78 to 92% of AHF, and 70 to 89% of GE) with pork peptone having the highest DM, AHF, and GE digestibility and calamari meal having the highest OM digestibility. True indispensable AA digestibility was highest for calamari meal, with all AA having a digestibility greater than 90%. Except for histidine, all indispensable AA had a digestibility over 85% for pork peptone. In contrast, true indispensable AA digestibility was lowest for lamb meal, with histidine having digestibility less than 70% and the other entire indispensable AA having digestibility between 72 and 88%. The TMEn of calamari meal (4.82 kcal/g DM and 86.9% of GE) was greater ( digestibility among protein sources intended for use in dog and cat foods and justifies further in vivo testing of novel

  11. Microarray analysis of gene expression profiles in ripening pineapple fruits.

    Science.gov (United States)

    Koia, Jonni H; Moyle, Richard L; Botella, Jose R

    2012-12-18

    Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit

  12. Proseek single-plex protein assay kit system to detect sAxl and Gas6 in serological material of brain tumor patients

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    Heidi Jaksch-Bogensperger

    2018-06-01

    Full Text Available • The receptor tyrosine kinase (RTK Axl and its ligand Gas6 are critically involved in the pathogenesis of high-grade glioma (HGG. Both proteins were found to be overexpressed e.g. in tumor cells, mediating cell proliferation and migration as well as tumor angiogenesis and neuroinflammation. The extracellular domain of Axl (sAxl and Gas6 were found in the peri-tumoral edema and blood of animals as well as in human glioma tissue. Therefore, we monitored the level of sAxl and Gas6 in human blood samples. To increase the sensitivity of protein detection beyond commonly used standard methods we preliminary tested the innovative Proseek Single-Plex Protein Assay Kit System from Olink Bioscience together with new antibodies against the soluble RTK sAxl and its ligand Gas6. We conclude that the Proseek method is a highly sensitive and fast procedure that can be used as a possible powerful tool compared to routinely used ELISA-methods.

  13. Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

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    Zhou, Lijun; Lv, Zhiqiang; Shao, Jing; Xu, Ying; Luo, Xiaohong; Zhang, Yuming; Hu, Yang; Zhang, Wenji; Luo, Shuhong; Fang, Jianmin; Wang, Ying; Duan, Chaohui; Huang, Ruopan

    2016-09-01

    The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas. © 2015 Wiley Periodicals, Inc.

  14. AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG.

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    Senga, Yukako; Imamura, Hiroshi; Miyafusa, Takamitsu; Watanabe, Hideki; Honda, Shinya

    2017-09-29

    Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

  15. Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

    Science.gov (United States)

    Mukherjee, Ali; Badal, Youssouf; Nguyen, Xuan-Thao; Miller, Johanna; Chenna, Ahmed; Tahir, Hasan; Newton, Alicia; Parry, Gordon; Williams, Stephen

    2011-01-01

    Background The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples. Methodology and Principal Findings Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation. Significance This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models. PMID:21297994

  16. Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

    Science.gov (United States)

    Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

    2003-10-01

    A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

  17. Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Niu, Qingli; Liu, Zhijie; Yang, Jifei; Yu, Peifa; Pan, Yuping; Zhai, Bintao; Luo, Jianxun; Guan, Guiquan; Yin, Hong

    2016-12-01

    Ovine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

  18. Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray.

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    Nathan D Grubaugh

    Full Text Available BACKGROUND: Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae, Alphavirus (Togaviridae, Orthobunyavirus (Bunyaviridae, and Phlebovirus (Bunyaviridae. METHODOLOGY/PRINCIPAL FINDINGS: The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish

  19. MARS: Microarray analysis, retrieval, and storage system

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    Scheideler Marcel

    2005-04-01

    Full Text Available Abstract Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS, a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at http://genome.tugraz.at.

  20. Annotating breast cancer microarray samples using ontologies

    Science.gov (United States)

    Liu, Hongfang; Li, Xin; Yoon, Victoria; Clarke, Robert

    2008-01-01

    As the most common cancer among women, breast cancer results from the accumulation of mutations in essential genes. Recent advance in high-throughput gene expression microarray technology has inspired researchers to use the technology to assist breast cancer diagnosis, prognosis, and treatment prediction. However, the high dimensionality of microarray experiments and public access of data from many experiments have caused inconsistencies which initiated the development of controlled terminologies and ontologies for annotating microarray experiments, such as the standard microarray Gene Expression Data (MGED) ontology (MO). In this paper, we developed BCM-CO, an ontology tailored specifically for indexing clinical annotations of breast cancer microarray samples from the NCI Thesaurus. Our research showed that the coverage of NCI Thesaurus is very limited with respect to i) terms used by researchers to describe breast cancer histology (covering 22 out of 48 histology terms); ii) breast cancer cell lines (covering one out of 12 cell lines); and iii) classes corresponding to the breast cancer grading and staging. By incorporating a wider range of those terms into BCM-CO, we were able to indexed breast cancer microarray samples from GEO using BCM-CO and MGED ontology and developed a prototype system with web interface that allows the retrieval of microarray data based on the ontology annotations. PMID:18999108

  1. Simulation of microarray data with realistic characteristics

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    Lehmussola Antti

    2006-07-01

    Full Text Available Abstract Background Microarray technologies have become common tools in biological research. As a result, a need for effective computational methods for data analysis has emerged. Numerous different algorithms have been proposed for analyzing the data. However, an objective evaluation of the proposed algorithms is not possible due to the lack of biological ground truth information. To overcome this fundamental problem, the use of simulated microarray data for algorithm validation has been proposed. Results We present a microarray simulation model which can be used to validate different kinds of data analysis algorithms. The proposed model is unique in the sense that it includes all the steps that affect the quality of real microarray data. These steps include the simulation of biological ground truth data, applying biological and measurement technology specific error models, and finally simulating the microarray slide manufacturing and hybridization. After all these steps are taken into account, the simulated data has realistic biological and statistical characteristics. The applicability of the proposed model is demonstrated by several examples. Conclusion The proposed microarray simulation model is modular and can be used in different kinds of applications. It includes several error models that have been proposed earlier and it can be used with different types of input data. The model can be used to simulate both spotted two-channel and oligonucleotide based single-channel microarrays. All this makes the model a valuable tool for example in validation of data analysis algorithms.

  2. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    International Nuclear Information System (INIS)

    Railo, Antti; Pajunen, Antti; Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka; Vainio, Seppo

    2009-01-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  3. Radioactive cDNA microarray in neurospsychiatry

    International Nuclear Information System (INIS)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon

    2003-01-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  4. Radioactive cDNA microarray in neurospsychiatry

    Energy Technology Data Exchange (ETDEWEB)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon [Korea University Medical School, Seoul (Korea, Republic of)

    2003-02-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  5. Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan.

    Science.gov (United States)

    Chen, Tsu-Han; Lee, Fan; Lin, Yeou-Liang; Pan, Chu-Hsiang; Shih, Chia-Ni; Tseng, Chun-Hsien; Tsai, Hsiang-Jung

    2016-04-01

    Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus. Copyright © 2014. Published by Elsevier B.V.