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Sample records for specific affinity reagent

  1. Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents.

    Science.gov (United States)

    Katilius, Evaldas; Carmel, Andrew B; Koss, Heidi; O'Connell, Dan; Smith, Breanna C; Sanders, Glenn M; LaBerge, Greggory S

    2018-03-27

    We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (forensic casework. Copyright © 2018. Published by Elsevier B.V.

  2. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  3. A community standard format for the representation of protein affinity reagents

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Orchard, Sandra; Bertinetti, Daniela

    2010-01-01

    Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often...... that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange....... Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of Proteome...

  4. Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics.

    Science.gov (United States)

    Hung, Lien-Yu; Wang, Chih-Hung; Fu, Chien-Yu; Gopinathan, Priya; Lee, Gwo-Bin

    2016-08-07

    Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

  5. 21 CFR 864.4020 - Analyte specific reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Analyte specific reagents. 864.4020 Section 864.4020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4020 Analyte specific...

  6. Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

    Science.gov (United States)

    Gray, A C; Sidhu, S S; Chandrasekera, P C; Hendriksen, C F M; Borrebaeck, C A K

    2016-12-01

    The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. N-tritioacetoxyphthalimide: A new high specific activity tritioacetylating reagent

    International Nuclear Information System (INIS)

    Saljoughian, M.; Morimoto, Hiromi; Than, Chit

    1996-01-01

    The authors' aim was to develop a nonvolatile, stable, and facile tritioacetylating reagent and to demonstrate its use on simple peptides. Accordingly, the authors made the synthesis of high specific activity N-(tritioacetoxy) derivatives of succinimide, phthalimide, and naphthalimide a major focus. As the preferred approach, N-(tritioacetoxy)phthalimide was prepared by radical dehalogenation of N-(iodoacetoxy)phthalimide using high specific activity tributyltin tritide. This tritiated acetylation reagent was characterized by 3 H and 1 H NMR spectroscopy and by radio-HPLC. Efficacy of the reagent was investigated by tritioacetylation of several peptides at their N-terminal amino group. 26 refs., 1 fig

  8. Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2014-01-01

    Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

  9. [Comparative measurement of urine specific gravity: reagent strips, refractometry and hydrometry].

    Science.gov (United States)

    Costa, Christian Elías; Bettendorff, Carolina; Bupo, Sol; Ayuso, Sandra; Vallejo, Graciela

    2010-06-01

    The urine specific gravity is commonly used in clinical practice to measure the renal concentration/dilution ability. Measurement can be performed by three methods: hydrometry, refractometry and reagent strips. To assess the accuracy of different methods to measure urine specific gravity. We analyzed 156 consecutive urine samples of pediatric patients during April and May 2007. Urine specific gravity was measured by hydrometry (UD), refractometry (RE) and reagent strips (TR), simultaneously. Urine osmolarity was considered as the gold standard and was measured by freezing point depression. Correlation between different methods was calculated by simple linear regression. A positive and acceptable correlation was found with osmolarity for the RE as for the UD (r= 0.81 and r= 0.86, respectively). The reagent strips presented low correlation (r= 0.46). Also, we found good correlation between measurements obtained by UD and RE (r= 0.89). Measurements obtained by TR, however, had bad correlation when compared to UD (r= 0.46). Higher values of specific gravity were observed when measured with RE with respect to UD. Reagent strips are not reliable for measuring urine specific gravity and should not be used as an usual test. However, hydrometry and refractometry are acceptable alternatives for measuring urine specific gravity, as long as the same method is used for follow-up.

  10. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  11. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

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    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  12. Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

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    Gualberto Gonzalez-Sapienza

    2017-08-01

    Full Text Available With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

  13. Specificity and affinity quantification of protein-protein interactions.

    Science.gov (United States)

    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  14. Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

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    Xiakun Chu

    2014-08-01

    Full Text Available Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less flexibility leads to weaker (stronger coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

  15. Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

    DEFF Research Database (Denmark)

    Tungatt, Katie; Bianchi, Valentina; Crowther, Michael D

    2015-01-01

    Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multim...

  16. ETAC reagents: A new class of sulfhydryl site-specific radiolabelling probes for antibodies

    International Nuclear Information System (INIS)

    del Rosario, R.B.; Brocchini, S.J.; Baron, L.A.; Smith, R.H.; Lawton, R.G.; Wahl, R.L.

    1990-01-01

    A new class of bis-alkylating Michael reagents, equilibrium transfer crosslink reagents, 'ETAC', which combine the techniques of crosslinking with tethering have been synthesized. Following a succession of Michael and retro-Michael additions and elimination of the arylsulfone groups, reduced heavy-heavy and heavy-light disulfide links of an anti-ovarian IgG2a monoclonal antibody, 5G6.4, were site-specifically re-annealed via a 3-carbon bridge having a tether branch containing a designated label

  17. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  18. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

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    George P Anderson

    Full Text Available Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

  19. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  20. 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.

  1. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  2. A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

    Science.gov (United States)

    Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

    2003-01-01

    A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

  3. A new class of photoactivatable and carbene generating reagents with extremely high specific radioactivity. Synthesis, characterization and applications

    International Nuclear Information System (INIS)

    Weber, T.

    1994-01-01

    The main objective of this work was the development of new photocrosslinking and labeling reagents which show favourable photochemical properties and can be synthesized in an extremely high specific radioactivity. A key compound in the synthesis of these reagents was 2-tributyltin-4-(3-trifluormethyl-3H-diazirin-3-yl)-benzyl alcohol. Esters of this alcohol can be easily radioiodinated at a specific radioactivity of >2000 Ci/mmol under mild conditions. By experiments with a model compound it was shown that 2-iodo-4-(3-trifluormethyl-3H-diazirin-3-yl)-benzyl esters, upon photolysis, generate highly reactive (singlet) carbenes capable of inserting into CH-bonds. Equally important, there is no indication of loss of iodine under the photolysis conditions applied. Therefore two key requirements for photolabeling reagents are fulfilled. Several photoactivatable radioiodinated phospholipids have been synthesized. Their properties have been evaluated by labeling of the the membranes of intact erythrocytes and influenza viruses. Currently these lipids are used to study the interaction of various proteins (shown are experiments with MARCKS) with membranes. Furthermore a new class of actual label transfer crosslinkers (two thiol specific and one amino specific) have been developed. The range of potentialities of these reagents is currently being investigated. Finally a photoactivatable radioiodinated ceramide analogue suitable as a photoaffinity crosslinker has been developed with the goal of identifying the putative receptor of this second messenger-like lipid. Preliminary studies towards this goal are described. We are convinced that the reagents and methods presented in this work are valuable tools, and that they will find widespread use in future cell-biological and biochemical research. (author) figs., tabs., refs

  4. Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization.

    Science.gov (United States)

    Ali, Firoj; Saha, Sukdeb; Maity, Arunava; Taye, Nandaraj; Si, Mrinal Kanti; Suresh, E; Ganguly, Bishwajit; Chattopadhyay, Samit; Das, Amitava

    2015-10-15

    A new rhodamine-based reagent (L1), trapped inside the micellar structure of biologically benign Triton-X 100, could be used for specific recognition of Cr(III) in aqueous buffer medium having physiological pH. This visible light excitable reagent on selective binding to Cr(III) resulted in a strong fluorescence turn-on response with a maximum at ∼583 nm and tail of that luminescence band extended until 650 nm, an optical response that is desired for avoiding the cellular autofluorescence. Interference studies confirm that other metal ions do not interfere with the detection process of Cr(III) in aqueous buffer medium having pH 7.2. To examine the nature of binding of Cr(III) to L1, various spectroscopic studies are performed with the model reagent L2, which tend to support Cr(III)-η(2)-olefin π-interactions involving two olefin bonds in molecular probe L1. Computational studies are also performed with another model reagent LM to examine the possibility of such Cr(III)-η(2)-olefin π-interactions. Presumably, polar functional groups of the model reagent LM upon coordination to the Cr(III) center effectively reduce the formal charge on the metal ion and this is further substantiated by results of the theoretical studies. This assembly is found to be cell membrane permeable and shows insignificant toxicity toward live colon cancer cells (Hct116). Confocal laser scanning microscopic studies further revealed that the reagent L1 could be used as an imaging reagent for detection of cellular uptake of Cr(III) in pure aqueous buffer medium by Hct116 cells. Examples of a specific reagent for paramagnetic Cr(III) with luminescence ON response are scanty in the contemporary literature. This ligand design helped us in achieving the turn on response by utilizing the conversion from spirolactam to an acyclic xanthene form on coordination to Cr(III).

  5. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  6. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

    Science.gov (United States)

    Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

    2012-10-26

    For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  7. Glycoproteins of axonal transport: affinity chromatography on fucose-specific lectins

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, S.; Ohlson, C.; Karlsson, J.O.

    1982-03-01

    Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.

  8. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka

    2018-01-01

    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

  9. Diagnostic reagent system

    International Nuclear Information System (INIS)

    1976-01-01

    A solid phase reagent for use in radioimmunoassay of antigens and antibodies is described. The reagent is prepared by mixing specific antibody or radiolabeled antigen with polyethylene glycol (4000-6000) and gamma globulin in a buffer at pH 4-10 and lyophilizing, the antigens being thyroxine, triiodothyronine, digoxin and digitoxin (1-1000 μCi 125 I/μg). The buffer consists of a 0.08 molar sodium barbital solution containing 0.1% of ox-serum albumin and 0.35% of 8-anilino-1-naftalene sulfonic acid

  10. Specificity and affinity motifs for Grb2 SH2-ligand interactions

    NARCIS (Netherlands)

    Kessels, Helmut W. H. G.; Ward, Alister C.; Schumacher, Ton N. M.

    2002-01-01

    Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains.

  11. MHC class II tetramers made from isolated recombinant α and β chains refolded with affinity-tagged peptides

    DEFF Research Database (Denmark)

    Braendstrup, Peter; Justesen, Sune Frederik Lamdahl; Osterbye, Thomas

    2013-01-01

    Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking...... effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding...... a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides...

  12. Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

    OpenAIRE

    Weise, C; Kreienkamp, H J; Raba, R; Pedak, A; Aaviksaar, A; Hucho, F

    1990-01-01

    Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270-278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be pre...

  13. Specific oligobodies against ERK-2 that recognize both the native and the denatured state of the protein.

    Science.gov (United States)

    Bianchini, M; Radrizzani, M; Brocardo, M G; Reyes, G B; Gonzalez Solveyra, C; Santa-Coloma, T A

    2001-06-01

    Oligonucleotide aptamer(s), obtained by using the SELEX procedure, has been used as reagents to recognize different molecules with high affinity and specificity. However, until recently, it was not possible to obtain oligonucleotide-based reagents able to recognize proteins with high specificity in assays typical of antibodies, such as immunohistochemistry, Western blotting and immunoprecipitations. Here, we show the results obtained by applying the strategy of "target switching" to obtain specific polyclonal and monoclonal oligobodies against the protein ERK2. We were able to develop highly specific polyclonal oligobodies by using only one selection step with a temporary target and one selection step with the final target (ERK2). Since only two selection steps were required, these results demonstrate that it is possible to obtain specific reagents against a protein without a need for an "in vitro evolution" using many selection steps, or error-prone polymerases. After one additional selection step, the polyclonal oligobodies were cloned to obtain a highly specific monoclonal oligobody.

  14. First-in-man open clinical trial of a combined rdESAT-6 and rCFP-10 tuberculosis specific skin test reagent.

    Directory of Open Access Journals (Sweden)

    Winnie Bergstedt

    2010-06-01

    Full Text Available Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity.In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1ratio1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 microg or 0.1 microg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN gamma response just below the Quantiferon(R cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded.The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease.ClinicalTrials.gov NCT00793702.

  15. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Sternberg, Claus

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents...... which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea...... epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent...

  16. Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

    Directory of Open Access Journals (Sweden)

    Michael Allevato

    Full Text Available The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX bind Enhancer box (E-box DNA elements (CANNTG and have the greatest affinity for the canonical MYC E-box (CME CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87% of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

  17. Colorimetric Detection of Specific DNA Segments Amplified by Polymerase Chain Reactions

    Science.gov (United States)

    Kemp, David J.; Smith, Donald B.; Foote, Simon J.; Samaras, N.; Peterson, M. Gregory

    1989-04-01

    The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.

  18. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    Science.gov (United States)

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  19. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  20. Development of versatile universal reagent immunoradiometric assay technique

    International Nuclear Information System (INIS)

    Hazra, D.K.

    1982-10-01

    Immunoradiometric assays, which make use of labelled antibodies, potentially offer better sensitivity and specificity than do radioimmunoassays, which use labelled antigens. In addition, they can in principle be performed in a particularly convenient scheme wherein the same labelled reagent may be used for many different analytes - thus serving as a ''universal'' labelled reagent. Thus if the specific antibody for every analyte is raised in rabbits, and an anti-rabbit antibody is labelled, the latter may be added after the specific antibody to quantify the amount of specific antibody bound to analyte and thereby the amount of analyte present. The potential greater sensitivity and specificity of the immunoradiometric procedure, coupled with the potential convenience of the ''universal'' labelled reagent, might allow such immunoradiometric techniques to be used effectively in the study of communicable diseases in developing countries. Development of these procedures was the subject of this investigation. Many components of these procedures had to be explored and provisionally optimized, including coating of assay tubes with ''extraction'' antibody, immunological purification of antibodies, labelling of antibodies, and intermediate steps toward these goals. Applications were thereupon tested using those provisionally optimized components. The ''universal'' labelled reagent, a donkey anti-rabbit antiserum, was successfully used in the assay of TSH; however, cross reactions of the reagent with non-rabbit immunoglobulins and other materials present seriously limited the sensitivity of the method. Using conventional immunoradiometric procedures, circulating TB and amoebic antibodies could be detected in patients suffering from these diseases. Similarly, circulating antigens in the same patients could also be detected, but not with sufficient sensitivity and specificity to provide a reliable analytical system. Numerous improvements will be required before these techniques

  1. Organization of a system of guarantee of quality for the control of specifications of chemical reagents

    International Nuclear Information System (INIS)

    Bustamante Gutierrez, M. G.

    2000-01-01

    Analytic methods were implemented based on valuations acid-base and redox for the quantitative determination of sodium hidroxid, iodine and iron chloride III, like part of a system of quality for the technical specifications of these reagents. The planning of its system of quality includes two fundamental parts: insurance and control of quality. In the insurance part the state of operation of the team that you uses settled down, gauges the one that achieve to maintain under the supervision of a single operator (electronic equip and glassware) and the design of the appropriate documents settled down to carry out the periodic supervision of the acting of the same ones and other aspects characteristic of the system (experimental results). Also protocolized and validated the analytic methods to use following the approaches of precision given by ASTM, organism whose methodologies are recognized in the country like official; other procedures, as that of calibration of the volumetric equipments, they were also protocolized. In the part of control of quality, the limits settled down and procedures were applied the scales and used other, to the necessary distilled water to carry out the determinations and to the used chemical reagents. With base in the obtained results you determines that the analyzed reagents fulfill the specifications of ACS (and with those reported by the maker) at the same time settled down that the laboratory 09 of the Chemistry School, used in a large part of development of this project, it didn't fulfill the necessary requirements so that it works as laboratory of control of quality. As an alternative, the administration of the School outlines as solution the use of the Laboratory of Insurance of the Quality from the Unit of Service to the Industry for such end [es

  2. [An evaluation of the China-made HIV antibody test reagents].

    Science.gov (United States)

    Zheng, X W; Zhu, D

    1990-06-01

    This paper reports the results of the evaluation of the China-made HIV antibody screening test reagents, including the IF and IE reagents prepared by the Institute of Virology, CAPM, the ELISA reagent prepared by the Shanghai Institute of Biological Products. Based on the results, the sensitivities of the IF and IE are from 91.2% to 96.9%; the specificities, from 94.6% to 97.3%. Due to the low HIV prevalence in China, the predictive values of negative of these reagents are up to 100%; but the predictive values of positive are very low. It is suggested that these reagents can be used for HIV antibody screen testing in China. The package of some reagents should be improved, the price of some reagents should be decreased.

  3. Evaluation of monoclonal anti-A and anti-B and affinity-purified Ulex europaeus lectin I for forensic blood grouping.

    Science.gov (United States)

    Gaensslen, R E; Lee, H C; Carroll, J E

    1984-01-01

    Two different monoclonal anti-A and anti-B and several different affinity purified Ulex europaeus lectin I reagents were evaluated and compared with conventional anti-A and anti-B sera and Ulex anti-H for serologic properties, in inhibition tests with secretor salivas, and in elution tests with bloodstains. The monoclonal and purified reagents were found to be comparable to conventional ones, and accordingly suitable for forensic inhibition and elution procedures.

  4. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  5. Characterization of Reagent Pencils for Deposition of Reagents onto Paper-Based Microfluidic Devices

    Directory of Open Access Journals (Sweden)

    Cheyenne H. Liu

    2017-08-01

    Full Text Available Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol.

  6. Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

    Science.gov (United States)

    Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

    2018-01-01

    The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

  7. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  8. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.

    Science.gov (United States)

    Dolman, Nick J; Kilgore, Jason A; Davidson, Michael W

    2013-07-01

    Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. © 2013 by John Wiley & Sons, Inc.

  9. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  10. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  11. Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

    International Nuclear Information System (INIS)

    Preston, D.A.; Wu, C.Y.; Blaszczak, L.C.; Seitz, D.E.; Halligan, N.G.

    1990-01-01

    Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. 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. We demonstrated the general equivalence of commercially obtained [ 3 H]penicillin G and locally synthesized [ 125 I]penicillin V (IPV) in their recognition of bacterial PBPs. Profiles of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or [3H]penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with [ 3 H]penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4 degrees C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4 degrees C. IPV represents a practical and stable reagent for rapid PBP assays

  12. Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds - Applications in acidic modification-specific proteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Larsen, Martin R

    2011-01-01

    biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field...... matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated....... The development of TiO(2)-based chromatographic strategies for separation of various biomolecules from its introduction for small molecules more than 20years ago until recent proteomics applications today will be reviewed here....

  13. A specific Tween-80-Rhodamine S-MWNTs phosphorescent reagent for the detection of trace calcitonin

    Energy Technology Data Exchange (ETDEWEB)

    Liu Jiaming, E-mail: zzsyliujiaming@163.com [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Huang Xiaomei; Zhang Lihong; Zheng Zhiyong [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou, 363000 (China); Lin Xuan; Zhang Xiaoyang; Jiao Li; Cui Malin [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Jiang Shulian [Fujian Provincial Bureau of Quality and Technical Supervision, Zhangzhou, 363000 (China); Lin Shaoqin [Department of Biochemistry, Fujian Education College, Fuzhou 350001 (China)

    2012-09-26

    Graphical abstract: A new Tween-80-Rhodamine S-water-soluble multi-walled carbon nanotubes (Tween-80-Rhod.S-MWNTs-EDC-NHS, TRMEN) phosphorescent labelling reagent was developed. High sensitive solid substrate room temperature phosphorescence immunoassay (SSRTPIA) for the determination of calcitonin (CT) in human serum and the prediction of human diseases based on the TRMEN could be used to label anti-calcitonin antibody (Ab{sub CT}) to form the TRMEN-Ab{sub CT} labelling product, which could take high specific immunoreaction with CT causing that the {Delta}I{sub p} of the system was linear to the content of CT. Moreover, the reaction mechanisms of both labelling Ab{sub CT} by TRMEN and SSRTPIA for the determination of trace CT were discussed. This research not only provides a new hormones analysis method, but also expands the application field of MWNTs and promotes the development of SSRTP and IA. --Highlights: Black-Right-Pointing-Pointer A Tween-80-Rhodamine S-multi-walled carbon nanotubes labelling reagent was developed. Black-Right-Pointing-Pointer The phosphorescence immunoassay was established for the determination of calcitonin. Black-Right-Pointing-Pointer This method has been applied to determine CT and the prediction of diseases. Black-Right-Pointing-Pointer The structure of MWNTs was characterized with SEM and IR. Black-Right-Pointing-Pointer The mechanisms for both determining trace CT and labelling Ab{sub CT} were discussed. - Abstract: The present study proposed a simple sensitive and specific immunoassay for the quantification of calcitonin (CT) in human serum with water-soluble multi-walled carbon nanotubes (MWNTs). The -COOH group of MWNTs could react with the -NH- group of rhodamine S (Rhod.S) molecules to form Rhod.S-MWNTs, which could emit room temperature phosphorescence (RTP) on acetate cellulose membrane (ACM) and react with Tween-80 to form micellar compound. Tween-80-Rhod.S-MWNTs (TRM), as a phosphorescent labelling reagent, could

  14. Affinity imaging mass spectrometry (AIMS): high-throughput screening for specific small molecule interactions with frozen tissue sections.

    Science.gov (United States)

    Yoshimi, T; Kawabata, S; Taira, S; Okuno, A; Mikawa, R; Murayama, S; Tanaka, K; Takikawa, O

    2015-11-07

    A novel screening system, using affinity imaging mass spectrometry (AIMS), has been developed to identify protein aggregates or organ structures in unfixed human tissue. Frozen tissue sections are positioned on small (millimetre-scale) stainless steel chips and incubated with an extensive library of small molecules. Candidate molecules showing specific affinity for the tissue section are identified by imaging mass spectrometry (IMS). As an example application, we screened over a thousand compounds against Alzheimer's disease (AD) brain tissue and identified several compounds with high affinity for AD brain sections containing tau deposits compared to age-matched controls. It should also be possible to use AIMS to isolate chemical compounds with affinity for tissue structures or components that have been extensively modified by events such as oxidation, phosphorylation, acetylation, aggregation, racemization or truncation, for example, due to aging. It may also be applicable to biomarker screening programs.

  15. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies

    Directory of Open Access Journals (Sweden)

    Jennifer D. Stone

    2013-08-01

    Full Text Available Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional alpha-beta T cell receptor (TCR against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR consisting of a single-chain antibody as an Fv fragment (scFv linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the alpha-beta TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

  16. Tau passive immunotherapy in mutant P301L mice: antibody affinity versus specificity.

    Directory of Open Access Journals (Sweden)

    Cristina d'Abramo

    Full Text Available The use of antibodies to treat neurodegenerative diseases has undergone rapid development in the past decade. To date, immunotherapeutic approaches to Alzheimer's disease have mostly targeted amyloid beta as it is a secreted protein that can be found in plasma and CSF and is consequently accessible to circulating antibodies. Few recent publications have suggested the utility of treatment of tau pathology with monoclonal antibodies to tau. Our laboratory has begun a systematic study of different classes of tau monoclonal antibodies using mutant P301L mice. Three or seven months old mutant tau mice were inoculated weekly with tau monoclonal antibodies at a dose of 10 mg/Kg, until seven or ten months of age were reached respectively. Our data strongly support the notion that in P301L animals treated with MC1, a conformational monoclonal antibody specific for PHF-tau, the rate of development of tau pathology is effectively reduced, while injecting DA31, a high affinity tau sequence antibody, does not exert such benefit. MC1 appears superior to DA31 in overall effects, suggesting that specificity is more important than affinity in therapeutic applications. Unfortunately the survival rate of the P301L treated mice was not improved when immunizing either with MC1 or PHF1, a high affinity phospho-tau antibody previously reported to be efficacious in reducing pathological tau. These data demonstrate that passive immunotherapy in mutant tau models may be efficacious in reducing the development of tau pathology, but a great deal of work remains to be done to carefully select the tau epitopes to target.

  17. Affinity reagent technology development and application to rapid immunochromatographic pathogen detection

    Science.gov (United States)

    Sooter, Letha J.; Stratis-Cullum, Dimitra N.; Zhang, Yanting; Daugherty, Patrick S.; Soh, H. Tom; Pellegrino, Paul; Stagliano, Nancy

    2007-09-01

    Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

  18. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  19. Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches.

    Science.gov (United States)

    Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis; Ricci, Francesco

    2013-12-23

    Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

  20. Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Williams, Stephanie M; Venkataraman, Lalitha; Tian, Huilai; Khan, Galam; Harris, Brent T; Sierks, Michael R

    2015-02-12

    Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

  1. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  2. Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

    Science.gov (United States)

    Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

    2005-01-01

    Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

  3. Specific capture of uranyl protein targets by metal affinity chromatography

    International Nuclear Information System (INIS)

    Basset, C.; Dedieu, A.; Guerin, P.; Quemeneur, E.; Meyer, D.; Vidaud, C.

    2008-01-01

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO 2 2+ ) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of amino-phosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. (authors)

  4. Handling Pyrophoric Reagents

    Energy Technology Data Exchange (ETDEWEB)

    Alnajjar, Mikhail S.; Haynie, Todd O.

    2009-08-14

    Pyrophoric reagents are extremely hazardous. Special handling techniques are required to prevent contact with air and the resulting fire. This document provides several methods for working with pyrophoric reagents outside of an inert atmosphere.

  5. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  6. Enzyme-Linked Immunosorbent Assay Specific for (1→6) Branched, (1→3)-β-d-Glucan Detection in Environmental Samples

    OpenAIRE

    Milton, Donald K.; Alwis, K. Udeni; Fisette, Leslie; Muilenberg, Michael

    2001-01-01

    (1→3)-β-d-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1→6) branched, (1→3)-β-d-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1→3)-β-d-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall β-d-glucans...

  7. Method for controlled introduction of reagent into a liquid

    Energy Technology Data Exchange (ETDEWEB)

    Newlove, J.C.; McDougall, L.A.

    1988-11-29

    It is an object of this invention to provide a method for using an article to enhance the production of hydrocarbons from geological reservoirs, more particularly from fractured formations. It is an additional object to devise a method for providing controlled release of a reagent downhole, in a pipeline, or in other oil-containing environments or fluids. Thus, there is provided a method for releasing a treating reagent, such as a wax crystal modifier, scale inhibitor, demulsifier, corrosion inhibitor, antioxidant, and biocide, into a liquid hydrocarbon stream. A plurality of porous, substantially wax-free, plastic particles having a softening point above 60/sup 0/C and being chemically resistant to the hydrocarbon stream are placed in the stream. The said particles contain the treating reagent in their pores, said reagent being insoluble in water and in the particles and being leachable on contact with the stream. The hydrocarbon stream is then flowed past said particles and the reagent is leached from the particle pores. In a specific aspect of this invention, a method is provided for recovering crude oil from an underground formation by means of: depositing the aforementioned particles, containing a suitable reagent, downhole in the oil-producing region of the formation; flowing the oil through the deposited particles, thereby leaching the reagent into the oil; and recovering the oil modified by the presence of an active amount of said reagent. Experiments are described to illustrate ways of producing the polymeric particles of the invention and to illustrate the processes of the invention.

  8. Labelling of HBV-DNA probe using reagent made in China

    International Nuclear Information System (INIS)

    Wang Quanshi

    1991-01-01

    The labelling hepatitis Bvirus DNA (HBV-DNA) probe was studied by using reagent made in China. The results showed that: (1) The dNTPs with high specific activity was necessary for the labelling of nigh specific activity HBV-DNA probe; (2) reaction of labelling HBV-DNA probe was completed in a few minutes; (3) 0.37 MBq 3 H dTTP (specific activity 1.554TBq/mmol) was enough to label 1 μg HBV-DNA and the specific activity of probe reached 3.4 x 10 cpm/μg; (4) 7 MBqα- 32 P dATP (specific activity > 111 TBq/mmol) can label HBV-DNA probe to specific activity 1.35 x 10 cpm/μg. It was concluded that the reagent made in China can be used for the study in molecular biology

  9. The universal statistical distributions of the affinity, equilibrium constants, kinetics and specificity in biomolecular recognition.

    Directory of Open Access Journals (Sweden)

    Xiliang Zheng

    2015-04-01

    Full Text Available We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity, the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics.

  10. Optimal lipofection reagent varies with the molecular modifications of the DNA.

    Science.gov (United States)

    Conrad, A H; Behlke, M A; Jaffredo, T; Conrad, G W

    1998-10-01

    Cationic lipid reagents differ in their cytofection efficacy with different cell types. No evidence has addressed whether the same lipid reagent is best for different DNAs in a single cell line. Immortalized avian embryonic cardiomyocytes cultured in vitro were tested with 15 cationic lipid reagents using (A) a beta-gal expression plasmid, (B) a fluorescein-tagged, phosphorothioate-modified ODN B, (C) a fluorescein-tagged, ethoxy-modified ODN C with the same nucleotide sequence as ODN B, and (D) a fluorescein-tagged, phosphorothioate-modified ODN D with a different nucleotide sequence from ODNs B and C. Cytofection was scored as percent of cells expressing beta-gal activity or showing diffuse cellular fluorescence. The best lipid reagents for the phosphorothioate-modified ODNs were ODN-specific and markedly different from the best lipid reagents for the expression plasmid or for the ethoxy-modified ODN. These results suggest that the best cationic lipid reagent for a particular cell type varies with the physical and chemical form of the DNA being transfected into the cells.

  11. Development of bacterial display peptides for use in biosensing applications

    Science.gov (United States)

    Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

    2012-06-01

    Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

  12. Polymer-supported reagents with enhanced metal ion recognition: Application to separations science

    International Nuclear Information System (INIS)

    Alexandratos, S.D.

    1993-01-01

    The design and development of polymer-supported reagents with ever-increasing specificities for targeted metal ions remains an important areas of research. The need for efficient separation schemes for both ions and molecules has been outlined in a report by the National Research Council (King) and will gain increased emphasis as environmental restoration is pursued. Polymer-supported reagents are unique in their ability to be applied in an environmentally benign manner to a host of challenges. Such reagents, in the form of beads, can be applied to continuous separation processes ranging from the removal of metal ions in water to the recovery of medicinal drugs produced through biotechnological means. The application of polymer-supported reagents to metal ion separations still requires developing a fundamental understanding of ligand-metal interactions, the role of the polymer in those interactions, and the methods of synthesizing such polymeric reagents in a readily applicable form. Ion exchange resins with sulfonic acid ligands are the prototypical polymer-supported reagents, and their properties have been exhaustively studied (Helfferich). The high acidity of the sulfonic acid group, however, precludes much selectivity, and it displays a very limited range of reaction free energy values with different metal ions (Boyd et al.). The carboxylic acid ligand, present in the acrylate resins, is more selective, though its weak acidity requires relatively high pH solutions for it to be effective. Research has thus been focused on the preparation of polymer-supported reagents with high levels of specificity for targeted metal ions

  13. Affine LIBOR Models with Multiple Curves

    DEFF Research Database (Denmark)

    Grbac, Zorana; Papapantoleon, Antonis; Schoenmakers, John

    2015-01-01

    are specified following the methodology of the affine LIBOR models and are driven by the wide and flexible class of affine processes. The affine property is preserved under forward measures, which allows us to derive Fourier pricing formulas for caps, swaptions, and basis swaptions. A model specification...... with dependent LIBOR rates is developed that allows for an efficient and accurate calibration to a system of caplet prices....

  14. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    International Nuclear Information System (INIS)

    Öberg, Christine; Belikov, Sergey

    2012-01-01

    Highlights: ► wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, ΔN-hH1.4, were compared. ► Both histones bind to chromatin, however, ΔN-hH1.4 displays lower binding affinity. ► Interaction of ΔN-hH1.4 with chromatin includes a significant unspecific component. ► N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). The ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that ΔN-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  15. Validity of HydraTrend reagent strips for the assessment of hydration status.

    Science.gov (United States)

    Abbey, Bryce M; Heelan, Kate A; Brown, Gregory A; Bartee, Rodrick T

    2014-09-01

    Hydration is used by athletic governing organizations for weight class eligibility. The measurement of urine specific gravity (USG) as a measure of hydration by reagent strips is a controversial issue. The purpose of this study was to determine the validity of HydraTrend reagent strips that facilitate the correction of USG for alkaline urine samples against refractometry for the assessment of USG. Fifty-one participants (33 males, age = 22.3 ± 1.3 years; 18 females, age = 22.4 ± 1.2 years) provided 84 urine samples. The samples were tested for USG using refractometry and reagent strips and for pH using reagent strips and a digital pH meter. Strong correlation coefficients were found between refractometry and reagent strips for USG (rs(82) = 0.812, p refractometry with USG >1.020, pass reagent strips with USG ≤1.020) occurred 39% (33/84) of the time and false negative results for National Federation of State High School Association (NFHS) requirements (fail refractometry with USG >1.025, pass reagent strips with USG ≤1.025) occurred 14% (12/84) of the time. There were no false positives (pass refractometry and fail reagent strips) for NCAA or NFHS requirements. These data show that refractometry and reagent strips have strong positive correlations. However, the risk of a false negative result leading to incorrect certification of euhydration status outweighs the benefits of the HydraTrend reagent strips for the measurement of USG.

  16. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  17. Diazo Reagents with Small Steric Footprints for Simultaneous Arming/SAR Studies of Alcohol-Containing Natural Products via O–H Insertion

    Science.gov (United States)

    Chamni, Supakarn; He, Qing-Li; Dang, Yongjun; Bhat, Shridhar; Liu, Jun O.; Romo, Daniel

    2011-01-01

    Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O–H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors. PMID:21894934

  18. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. High affinity, ligand specific uptake of complexed copper-67 by brain tissue incubated in vitro

    International Nuclear Information System (INIS)

    Barnea, A.; Hartter, D.E.

    1987-01-01

    Copper is an essential metal that is highly concentrated in the brain. The blood, the sole source of tissue Cu, contains 16-20 μM Cu, of which >95% is complexed to proteins and 2 was 10 times greater than that of CuAlbumin or Cu(II). Within the range of 0.2-150μM Cu, multiple uptake sites for CuHis were apparent. Increasing the molar ratio of His:Cu had a differential effect on Cu uptake: enhancing uptake at [Cu] 1 μM. Thus, using a His:Cu ratio of 1000, they observed a high affinity process exhibiting saturating and half saturating values of 5 μM and 1.5 μM Cu, respectively; using a His:Cu ratio of 2, they observed a low affinity process exhibiting saturating and half-saturating values of 100 μM and 40 μM Cu, respectively. Both processes required thermic but not metabolic energy, suggestive of facilitated diffusion. Considering the blood brain barrier for proteins, CuHis appears to be the major substrate for Cu uptake by neuronal tissue. They demonstrate the existence of a ligand specific, high affinity (apparent Km about 1.5 μM Cu) uptake process for CuHis in the brain, operative at the physiological concentration range of CuHis and histidine

  20. 21 CFR 866.3500 - Rickettsia serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rickettsia serological reagents. 866.3500 Section... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3500 Rickettsia serological reagents. (a) Identification. Rickettsia serological reagents are devices that consist of antigens...

  1. 21 CFR 866.3405 - Poliovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Poliovirus serological reagents. 866.3405 Section... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405 Poliovirus serological reagents. (a) Identification. Poliovirus serological reagents are devices that consist of antigens...

  2. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  3. Production of lipase from Geotrichum sp and adsorption studies on affinity resin

    Directory of Open Access Journals (Sweden)

    E. S. KAMIMURA

    1999-06-01

    Full Text Available There is a growing interest in microbial lipase production due to its great potential for industrial applications such as food additives, industrial reagents and stain removers, as well as for medical applications. Specially for medical applications a high degree of purity is required, which is accomplished with high resolution chromatographic techniques. Affinity chromatography is considered a very high resolution chromatographic technique. In this work the adsorption isotherms and kinetics of the adsorption of lipase from Geotrichum sp on biospecific resin were determined. The resin was prepared using EAH sepharose 4B gel (Pharmacia, made to react with oleic acid as the specific ligand.The lipase was produced in a five-liter fermenter, with both complex and synthetic media. Fermentation conditions were a temperature of 30°C, an aeration of 1VVM and an agitation of 400 rpm. Maximum lipase activity was around 28 U/ml after 10 hours of fermentation for the complex medium. The kinetic model and parameters were determined by dynamic fitting to experimental results using the fourth-order Runge-Kutta method.

  4. 21 CFR 866.3510 - Rubella virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3510 Rubella virus... Clinical Laboratory Standards': (i) 1/LA6 “Detection and Quantitation of Rubella IgG Antibody: Evaluation... Products in the Clinical Laboratory, October 1997,” (ii) 1/LA18 “Specifications for Immunological Testing...

  5. Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides.

    Science.gov (United States)

    Lohse, Jonas; Swier, Lotteke J Y M; Oudshoorn, Ruben C; Médard, Guillaume; Kuster, Bernhard; Slotboom, Dirk-Jan; Witte, Martin D

    2017-04-19

    In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.

  6. Reagent strip testing is not sensitive for the screening of asymptomatic bacteriuria in pregnant women.

    Science.gov (United States)

    Lumbiganon, Pisake; Chongsomchai, Chompilas; Chumworathayee, Bundit; Thinkhamrop, Jadsada

    2002-08-01

    The objective of the study was to assess the diagnostic performance of the reagent strip in screening for asymptomatic bacteriuria in pregnant women using urine culture as a gold standard. This study comprised 204 asymptomatic pregnant women who attended their first antenatal care at Srinagarind Hospital, Khon Kaen University from April 1, 1999 to June 30, 1999. Women with symptoms of urinary tract infection, antibiotic treatment within the previous 7 days, pregnancy-induced hypertension, bleeding per vagina and history of urinary tract diseases were excluded. Urine specimens were collected by clean catched midstream urine technique for urinalysis, reagent strip test and urine culture. Diagnostic performance of reagent strip in terms of sensitivity, specificity, positive and negative predictive value was analyzed. Urine reagent strip test had a sensitivity of 13.9 per cent, a specificity of 95.6 per cent, a positive predictive value of 46.1 per cent, a negative predictive value of 80.6 per cent in detecting asymptomatic bacteriuria in pregnant women.

  7. Characterization and enzyme-conjugation of a specific anti-L1 nanobody.

    Science.gov (United States)

    Minaeian, Sara; Rahbarizadeh, Fatemeh; Zarkesh Esfahani, Sayyed Hamid; Ahmadvand, Davoud

    2012-01-01

    Persistent infection of the human papillomaviruses (HPV) has been shown to result in cervical cancer and intraepithelial neoplasia. Early detection and screening programs are essential strategies against cervical cancer. A nanobody is the smallest antigen-binding fragment known and is derived from a camelid heavy-chain antibody. This tiny protein shows high solubility and stability. It can be produced cost-effectively with high yield production. In this study, we enriched a nanobody library against the L1 protein of HPV. Several colons were selected from this enriched library using monoclonal phage-enzyme linked immunosorbent assay (phage-ELISA) and analyzed for identification of nanobody genes. The expression of nanobody fragments was performed in Rosetta gami2. The C74 nanobody that showed strong binding to the L1 protein of HPV16 was selected, purified, and characterized by Western blotting and ELISA. The selected nanobody was tested for sensitivity, specificity, and affinity. A nanobody conjugated to horseradish peroxidase (HRP) was selected and used for detection of L1 protein of HPV16. This study demonstrates that the C74-HRP, due to its specificity and good binding affinity for a specific viral antigen, is a potential diagnostic tool that can be used as a promising reagent for the new generation of HPV diagnosis approaches.

  8. 21 CFR 866.3350 - Leptospira spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leptospira spp. serological reagents. 866.3350... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350 Leptospira spp. serological reagents. (a) Identification. Leptospira spp. serological reagents are devices that...

  9. 21 CFR 866.3200 - Echinococcus spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Echinococcus spp. serological reagents. 866.3200... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3200 Echinococcus spp. serological reagents. (a) Identification. Echinococcus spp. serological reagents are devices that...

  10. 21 CFR 866.3415 - Pseudomonas spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pseudomonas spp. serological reagents. 866.3415... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3415 Pseudomonas spp. serological reagents. (a) Identification. Pseudomonas spp. serological reagents are devices that...

  11. Affine and quasi-affine frames for rational dilations

    DEFF Research Database (Denmark)

    Bownik, Marcin; Lemvig, Jakob

    2011-01-01

    In this paper we extend the investigation of quasi-affine systems, which were originally introduced by Ron and Shen [J. Funct. Anal. 148 (1997), 408-447] for integer, expansive dilations, to the class of rational, expansive dilations. We show that an affine system is a frame if, and only if......, the corresponding family of quasi-affine systems are frames with uniform frame bounds. We also prove a similar equivalence result between pairs of dual affine frames and dual quasi-affine frames. Finally, we uncover some fundamental differences between the integer and rational settings by exhibiting an example...

  12. Na+-K+-ATPase in rat skeletal muscle: muscle fiber-specific differences in exercise-induced changes in ion affinity and maximal activity

    DEFF Research Database (Denmark)

    Juel, Carsten

    2008-01-01

    It is unclear whether muscle activity reduces or increases Na(+)-K(+)-ATPase maximal in vitro activity in rat skeletal muscle, and it is not known whether muscle activity changes the Na(+)-K(+)-ATPase ion affinity. The present study uses quantification of ATP hydrolysis to characterize muscle fiber...... membranes of glycolytic muscle, which abolished the fiber-type difference in Na(+) affinity. K(m) for K(+) (in the presence of Na(+)) was not influenced by running. Running only increased the maximal in vitro activity (V(max)) in total membranes from soleus, whereas V(max) remained constant in the three...... other muscles tested. In conclusion, muscle activity induces fiber type-specific changes both in Na(+) affinity and maximal in vitro activity of the Na(+)-K(+)-ATPase. The underlying mechanisms may involve translocation of subunits and increased association between PLM units and the alphabeta complex...

  13. Production of reagents for cleaning fluids

    Energy Technology Data Exchange (ETDEWEB)

    Grunberg, I V; Korostyleva, R N; Pytel, S P; Spasskii, P I; Titarenko, N K; Trachtenberg, S I; Yushkevich, V I

    1980-10-25

    A method for producing reagents for cleaning fluids is proposed using polymerization of acrylonitril, metachrylate or a mixture of the two in water and saponification of the polymers with alakali. To reduce the consumption of monomers and increase the quality of the reagents, 0.4-1.0 parts humic substances, 0.2-1.0 parts hydrolizate from tanning waste products and 1.2-4.0 parts monomers are added to the reaction medium, followed by copolymerization in an acid medium. The proposed method ensures quality reagents which combine lower water yield with a moderate increase in viscosity when acting on clay solutions. Compared with the current method, this method lowers the consumption of an expensive and hard-to-find monomer 1.2-1.4X for one ton of reagent, which lowers the cost of raw material by 1.3-1.7X. This results in a savings of 195-385 rubles per ton of reagent, 600-1200 thousand at 3000 tons/yr.

  14. A reagent for processing drilling muds

    Energy Technology Data Exchange (ETDEWEB)

    Polyakov, G.A.; Khon-Pak, A.T.; Khon, A.V.; Normatov, L.N.; Telegin, B.V.

    1983-01-01

    A reagent is proposed for processing drilling muds. It contains an acrylic polymer and potassium permanganate. The reagent is distinguished by the fact that in order to improve the quality of the drilling muds by increasing their salt resistance, the reagent contains hydrolized nitron fiber as the acrylic polymer with the following component relationship (in percent by weight): potassium permanganate, 0.015 to 0.065 and hydrolyzed nitron fiber, the remainder.

  15. Fast biosensor with reagent layer

    NARCIS (Netherlands)

    2008-01-01

    A detection system and a sensor chip for detecting target mols., and thus corresponding analytes in a sample is described. Typically the detection system includes a sensor chip. The sensor chip (1) comprises on its detection surface a dissolvable reagent layer. When the dissolvable reagent layer is

  16. High-throughput bioscreening system utilizing high-performance affinity magnetic carriers exhibiting minimal non-specific protein binding

    International Nuclear Information System (INIS)

    Hanyu, Naohiro; Nishio, Kosuke; Hatakeyama, Mamoru; Yasuno, Hiroshi; Tanaka, Toshiyuki; Tada, Masaru; Nakagawa, Takashi; Sandhu, Adarsh; Abe, Masanori; Handa, Hiroshi

    2009-01-01

    For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

  17. 21 CFR 864.8100 - Bothrops atrox reagent.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

  18. Development of national immunoassay reagent programmes

    International Nuclear Information System (INIS)

    Sufi, S.B.; Micallef, J.V.; Ahsan, R.; Goncharov, N.P.

    1992-01-01

    Despite the existence of networks of fully equipped laboratories with well-trained staff, the availability of immunodiagnostic services in developing countries is often limited by the high cost of imported kits. There are a number of ways of tackling this problem, ranging from bulk purchase of kits or reagents to local development and production of assay systems. Argentina/Chile, China, Cuba/Mexico, and Thailand are amongst the countries which have established local immunoassay reagent programmes to manufacture low cost, high quality immunoassay reagents. Kits from these projects are now beginning to become available, and it is hoped that they will promote national diagnostic services and research, as well as stimulating the development of reagent programmes for other analytes. (author). 4 refs, 1 tab

  19. Diagnóstico das meningites através de fitas reagentes Diagnosis of meningitis with reagent strips

    Directory of Open Access Journals (Sweden)

    Roberta M.C. Romanelli

    2001-06-01

    ção Hospitalar do Estado de Minas Gerais, CGP-FHEMIG during the daytime hours from May of 1997 to May 1999, and who presented with clinical suspicion of meningitis. Patients ranged in age from one month to 12 years (median 12 months. Results from the cytological and biochemical assay (cellularity, cerebrospinal fluid glucose and protein levels were obtained from 154 patients. These results were subsequently compared with the reaction of cerebrospinal fluid in reagent strips. RESULTS: The cytological and biochemical assay identified 43 cases of probable bacterial meningitis, 19 of probable viral meningitis, and 83 with no alterations. According to the reagent strips, there were 41 cases of probable bacterial meningitis, 2 of probable viral meningitis, and 71 with no alterations. By comparing the results of reagent strips and those of the cytological and biochemical assay, we obtained values for sensitivity, specificity, positive and negative predictive values, and accuracy (respectively 90.7; 98.1; 95.1; 96.4; and 96.1. Statistical analysis using McNemer test did not indicate significant differences between the two methods in the diagnosis of bacterial meningitis (P=0.68. Kappa statistics indicated a high level of agreement between the tests (P<0.0001. CONCLUSIONS: Our results suggest that reagent strips may be a useful additional resource in the diagnosis of bacterial meningitis, especially when it is difficult to collect a sufficient amount of cerebrospinal fluid or to indicate the initial treatment.

  20. Inventory management and reagent supply for automated chemistry.

    Science.gov (United States)

    Kuzniar, E

    1999-08-01

    Developments in automated chemistry have kept pace with developments in HTS such that hundreds of thousands of new compounds can be rapidly synthesized in the belief that the greater the number and diversity of compounds that can be screened, the more successful HTS will be. The increasing use of automation for Multiple Parallel Synthesis (MPS) and the move to automated combinatorial library production is placing an overwhelming burden on the management of reagents. Although automation has improved the efficiency of the processes involved in compound synthesis, the bottleneck has shifted to ordering, collating and preparing reagents for automated chemistry resulting in loss of time, materials and momentum. Major efficiencies have already been made in the area of compound management for high throughput screening. Most of these efficiencies have been achieved with sophisticated library management systems using advanced engineering and data handling for the storage, tracking and retrieval of millions of compounds. The Automation Partnership has already provided many of the top pharmaceutical companies with modular automated storage, preparation and retrieval systems to manage compound libraries for high throughput screening. This article describes how these systems may be implemented to solve the specific problems of inventory management and reagent supply for automated chemistry.

  1. Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

    Science.gov (United States)

    Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

    2005-10-01

    Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

  2. 21 CFR 866.3720 - Streptococcus spp. exo-enzyme reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Streptococcus spp. exo-enzyme reagents. 866.3720... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3720 Streptococcus spp. exo-enzyme reagents. (a) Identification. Streptococcus spp. exoenzyme reagents are devices used...

  3. Inactivation of rabies diagnostic reagents by gamma radiation

    International Nuclear Information System (INIS)

    Gamble, W.C.; Chappell, W.A.; George, E.H.

    1980-01-01

    Treatment of CVS-11 rabies adsorbing suspensions and street rabies infected mouse brains with gamma radiation resulted in inactivated reagents that are safer to distribute and use. These irradiated reagents were as sensitive and reactive as the nonirradiated control reagents

  4. Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

    Directory of Open Access Journals (Sweden)

    Feifan Yu

    Full Text Available Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁. Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs. The best variant, denoted Z(F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe

  5. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    Science.gov (United States)

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  6. 21 CFR 866.3255 - Escherichia coli serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

  7. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    Science.gov (United States)

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-05

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

  8. Characterizing complex polysera produced by antigen-specific immunization through the use of affinity-selected mimotopes.

    Directory of Open Access Journals (Sweden)

    Galina Denisova

    Full Text Available BACKGROUND: Antigen-based (as opposed to whole organism vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.

  9. Molecular electron affinities

    International Nuclear Information System (INIS)

    Fukuda, E.K.

    1983-01-01

    Molecular electron affinities have historically been difficult quantities to measure accurately. These difficulties arise from differences in structure between the ion and neutral as well as the existence of excited negative ion states. To circumvent these problems, relative electron affinities were determined in this dissertation by studying equilibrium electron transfer reactions using a pulsed ion cyclotron resonance (ICR) spectrometer. Direct measurement of ion and neutral concentrations for reactions of the general type, A - + B = B - + A, allow calculation of the equilibrium constant and, therefore, the free energy change. The free energy difference is related to the difference in electron affinities between A and B. A relative electron affinity scale covering a range of about 45 kcal/mol was constructed with various substituted p-benzoquinones, nitrobenzenes, anhydrides, and benzophenones. To assign absolute electron affinities, various species with accurately known electron affinities are tied to the scale via ion-cyclotron double resonance bracketing techniques. After the relative scale is anchored to these species with well-known electron affinities, the scale is then used as a check on other electron affinity values as well as generating new electron affinity values. Many discrepancies were found between the electron affinities measured using the ICR technique and previous literature determinations

  10. Possible site-specific reagent for the general amino acid transport system of Saccharomyces cerevisiae.

    Science.gov (United States)

    Larimore, F S; Roon, R J

    1978-02-07

    The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids. The amino acid analogue N-delta-chloroacetyl-L-ornithine (NCAO) has been tested as potential site specific reagent for this system. L-Tryptophan, which is transported exclusively by the general transport system, was used as a substrate. In the presence of glucose as an energy source, NCAO inhibited tryptophan transport competitively (Ki = 80 micrometer) during short time intervals (1-2 min), but adding 100 micrometer NCAO to a yeast cell suspension resulted in a time-dependent activation of tryptophan transport during the first 15 min of treatment. Following the activation a time-dependent decay of tryptophan transport activity occurred. Approximately 80% inactivation of the system was observed after 90 min. When a yeast cell suspension was treated with NCAO in the absence of an energy source, an 80% inactivation of tryptophan transport occurred in 90 min. The inactivation was noncompetitive (Ki congruent to 60 micrometer) and could not be reversed by the removal of the NCAO. Addition of a five-fold excess of L-lysine during NCAO treatment or prevented inactivation of tryptophan transport. Under parallel conditions of incubation, other closely related transport systems were not inhibited by NCAO.

  11. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  12. 21 CFR 864.8540 - Red cell lysing reagent.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent...

  13. Synthesis and characterization of N-hydroxysuccinimide ester chemical affinity derivatives of asialoorosomucoid that covalently cross-link to galactosyl receptors on isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Herzig, M.C.S.; Weigel, P.H.

    1989-01-01

    The authors have developed chemical affinity reagents for the hepatic galactosyl receptor. Asialoorosomucoid (ASOR) was derivatized with five homobifunctional N-hydroxysuccinimide (NHS) ester cross-linkers. NHS/ASOR derivatives were synthesized, purified, and applied within 10 min to isolated rat hepatocytes at 4 degree C. Specific binding of these 125 I-labeled derivatives was ∼90% in the presence of either EGTA or excess ASOR. Specific cross-linking assessed by the resistance of specifically bound NHS/ 125 I-ASOR to release by EGTA, was 50-75% of the specifically bound ligand. The extent of specific cross-linking correlated with the average number of NHS groups per ASOR and was controlled by varying the molar ratio of cross-linker to ASOR during the synthesis. After being cross-linked with any of the NHS/ 125 I-ASOR derivatives, cells were washed with EGTA, solubilized in Triton X-100, and analyzed by SDA-PAGE and autoradiography. They conclude that all three receptor subunits can cross-link to ligand. They propose a model in which the native receptor is a heterohexamer composed of four subunits of RHL 1 and two subunits of RHL 2 and/or RHL 3

  14. Reagent-free bacterial identification using multivariate analysis of transmission spectra

    Science.gov (United States)

    Smith, Jennifer M.; Huffman, Debra E.; Acosta, Dayanis; Serebrennikova, Yulia; García-Rubio, Luis; Leparc, German F.

    2012-10-01

    The identification of bacterial pathogens from culture is critical to the proper administration of antibiotics and patient treatment. Many of the tests currently used in the clinical microbiology laboratory for bacterial identification today can be highly sensitive and specific; however, they have the additional burdens of complexity, cost, and the need for specialized reagents. We present an innovative, reagent-free method for the identification of pathogens from culture. A clinical study has been initiated to evaluate the sensitivity and specificity of this approach. Multiwavelength transmission spectra were generated from a set of clinical isolates including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Spectra of an initial training set of these target organisms were used to create identification models representing the spectral variability of each species using multivariate statistical techniques. Next, the spectra of the blinded isolates of targeted species were identified using the model achieving >94% sensitivity and >98% specificity, with 100% accuracy for P. aeruginosa and S. aureus. The results from this on-going clinical study indicate this approach is a powerful and exciting technique for identification of pathogens. The menu of models is being expanded to include other bacterial genera and species of clinical significance.

  15. Design, synthesis, and activity of 2,3-diphosphoglycerate analogs as allosteric modulators of hemoglobin O2 affinity.

    Science.gov (United States)

    Kassa, Tigist W; Zhang, Ning; Palmer, Andre F; Matthews, Jason Shastri

    2013-04-01

    Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9-12.8 with Hill coefficients in the range of 1.5-2.4.

  16. The utility of affine variables and affine coherent states

    International Nuclear Information System (INIS)

    Klauder, John R

    2012-01-01

    Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when appropriate. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’. (review)

  17. 21 CFR 660.30 - Reagent Red Blood Cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

  18. New polymer-supported ion-complexing agents: Design, preparation and metal ion affinities of immobilized ligands

    International Nuclear Information System (INIS)

    Alexandratos, Spiro D.

    2007-01-01

    Polymer-supported reagents are comprised of crosslinked polymer networks that have been modified with ligands capable of selective metal ion complexation. Applications of these polymers are in environmental remediation, ion chromatography, sensor technology, and hydrometallurgy. Bifunctional polymers with diphosphonate/sulfonate ligands have a high selectivity for actinide ions. The distribution coefficient for the uranyl ion from 1 M nitric acid is 70,000, compared to 900 for the monophosphonate/sulfonate polymer and 200 for the sulfonic acid ion-exchange resin. A bifunctional trihexyl/triethylammonium polymer has a high affinity and selectivity for pertechnetate and perchlorate anions from groundwater. In one example, its distribution coefficient for perchlorate ions in the presence of competing anions is 3,300,000, compared to 203,180 for a commercially available anion-exchange resin. Polystyrene modified with N-methyl-D-glucamine ligands is capable of selectively complexing arsenate from groundwater. It complexes 99% of the arsenate present in a solution of 100 mg/L arsenate with 560 mg/L sulfate ions. Its selectivity is retained even in the presence of 400 mg/L phosphate. There is no affinity for arsenate above pH 9, allowing for the polymer to be regenerated with moderate alkali solution. In studies aimed at developing a Hg(II)-selective resin, simple amine resins were found to have a high Hg(II) affinity and that affinity is dependent upon the solution pH and the counterion

  19. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Williams

    2017-07-01

    Full Text Available The discovery of naturally occurring T cell receptors (TCRs that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds rather than synergy (total CD8 cooperation alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our

  20. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  1. Affine coherent states and Toeplitz operators

    Science.gov (United States)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  2. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  3. Report: Affinity Chromatography.

    Science.gov (United States)

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  4. Spectrophotometric determination of tannins by phosphotungstic-phosphomolybdic reagent

    Energy Technology Data Exchange (ETDEWEB)

    Reicher, F; Sierakowski, M R; Correa, J B.C. [Parana Univ., Curitiba (Brazil). Dept. de Bioquimica

    1981-01-01

    There are several colorimetric techniques to determine tannins in plant extracts. One frequently used is the Folin method (phosphotungstic acid reagent) that procedures a blue color with phenolic compounds. However, this coloured complex is unstable. With the Folin-Ciocalteau reagent, used in protein determination (Lowry et al. J.B.C. 193: 265, 1951) good results were obtained, even in the absence of cooper solution. Using phosphotungstic-phosphomolybdic reagent (Folin-Denis), it was obtained maximum color with 1,0 ml of the reagent in 20 minutes, after the additon of 10 ml 20% sodium carbonate solution. Tannins samples containing 10 to 200 ..mu..g/ml were analysed. Absorbances are determined at 720 or 600 nm. Tannins of commercial preparations from Acacia negra were analysed by the phosphotungstic-phosphomolybdic reagent before (A) and after (B) treatment with chromate hyde powder. By this procedure hydrolysible tannins were determined (A-B).

  5. Spectrophotometric determination of tannins by phosphotungstic-phosphomolibdic reagent

    International Nuclear Information System (INIS)

    Reicher, F.; Sierakowski, M.R.; Correa, J.B.C.

    1981-01-01

    There are several colorimetric techniques to determine tannins in plant extracts. One frequently used is the Folin method (phosphotungstic acid reagent) that procedures a blue color with phenolic compounds. However, this coloured complex is unstable. With the Folin-Ciocalteau reagent, used in protein determination (Lowry et al. J.B.C. 193: 265, 1951) good results were obtained, even in the absence of cooper solution. Using phosphotungstic-phosphomolibdic reagent (Folin-Denis), it was obtained maximum color with 1,0 ml of the reagent in 20 minutes, after the adition of 10 ml 20% sodium carbonate solution. Tannins samples containing 10 to 200 μg/ml were analysed. Absorbances are determined at 720 or 600 nm. Tannins of commercial preparations from Acacia negra were analysed by the phosphotungstic-phosphomolibdic reagent before (A) and after (B) treatment with chromate hyde powder. By this procedure hydrolysible tannins were determined (A-B). (Author) [pt

  6. Constructing New Bioorthogonal Reagents and Reactions.

    Science.gov (United States)

    Row, R David; Prescher, Jennifer A

    2018-05-15

    Chemical tools are transforming our understanding of biomolecules and living systems. Included in this group are bioorthogonal reagents-functional groups that are inert to most biological species, but can be selectively ligated with complementary probes, even in live cells and whole organisms. Applications of these tools have revealed fundamental new insights into biomolecule structure and function-information often beyond the reach of genetic approaches. In many cases, the knowledge gained from bioorthogonal probes has enabled new questions to be asked and innovative research to be pursued. Thus, the continued development and application of these tools promises to both refine our view of biological systems and facilitate new discoveries. Despite decades of achievements in bioorthogonal chemistry, limitations remain. Several reagents are too large or insufficiently stable for use in cellular environments. Many bioorthogonal groups also cross-react with one another, restricting them to singular tasks. In this Account, we describe our work to address some of the voids in the bioorthogonal toolbox. Our efforts to date have focused on small reagents with a high degree of tunability: cyclopropenes, triazines, and cyclopropenones. These motifs react selectively with complementary reagents, and their unique features are enabling new pursuits in biology. The Account is organized by common themes that emerged in our development of novel bioorthogonal reagents and reactions. First, natural product structures can serve as valuable starting points for probe design. Cyclopropene, triazine, and cyclopropenone motifs are all found in natural products, suggesting that they would be metabolically stable and compatible with a variety of living systems. Second, fine-tuning bioorthogonal reagents is essential for their successful translation to biological systems. Different applications demand different types of probes; thus, generating a collection of tools that span a continuum of

  7. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    Science.gov (United States)

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  8. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3220... fluorescent dye (immunofluorescent reagents) used to identify Entamoeba histolytica directly from clinical...

  9. Reagent-loaded plastic microfluidic chips for detecting homocysteine

    International Nuclear Information System (INIS)

    Suk, Ji Won; Jang, Jae-Young; Cho, Jun-Hyeong

    2008-01-01

    This report describes the preliminary study on plastic microfluidic chips with pre-loaded reagents for detecting homocysteine (Hcy). All reagents needed in an Hcy immunoassay were included in a microfluidic chip to remove tedious assay steps. A simple and cost-effective bonding method was developed to realize reagent-loaded microfluidic chips. This technique uses an intermediate layer between two plastic substrates by selectively patterning polydimethylsiloxane (PDMS) on the embossed surface of microchannels and fixing the substrates under pressure. Using this bonding method, the competitive immunoassay for SAH, a converted form of Hcy, was performed without any damage to reagents in chips, and the results showed that the fluorescent signal from antibody antigen binding decreased as the SAH concentration increased. Based on the SAH immunoassay, whole immunoassay steps for Hcy detection were carried out in plastic microfluidic chips with all necessary reagents. These experiments demonstrated the feasibility of the Hcy immunoassay in microfluidic devices

  10. 21 CFR 866.3375 - Mycoplasma spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3375 Mycoplasma... fluorescent dye (immunofluorescent reagents) used to identify Mycoplasma spp. directly from clinical specimens...

  11. 21 CFR 866.3780 - Toxoplasma gondii serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3780 Toxoplasma... (immunofluorescent reagents) used to identify Toxoplasma gondii from clinical specimens. The identification aids in...

  12. Effect of reagent charge on the labeling of erythrocyte membrane proteins by photoactivated reagents

    International Nuclear Information System (INIS)

    Schaeffer, J.C.; Hakimian, R.; Shimer, M.L.

    1986-01-01

    Leaky erythrocyte ghosts were labeled with 3 H-[2-(4-azido-2-nitroanilino)ethyl]trimethylammonium iodide (cationic label) or 3 H-N-(4-azido-2-nitrophenyl)-β-alanine (anionic label). After the membranes were thoroughly washed, seven times as much cationic label was associated with the membranes as anionic label at 5 μM, whereas at 50 μM the cationic label was favored 15-fold. The distribution of label in the membrane proteins was ascertain by SDS-gel electrophoresis followed by autoradiography. At 50 μM cationic label, erythrocyte membrane protein bands 1,2,3,4.2, and 5 were intensely labeled, while band 6 was labeled weakly. At 5 μM cationic label, bands 1 and 4.2 were heavily labeled, while 2,3 and 5 were labeled less well. At both 50 μM and 5 μM anionic label, bands 1 and 6 were most prominently labeled. Bands 2,3,4.2 and 5 were labeled also at 50 μM, but they were labeled only very weakly at 5 μM. Band 4.1 was labeled very poorly if at all by either reagent. A mixture of the reagents gave an additive pattern. Thus, the charge and concentration of these reagents appear to play a major role in their ability to label membrane proteins indiscriminately. Because these reagents contain the same chromophore, 4-azido-2-nitroaniline, and differ mainly only in their charge, they may prove useful in assessing the location of charged sites on proteins in supramolecular complexes

  13. Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

    Science.gov (United States)

    Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

    2013-04-08

    Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity

  14. Development of IRMA reagent and methodology for PSA

    International Nuclear Information System (INIS)

    Najafi, R.

    1997-01-01

    The PSA test is a solid phase two-site immunoassay. Rabbit anti PSA is coated or bound on surface of solid phase and monoclonal anti PSA labeled with 1-125. The PSA molecules present in the standard solution or serum are 'Sandwiched' between the two antibodies. After formation of coated antibody-antigen-labeled antibody complex, the unbound labeled antibody will removed by washing. The complex is measured by gamma counter. The concentration of analyte is proportional to the counts of test sample. In order to develop kits for IRMA PSA, it should be prepared three essential reagents Antibody coated solid phase, labeled antibody, standards and finally optimizing them to obtain an standard curve fit to measure specimen PSA in desired range of concentration. The type of solid phase and procedure(s) to coat or bind to antibody, is still main debatable subject in development and setting up RIA/IRMA kits. In our experiments, polystyrene beads, because of their easy to coat with antibody as well as easy to use, can be considered as a desired solid phase. Most antibodies are passively adsorbed to a plastic surface (e.g. Polystyrene, Propylene, and Polyvinyl chloride) from a diluted buffer. The antibody coated plastic surface, then acts as solid phase reagent. Poor efficiency and time required to reach equilibrium and also lack of reproducibility especially batch-to-batch variation between materials, are disadvantages in this simple coating procedure. Improvements can be made by coating second antibody on surface of beads, and reaction between second and primary antibodies. There is also possible to enhance more coating efficiency of beads by using Staphylococcus ureus-Protein A. Protein A is a major component of staphylococcus aureus cell wall which has an affinity for FC segment of immunoglobulin G (IgG) of some species, including human; rabbit; and mice. This property of Staphylococcal Protein A has made it a very useful tool in the purification of classes and subclasses

  15. 21 CFR 864.4010 - General purpose reagent.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false General purpose reagent. 864.4010 Section 864.4010 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4010 General purpose...

  16. The blocking reagent optimization for the magnetoelastic biosensor

    Science.gov (United States)

    Hu, Jiajia; Chai, Yating; Horikawa, Shin; Wikle, Howard C.; Wang, Feng'en; Du, Songtao; Chin, Bryan A.; Hu, Jing

    2015-06-01

    The wireless phage-based magnetoelastic (ME) biosensor has proven to be promising for real-time detection of pathogenic bacteria on fresh produces. The ME biosensor consists of a freestanding ME resonator as the signal transducer and filamentous phage as the biomolecular-recognition element, which can specifically bind to a pathogen of interest. Due to the Joule magnetostriction effect, the biosensors can be placed into mechanical resonance when subjected to a time-varying magnetic field alternating at the sensor's resonant frequency. Upon the attachment of the target pathogen, the mass of the biosensor increases, thereby decreasing its resonant frequency. This paper presents an investigation of blocking reagents immobilization for detecting Salmonella Typhimurium on fresh food surfaces. Three different blocking reagents (BSA, SuperBlock blocking buffer, and blocker BLOTTO) were used and compared. The optical microscope was used for bacterial cells binding observation. Student t-test was used to statistically analysis the experiment results. The results shows that SuperBlock blocking buffer and blocker BLOTTO have much better blocking performance than usually used BSA.

  17. Roles of iron species and pH optimization on sewage sludge conditioning with Fenton's reagent and lime.

    Science.gov (United States)

    Yu, Wenbo; Yang, Jiakuan; Shi, Yafei; Song, Jian; Shi, Yao; Xiao, Jun; Li, Chao; Xu, Xinyu; He, Shu; Liang, Sha; Wu, Xu; Hu, Jingping

    2016-05-15

    Conditioning sewage sludge with Fenton's reagent could effectively improve its dewaterability. However, drawbacks of conditioning with Fenton's reagent are requirement of acidic conditions to prevent iron precipitation and subsequent neutralization with alkaline additive to obtain the pH of the filtrate close to neutrality. In this study, roles of pH were thoroughly investigated in the acidification pretreatment, Fenton reaction, and the final filtrate after conditioning. Through the response surface methodology (RSM), the optimal dosages of H2SO4, Fe(2+), H2O2, and lime acted as a neutralizer were found to be 0 (no acidification), 47.9, 34.3 and 43.2 mg/g DS (dry solids). With those optimal doses, water content of the dewatered sludge cakes could be reduced to 55.8 ± 0.6 wt%, and pH of the final filtrate was 6.6 ± 0.2. Fenton conditioning without initial acidification can simplify the conditioning process and reduce the usage of lime. The Fe(3+) content in the sludge cakes showed a close correlation with the dewaterability of conditioned sludge, i.e., the water content of sludge cakes, SRF (specific resistance to filtration), CST (capillary suction time), bound water content, and specific surface area. It indicated that the coagulation by Fe(3+) species in Fenton reaction could play an important role, compared to traditional Fenton oxidation effect on sludge conditioning. Thus, a two-step mechanism of Fenton oxidation and Fe(III) coagulation was proposed in sewage sludge conditioning. The mechanisms include the following: (1) extracellular polymeric substances (EPS) were firstly degraded into dissolved organics by Fenton oxidation; (2) bound water was converted to free water due to degradation of EPS; (3) the sludge particles were disintegrated into small ones by oxidation; (4) Fe(3+) generated from Fenton reaction acted as a coagulant to agglomerate smaller sludge particles into larger dense particles with less bond water; (5) finally, the dewatered

  18. Calculation of protein-ligand binding affinities.

    Science.gov (United States)

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  19. Industrial detergent wastewater treatment via fenton reagent

    International Nuclear Information System (INIS)

    Mohd Zairie Mohd Yusuff; Mohd Zulkifli Mohamad Noor; Izirwan Izhab

    2010-01-01

    Production of detergent can generates wastewater containing an organic matter with will consume an oxidation demand, surfactants, suspended solids, fat and oil. Besides, sulfate concentration is high in the most detergent plant effluent because of the sulphonation process that has physiological and toxic effects on marine organisms. Therefore, a research must be conducted to find the solution for this problem. The feasibility of Fentons reagent to treat detergent waste was investigated in this study. The sample of detergent wastewater was taken from FPG Oleo chemicals Sdn. Bhd. This experiment studied the effect of temperature towards the feasibility of Fentons reagent process besides the dosage between hydrogen peroxide (H 2 O 2 ) and ferrous ion (Fe 2+ ) in the reagent. While, evaluated efficiency of Fentons reagent in term of chemical oxygen demand (COD), total suspended solid (TSS) and the turbidity reduction within the experimental design. The result found that overall removal was achieved until 96.2 % in term of COD, 98.1 % in term of TSS and 99.6 % in term of turbidity using Fentons reagent process. Besides, also found that this process is optimum at temperature 35 degree Celsius are able to achieve the Standard A of Parameter Limit of Effluent of Standard A and Standard B were outlined by Department of Environment Malaysia (DOE) based on Environment Quality Act 1974. (author)

  20. Hirota's solitons in the affine and the conformal affine Toda models

    International Nuclear Information System (INIS)

    Aratyn, H.; Constantinidis, C.P.; Ferreira, L.A.; Gomes, J.F.; Zimerman, A.H.

    1993-01-01

    We use Hirota's method formulated as a recursive scheme to construct a complete set of soliton solutions for the affine Toda field theory based on an arbitrary Lie algebra. Our solutions include a new class of solitons connected with two different types of degeneracies encountered in Hirota's perturbation approach. We also derive an universal mass formula for all Hirota's solutions to the affine Toda model valid for all underlying Lie groups. Embedding of the affine Toda model in the conformal affine Toda model plays a crucial role in this analysis. (orig.)

  1. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    Science.gov (United States)

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  2. Development of reagents for radioimmunoassay of: triiodothyronine, thyroxine and thyrotrophin

    International Nuclear Information System (INIS)

    Delgado S, B.; Lavalley E, C.; Ruiz J, A.; Garcia F, C.; Zamorano A, F.

    1991-12-01

    The radioimmunoassay (RIA) of thyroid hormones it is the but it frequents of all the studies carried out by RIA in the laboratories of Nuclear Medicine, these essays are carried out with imported reagents. In the ININ the reagents and the necessary methodology have been developed for the triiodothyronine (T3), thyroxine (T4) and thyrotrophin (TSH). The good titles of the antibodies (Ac) primary for each hormone were of 1:4,000; 1:750 and 1:1,500. The used separation system was of double Ac with PEG to 10%, with titles of 1:10 for the second Ac of lamb. The specific activity for 125-I-T3 and 125-I-T4 oscillate between 850 at 900 μCi / μ g: being this of 90 μ Ci /μg for TSH. To the first two hormones they were added 1-8 aniline naftalen sulfonic acid (ANS) to concentrations of 3 and 2 mg/ml respectively. As buffer for T3 and T4 it was used Tris-HCl pH 8.6 and PBS with normal serum of rabbit (SNC) for TSH. The standards got ready in buffer or free serum of thyroid hormones. The slope of the standard curves varied between -2.3 to -2.7 and the variation intra and inter assay among 4 to 10%. It is had at the moment in the ININ with standardized reagents for the RIA of T3, T4 and TSH, it is hoped to carry out tests in other laboratories and to establish the conditions of stability more appropriate to begin the preparation of pilot reagents. (Author)

  3. Compound immobilization and drug-affinity chromatography.

    Science.gov (United States)

    Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

    2012-01-01

    Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

  4. The solutions of affine and conformal affine Toda field theory

    International Nuclear Information System (INIS)

    Papadopoulos, G.; Spence, B.

    1994-02-01

    We give new formulations of the solutions of the field equations of the affine Toda and conformal affine Toda theories on a cylinder and two-dimensional Minkowski space-time. These solutions are parameterised in terms of initial data and the resulting covariant phase spaces are diffeomorphic to the Hamiltonian ones. We derive the fundamental Poisson brackets of the parameters of the solutions and give the general static solutions for the affine theory. (authors). 10 refs

  5. A Snippet of Grignard Reagent's Histroy There are very few reagents ...

    Indian Academy of Sciences (India)

    IAS Admin

    with bromine, (eq.1). But he failed to notice the formation of phenylmagnesium bromide, because he was using excess bromine which destroyed it. Hence, he could isolate only bromobenzene,. (eq.2). Had he used only one molar equivalent of bromine, perhaps the organomagnesium reagent would bear his name today.

  6. Physically absorbable reagents-collectors in elementary flotation

    Energy Technology Data Exchange (ETDEWEB)

    S.A. Kondrat' ev; I.G. Bochkarev [Russian Academy of Sciences, Novosibirsk (Russian Federation). Institute of Mining

    2007-09-15

    Based on the reviewed researches held at the Institute of Mining, Siberian Branch, Russian Academy of Sciences, the effect of physically absorbable reagents-collectors on formation of a flotation complex and its stability in turbulent pulp flows in flotation machines of basic types is considered. The basic requirements for physically absorbable reagents-collectors at different flotation stages are established.

  7. Accurate and sensitive quantification of protein-DNA binding affinity.

    Science.gov (United States)

    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  8. STUDY ON OIL WASTEWATER TREATMENT WITH POLYMERIC REAGENTS

    Directory of Open Access Journals (Sweden)

    RODICA BUCUROIU

    2016-04-01

    Full Text Available Used the polymeric reagents in oil wastewater treatment is an effective method of eliminate hydrocarbons. The present study aims to finding reagents that lead to lowering of extractible (EXT, suspended solids (SS and chemical oxygen demand (COD of industrial wastewater from washing cars in loading ramps petroleum products. For this purpose five reagents were tested, namely: polyamines, cationic polyacrylamides, polydiallydimethyl ammonium chloride (PolyDADMAC, melamine formaldehyde polymer resin and polydicyandiamide polymer resin. Obtaining removal degrees over 80 % justifies using this method in the industrial practice.

  9. 21 CFR 866.3550 - Salmonella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3550 Salmonella... isolates derived from clinical specimens. Additionally, some of these reagents consist of antisera... clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the...

  10. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  11. Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors

    Directory of Open Access Journals (Sweden)

    Simmonds Peter

    2008-01-01

    Full Text Available Abstract Background HIV-1 R5 viruses cause most of the AIDS cases worldwide and are preferentially transmitted compared to CXCR4-using viruses. Furthermore, R5 viruses vary extensively in capacity to infect macrophages and highly macrophage-tropic variants are frequently identified in the brains of patients with dementia. Here, we investigated the sensitivity of R5 envelopes to a range of inhibitors and antibodies that block HIV entry. We studied a large panel of R5 envelopes, derived by PCR amplification without culture from brain, lymph node, blood and semen. These R5 envelopes conferred a wide range of macrophage tropism and included highly macrophage-tropic variants from brain and non-macrophage-tropic variants from lymph node. Results R5 macrophage-tropism correlated with sensitivity to inhibition by reagents that inhibited gp120:CD4 interactions. Thus, increasing macrophage-tropism was associated with increased sensitivity to soluble CD4 and to IgG-CD4 (PRO 542, but with increased resistance to the anti-CD4 monoclonal antibody (mab, Q4120. These observations were highly significant and are consistent with an increased affinity of envelope for CD4 for macrophage-tropic envelopes. No overall correlations were noted between R5 macrophage-tropism and sensitivity to CCR5 antagonists or to gp41 specific reagents. Intriguingly, there was a relationship between increasing macrophage-tropism and increased sensitivity to the CD4 binding site mab, b12, but decreased sensitivity to 2G12, a mab that binds a glycan complex on gp120. Conclusion Variation in R5 macrophage-tropism is caused by envelope variation that predominantly influences sensitivity to reagents that block gp120:CD4 interactions. Such variation has important implications for therapy using viral entry inhibitors and for the design of envelope antigens for vaccines.

  12. 21 CFR 866.3355 - Listeria spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3355 Listeria spp... from clinical specimens. Additionally, some of these reagents consist of Listeria spp. antisera... clinical specimens. The identification aids in the diagnosis of listeriosis, a disease caused by bacteria...

  13. IN-PLACE REGENERATION OF GAC USING FENTON'S REAGENTS

    Science.gov (United States)

    This paper evaluates the feasibility of using Fenton’s reagents for in-place recovery of spent granular activated carbon (GAC). Fenton’s reagents are cycled through spent GAC to degrade sorbed chlorinated hydrocarbons with little loss of carbon capacity. Seven chlorinated compou...

  14. Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays

    Directory of Open Access Journals (Sweden)

    Robert J. Kubiak

    2016-01-01

    Full Text Available Consistent performance of anti-drug antibody (ADA assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

  15. Lp-dual affine surface area

    Science.gov (United States)

    Wei, Wang; Binwu, He

    2008-12-01

    According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

  16. The effect of label affinity on the sensitivity and specificity of a hapten radioimmunoassay: A comparison of three [125I]diphenylhydantoin radioligands with the 14C-labelled drug

    International Nuclear Information System (INIS)

    Rowell, F.J.

    1979-01-01

    The effects on the sensitivity and specificity of a radioimmunoassay for diphenylhydantoin (DPH) has been investigated using three 125 I-labelled tyrosine ester derivatives of DPH having different bridge lengths between the tyrosine moiety and the DPH moeity and 14 C-labelled DPH. The results demonstrate that for a hapten which does not completely fill the antibody binding sites, greatest sensitivity is achieved when the bridge of the iodine label is most dissimilar to that present in the original immunogen, when the hapten and label affinities are nearly equivalent. Greatest specificity is achieved with the label which most resembles the original immunogen. These results illustrate the difficulty of designing satisfactory labels for assays of both high specificity and sensitivity since minimal changes in label structure may produce greatly amplified changes in the subsequent affinity of the label for the antiserum. (Auth.)

  17. Inactivation and stability of viral diagnostic reagents treated by gamma radiation

    International Nuclear Information System (INIS)

    White, L.A.; Freeman, C.Y.; Hall, H.E.; Forrester, B.D.

    1990-01-01

    The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with >2.4 Mrad radiation at temperatures above 4 0 C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is a reliable and effective replacement for BPL in preparing diagnostic reagents. (author)

  18. Inactivation and stability of viral diagnostic reagents treated by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    White, L A; Freeman, C Y; Hall, H E; Forrester, B D [Department of Health and Human Services, Atlanta, GA (USA)

    1990-10-01

    The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with >2.4 Mrad radiation at temperatures above 4{sup 0}C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is a reliable and effective replacement for BPL in preparing diagnostic reagents. (author).

  19. Systematic trends in photonic reagent induced reactions in a homologous chemical family.

    Science.gov (United States)

    Tibbetts, Katharine Moore; Xing, Xi; Rabitz, Herschel

    2013-08-29

    The growing use of ultrafast laser pulses to induce chemical reactions prompts consideration of these pulses as "photonic reagents" in analogy to chemical reagents. This work explores the prospect that photonic reagents may affect systematic trends in dissociative ionization reactions of a homologous family of halomethanes, much as systematic outcomes are often observed for reactions between homologous families of chemical reagents and chemical substrates. The experiments in this work with photonic reagents of varying pulse energy and linear spectral chirp reveal systematic correlations between observable ion yields and the following set of natural variables describing the substrate molecules: the ionization energy of the parent molecule, the appearance energy of each fragment ion, and the relative strength of carbon-halogen bonds in molecules containing two different halogens. The results suggest that reactions induced by photonic reagents exhibit systematic behavior analogous to that observed in reactions driven by chemical reagents, which provides a basis to consider empirical "rules" for predicting the outcomes of photonic reagent induced reactions.

  20. 21 CFR 864.8950 - Russell viper venom reagent.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Russell viper venom reagent. 864.8950 Section 864.8950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8950 Russell viper venom...

  1. IA-2 autoantibody affinity in children at risk for type 1 diabetes.

    Science.gov (United States)

    Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

    2012-12-01

    Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Evaluation of novel derivatisation reagents for the analysis of oxysterols

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Peter J., E-mail: p.j.crick@swansea.ac.uk [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Aponte, Jennifer; Bentley, T. William [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Matthews, Ian [College of Engineering, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Wang, Yuqin [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Griffiths, William J., E-mail: w.j.griffiths@swansea.ac.uk [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom)

    2014-04-11

    Graphical abstract: - Highlights: • New derivatisation reagents for LC–MS analysis of oxysterols. • New reagents based on Girard P give high ion-currents and informative LC–MS{sup n} spectra. • Permanent charge is vital for efficient MS{sup n} fragmentation. • New reagents offer greater scope for incorporation of isotope labels. - Abstract: Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MS{sup n} fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.

  3. Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

    Science.gov (United States)

    He, Mingyue; Hamon, Maureen; Liu, Hong; Corper, Adam L; Taussig, Michael J

    2006-09-01

    The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 10(4)-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

  4. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    Science.gov (United States)

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  5. Reagent and process for detaching furfural in petroleum products

    Energy Technology Data Exchange (ETDEWEB)

    Orelup, R.B.

    1987-07-14

    A two-component liquid reagent and process is provided for detecting the presence of furfural in petroleum products. The reagent comprises two components which are stored separately from each other and are combined prior to admixture with the petroleum product. The process comprises combining the two separate components of the liquid reagent with each other prior to use, admixing the combined components with a petroleum product containing furfural, shaking the resultant mixture, allowing the mixture to separate and observing a red color characteristic of furfural in the lower layer. Alternately, the process may be carried out by combining the second component of the two-component liquid reagent with the petroleum product, followed by admixture with the first component to obtain two separate layers in which the red color characteristic of furfural is observed in the lower layer.

  6. Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents

    Directory of Open Access Journals (Sweden)

    Alban Silvana M

    2013-01-01

    Full Text Available Abstract Background An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients’ sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Methods Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Results Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5 and peptide 1B (1/5. In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. Conclusions The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

  7. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  8. The reaction of organocerium reagents with easily enolizable ketones

    International Nuclear Information System (INIS)

    Imamoto, Tsuneo; Kusumoto, Tetsuo; Sugiura, Yasushi; Suzuki, Nobuyo; Takiyama, Nobuyuki

    1985-01-01

    Organocerium (III) reagents were conveniently generated by the reaction of organolithium compounds with anhydrous cerium (III) chloride. The reagents are less basic than organolithiums and Grignard reagents, and they react readily at -78 deg C with easily enolizable ketones such as 2-tetralone to afford addition products in high yields. Cerium (III) enolates were also generated from lithium enolates and cerium (III) chloride. The cerium (III) enolates undergo aldol addition with ketones or sterically crowded aldehyde to give the corresponding β-hydroxy ketones in good to high yields. (author)

  9. YehZYXW of Escherichia coli Is a Low-Affinity, Non-Osmoregulatory Betaine-Specific ABC Transporter.

    Science.gov (United States)

    Lang, Shenhui; Cressatti, Marisa; Mendoza, Kris E; Coumoundouros, Chelsea N; Plater, Samantha M; Culham, Doreen E; Kimber, Matthew S; Wood, Janet M

    2015-09-22

    Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.

  10. Hydrazine reagents as derivatizing agents in environmental analysis--a critical review.

    Science.gov (United States)

    Vogel, M; Büldt, A; Karst, U

    2000-04-01

    Hydrazine reagents are a well-known group of derivatizing agents for the determination of aldehydes and ketones in liquid and gaseous samples. Within this article, the most important hydrazine reagents are critically summarized, and their major applications in different fields, including environmental analysis, food chemistry and industrial analysis are introduced. As 2,4-dinitrophenylhydrazine (DNPH) is the basic reagent for several international standard procedures, its properties are discussed in detail. Particular focus is directed on the chemistry of the hydrazine reagents, and chemical interferences are considered. Recent methods for the determination of various oxidants using hydrazine reagents are presented as well. Due to limited space, this review does not cover the related field of carbohydrate analysis, although many chemical aspects are similar.

  11. Reagent for treating clay drilling muds

    Energy Technology Data Exchange (ETDEWEB)

    Tkachenko, P V; Leshchinskiy, P A; Shnaper, B I; Zinchuk, I F; Zlobin, V P

    1982-01-01

    A reagent is proposed for treating clay drilling muds. It contains lignite, caustic soda and modifying agent. It is distinguished by the fact that in order to reduce the cost of the reagent with simultaneous decrease in the viscosity and static shear stress of the drilling mud, it additionally contains iron sulfate, and the modifying agent contained is wastes of carbonic acid production with the following ratio of components (parts by weight): lignite 10.0-15.0, caustic soda 2.0-3.0, wastes of carbonic acid production 0.5-0.75; iron sulfate 1.0-2.0.

  12. Evaluation of sensitivity and specificity of a standardized procedure using different reagents for the detection of lupus anticoagulants. The Working Group on Hemostasis of the Société Française de Biologie Clinique and for the Groupe d'Etudes sur I'Hémostase et la Thrombose.

    Science.gov (United States)

    Goudemand, J; Caron, C; De Prost, D; Derlon, A; Borg, J Y; Sampol, J; Sié, P

    1997-02-01

    This study was designed to test the sensitivity and specificity of a combination of 3 phospholipid-dependent assays performed with various reagents, for the detection of lupus anticoagulant (LA). Plasmas containing an LA (n = 56) or displaying various confounding pathologies [58 intrinsic pathway factor deficiencies, 9 factor VIII inhibitors, 28 plasmas from patients treated with an oral anticoagulant (OAC)] were selected. In a first step, the efficiency of each assay and reagent was assessed using the Receiving Operating Characteristic (ROC) method. Optimal cut-offs providing both sensitivity and specificity > or = 80% were determined. The APTT assay and most of the phospholipid neutralization assays failed to discriminate factor VIII inhibitors from LA. In a second step, using the optimal cut-offs determined above, the results of all the possible combinations of the 3 assays performed with 4 different reagents were analyzed. Thirteen combinations of reagents allowed > or = 80% of plasmas of each category (LA, factor deficiency or OAC) to be correctly classified (3/3 positive test results in LA-containing plasmas and 0/3 positive results in LA-negative samples).

  13. A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

    Science.gov (United States)

    Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

    2014-01-02

    Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.

  14. Simple questionnaire and urine reagent strips compared to microscopy for the diagnosis of Schistosoma haematobium in a community in northern Ghana.

    Science.gov (United States)

    Bogoch, Isaac I; Andrews, Jason R; Dadzie Ephraim, Richard K; Utzinger, Jürg

    2012-10-01

    To evaluate the utility of a simple questionnaire and urine reagent strip testing for the rapid diagnosis of Schistosoma haematobium in rural northern Ghana. Cross-sectional parasitological and questionnaire survey in a community in northern Ghana. Participants provided two urine specimens that were examined under a microscope using a centrifugation method. The first urine sample was additionally subjected to reagent strip testing. A short questionnaire was administered to all participants. Microscopy of urine samples obtained from 208 individuals aged 1-77 years revealed an S. haematobium prevalence of 6.8%. The presence of any blood or protein on a urine reagent strip was 100% and 42% sensitive, and 93% and 80% specific for S. haematobium diagnosis. Questionnaires were completed by 198 individuals. Self-reported haematuria showed a sensitivity of 53% and a specificity of 85%. A dichotomous two-question panel was helpful in S. haematobium diagnosis, with working and playing near the river significantly associated with S. haematobium infection (P < 0.001). The use of urine reagent strips, coupled with questions pertaining to water contact patterns, might be considered for point-of-contact diagnosis of S. haematobium where microscopy is unavailable. © 2012 Blackwell Publishing Ltd.

  15. Continuous affine processes

    DEFF Research Database (Denmark)

    Buchardt, Kristian

    2016-01-01

    Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general transformati...

  16. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    DEFF Research Database (Denmark)

    Botkjaer, Kenneth A; Kwok, Hang Fai; Terp, Mikkel G

    2016-01-01

    therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown......The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential...... to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332...

  17. 21 CFR 866.3520 - Rubeola (measles) virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rubeola (measles) virus serological reagents. 866... Rubeola (measles) virus serological reagents. (a) Identification. Rubeola (measles) virus serological... to rubeola virus in serum. The identification aids in the diagnosis of measles and provides...

  18. Affinity in electrophoresis.

    Science.gov (United States)

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  19. Rapid discovery of peptide capture candidates with demonstrated specificity for structurally similar toxins

    Science.gov (United States)

    Sarkes, Deborah A.; Hurley, Margaret M.; Coppock, Matthew B.; Farrell, Mikella E.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    Peptides have emerged as viable alternatives to antibodies for molecular-based sensing due to their similarity in recognition ability despite their relative structural simplicity. Various methods for peptide capture reagent discovery exist, including phage display, yeast display, and bacterial display. One of the primary advantages of peptide discovery by bacterial display technology is the speed to candidate peptide capture agent, due to both rapid growth of bacteria and direct utilization of the sorted cells displaying each individual peptide for the subsequent round of biopanning. We have previously isolated peptide affinity reagents towards protective antigen of Bacillus anthracis using a commercially available automated magnetic sorting platform with improved enrichment as compared to manual magnetic sorting. In this work, we focus on adapting our automated biopanning method to a more challenging sort, to demonstrate the specificity possible with peptide capture agents. This was achieved using non-toxic, recombinant variants of ricin and abrin, RiVax and abrax, respectively, which are structurally similar Type II ribosomal inactivating proteins with significant sequence homology. After only two rounds of biopanning, enrichment of peptide capture candidates binding abrax but not RiVax was achieved as demonstrated by Fluorescence Activated Cell Sorting (FACS) studies. Further sorting optimization included negative sorting against RiVax, proper selection of autoMACS programs for specific sorting rounds, and using freshly made buffer and freshly thawed protein target for each round of biopanning for continued enrichment over all four rounds. Most of the resulting candidates from biopanning for abrax binding peptides were able to bind abrax but not RiVax, demonstrating that short peptide sequences can be highly specific even at this early discovery stage.

  20. Efficacy comparative of different laboratory test reagents for hepatitis C virus antibody

    Directory of Open Access Journals (Sweden)

    GUO Feibo

    2016-09-01

    Full Text Available Objective To investigate the effects of different laboratory test reagents for hepatitis C virus (HCV antibody through a comparative analysis. Methods A total of 207 samples which tested positive by four anti-HCV screening reagents commonly used in the laboratories in China (Kehua, Xinchuang, Wantai, and Abbott were included. HCV RNA nucleic acid amplification (NAT was performed, and if NAT results were negative, recombinant immunoblot assay (RIBA was performed for further confirmation. The test results of these four screening reagents were compared, and their S/CO values and true positive rates were analyzed. Results Of all the 205 samples testing positive by any one reagent, 191 (93.2% tested positive by the four reagents, and 14 (6.8% were tested inconsistently by the four reagents. The positive predictive values of Xinchuang, Kehua, Wantai, and Abbott reagents were 88.2% (180/204, 93.8% (180/192, 91.4% (180/197, and 90.0% (180/200, respectively. The S/CO thresholds with a positive predictive value of ≥95% for Xinchuang, Kehua, Wantai, and Abbott reagents were 9.0, 4.0, 5.0, and 7.0, respectively. Conclusion Xinchuang, Kehua, Wantai, and Abbott reagents have significantly different S/CO thresholds with a positive predictive value of ≥95%, which are significantly different from those in other domestic laboratories. Each laboratory should establish an applicable S/CO threshold with a positive predictive value of ≥95%, in order to reduce the sample size for confirmatory test.

  1. [Research of regional medical consumables reagent logistics system in the modern hospital].

    Science.gov (United States)

    Wu, Jingjiong; Zhang, Yanwen; Luo, Xiaochen; Zhang, Qing; Zhu, Jianxin

    2013-09-01

    To explore the modern hospital and regional medical consumable reagents logistics system management. The characteristics of regional logistics, through cooperation between medical institutions within the region, and organize a wide range of special logistics activities, to make reasonable of the regional medical consumable reagents logistics. To set the regional management system, dynamic management systems, supply chain information management system, after-sales service system and assessment system. By the research of existing medical market and medical resources, to establish the regional medical supplies reagents directory and the initial data. The emphasis is centralized dispatch of medical supplies reagents, to introduce qualified logistics company for dispatching, to improve the modern hospital management efficiency, to costs down. Regional medical center and regional community health service centers constitute a regional logistics network, the introduction of medical consumable reagents logistics services, fully embodies integrity level, relevance, purpose, environmental adaptability of characteristics by the medical consumable reagents regional logistics distribution. Modern logistics distribution systems can increase the area of medical consumables reagent management efficiency and reduce costs.

  2. Treatment of laundrette wastewater using Starbon and Fenton's reagent.

    Science.gov (United States)

    Tony, Maha A; Parker, Helen L; Clark, James H

    2016-09-18

    The use of grey water for a variety of purposes is gaining increased popularity as a means of preserving scarce freshwater resources. In this work, catalytic oxidation over Fenton's reagent and adsorption techniques using Starbon (mesoporous material derived from polysaccharides) has been applied. These novel techniques are used as an alternative to already studied treatments of grey water such as filtration and/or biological processes. In this study, grey water, collected from a commercial laundrette, has been used. Treatment efficiency was determined by changes in the chemical oxygen demand (COD) of the grey water. Experiments using Fenton's reagent at optimum conditions of Fe(3+) = 40 mg L(-1); H2O2 = 400 mg L(-1) and pH 3 were very successful, resulting in a 95% COD removal after 15 min. Treatment with Starbon adsorption was also effective, reaching up to 81% COD removal at pH 3 within 1 h. The combined treatment with Fenton's reagent and Starbon resulted in a 93% COD removal at a significantly reduced concentration of Fenton's reagent compared to the treatment with solo Fenton's reagent. This lower chemical dose has the advantage of reducing costs and lowering sludge generation.

  3. 2D Affine and Projective Shape Analysis.

    Science.gov (United States)

    Bryner, Darshan; Klassen, Eric; Huiling Le; Srivastava, Anuj

    2014-05-01

    Current techniques for shape analysis tend to seek invariance to similarity transformations (rotation, translation, and scale), but certain imaging situations require invariance to larger groups, such as affine or projective groups. Here we present a general Riemannian framework for shape analysis of planar objects where metrics and related quantities are invariant to affine and projective groups. Highlighting two possibilities for representing object boundaries-ordered points (or landmarks) and parameterized curves-we study different combinations of these representations (points and curves) and transformations (affine and projective). Specifically, we provide solutions to three out of four situations and develop algorithms for computing geodesics and intrinsic sample statistics, leading up to Gaussian-type statistical models, and classifying test shapes using such models learned from training data. In the case of parameterized curves, we also achieve the desired goal of invariance to re-parameterizations. The geodesics are constructed by particularizing the path-straightening algorithm to geometries of current manifolds and are used, in turn, to compute shape statistics and Gaussian-type shape models. We demonstrate these ideas using a number of examples from shape and activity recognition.

  4. The metric-affine gravitational theory as the gauge theory of the affine group

    International Nuclear Information System (INIS)

    Lord, E.A.

    1978-01-01

    The metric-affine gravitational theory is shown to be the gauge theory of the affine group, or equivalently, the gauge theory of the group GL(4,R) of tetrad deformations in a space-time with a locally Minkowskian metric. The identities of the metric-affine theory, and the relationship between them and those of general relativity and Sciama-Kibble theory, are derived. (Auth.)

  5. A Catalyst-Free Amination of Functional Organolithium Reagents by Flow Chemistry.

    Science.gov (United States)

    Kim, Heejin; Yonekura, Yuya; Yoshida, Jun-Ichi

    2018-04-03

    Reported is the electrophilic amination of functional organolithium intermediates with well-designed aminating reagents under mild reaction conditions using flow microreactors. The aminating reagents were optimized to achieve efficient C-N bond formation without using any catalyst. The electrophilic amination reactions of functionalized aryllithiums were successfully conducted under mild reaction conditions, within 1 minute, by using flow microreactors. The aminating reagent was also prepared by the flow method. Based on stopped-flow NMR analysis, the reaction time for the preparation of the aminating reagent was quickly optimized without the necessity of work-up. Integrated one-flow synthesis consisting of the generation of an aryllithium, the preparation of an aminating reagent, and their combined reaction was successfully achieved to give the desired amine within 5 minutes of total reaction time. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Comparação de bulas de duas marcas de tiras reagentes utilizadas no exame químico de urina Comparison of product labelings of two marks of reagent strips for the chemical examination of urine

    Directory of Open Access Journals (Sweden)

    Adriana Scotti da Silva Colombeli

    2006-04-01

    a quick, simple and economic analyze. OBJECTIVES: To compare drug labelings of two marks of reagent strips in urinalysis (Roche Combur10 Test® UX and Bayer Multistix® 10 SG. MATERIAL AND METHOD: The principles of pH, protein, glucose, ketone bodies, hemoglobin, bilirubin, urobilinogen, nitrite, specific gravity and leukocytes determinations, as well as the information about possible interferences were compared. RESULTS: There were differences in the detection’s reagents of some parameters, like urobilinogen (The Ehrlich’s reagent present in Multistix is less specific and more propense to analytical interferences than diazonium salt derived of methoxybenzene, used by Roche strip; the differences in the reagents used for nitrite, protein, glucose, bilirubin and hemoglobin determinations were relatively subtle. DISCUSSION: There was diversity of information about the potential interferences, which could be justified (at least partially by differences in the reagents. Differences in the information about interferences had been detected between Spanish and Portuguese versions in the same product labeling, like the omission of some interferences in the product labeling in Portuguese. Great disparity in the reaction’s intensity and its expression in crosses were observed (e.g. for glucose, and it could lead to misunderstanding in the interpretation of laboratorial findings. CONCLUSION: These data reinforce the importance of standardizations in the urinalysis.

  7. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    Directory of Open Access Journals (Sweden)

    Helena Sork

    2016-01-01

    Full Text Available The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.

  8. Improved multiple displacement amplification (iMDA) and ultraclean reagents.

    Science.gov (United States)

    Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

    2014-06-06

    Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance

  9. Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2014-06-17

    Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

  10. Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

    Directory of Open Access Journals (Sweden)

    Heger Christopher D

    2010-12-01

    Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

  11. Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

    Science.gov (United States)

    Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

    2001-10-30

    The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

  12. Determination of tin(II) in reagents for radiopharmaceuticals

    International Nuclear Information System (INIS)

    Morosanova, E.I.; Loginova, K. A.; Epstein, N.B.

    2003-01-01

    The goal of this work is to elaborate a procedure for rapid and simple determination of tin(II) in reagents for preparation of radiopharmaceuticals based on a system albumin-Tc-99m. Original test tools for the determination of various analytes have been suggested in our lab based on the use of small glass tubes (1-2 mm i.d. - 50-70 mm) filled with indicator powders containing suitable immobilized chromogenic reagents. An analytical signal (a length of colored zone which is proportional to the concentration of an analyte) is detected after a sample passing through the indicator tube. Heteropoly compounds are well-known analytical reagents for a photometric determination of various reductants. For elaboration of indicator tubes abilities of Mo,P-heteropoly compounds to give deeply colored blue compounds after reduction were used. (authors)

  13. A Generalized Affine Isoperimetric Inequality

    OpenAIRE

    Chen, Wenxiong; Howard, Ralph; Lutwak, Erwin; Yang, Deane; Zhang, Gaoyong

    2004-01-01

    A purely analytic proof is given for an inequality that has as a direct consequence the two most important affine isoperimetric inequalities of plane convex geometry: The Blaschke-Santalo inequality and the affine isoperimetric inequality of affine differential geometry.

  14. Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

    Science.gov (United States)

    Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

    2014-06-01

    We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both

  15. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

  16. Stability study for magnetic reagent assaying Hb and HbA1c

    International Nuclear Information System (INIS)

    Hsieh, Wen-Pin; Chieh, J.J.; Yang, C.C.; Yang, S.Y.; Chen, Po-Yu; Huang, Yu-Hao; Hong, Y.W.; Horng, H.E.

    2013-01-01

    Reagents for magnetically labeled immunoassay on human Hb and human HbA1c have been synthesized. The reagents consist of Fe 3 O 4 magnetic particles biofunctionalized with antibodies against Hb and HbA1c. It has been demonstrated that the reagents can be applied to quantitatively detect Hb and HbA1c by using immunomagnetic reduction assay. In addition to characterizing the assay properties, such as the standard curve and the low-detection limit, the stability of reagents is investigated. To do this, the temporal dependence of particle sizes and the bio-activity of reagents are monitored. The results show that the reagents are highly stable when stored at 2–8 °C. This means that the reagents synthesized in this work are promising for practical applications. - Highlights: ► The properties of assaying Hb and HbA1c using immunomagnetic reduction are studied. ► The magnetic nanoparticles with antibodies are highly stable in solutions. ► No significant mutual interference between Hb and HbA1c in assays is observed. ► High-sensitivity assays on Hb and HbA1c using immunomagnetic reduction are achieved.

  17. Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

    DEFF Research Database (Denmark)

    Prentø, Jannick Cornelius; Bukh, Jens

    2011-01-01

    to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~30% recovery of infectious particles. The full viability and unaltered...

  18. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    Energy Technology Data Exchange (ETDEWEB)

    Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  19. The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

    Science.gov (United States)

    Baliova, Martina; Juhasova, Anna; Jursky, Frantisek

    2015-12-01

    Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Lp-mixed affine surface area

    Science.gov (United States)

    Wang, Weidong; Leng, Gangsong

    2007-11-01

    According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

  1. Stability study for magnetic reagent assaying Hb and HbA1c

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, Wen-Pin [Actherm Inc., Hsinchu 200, Taiwan (China); Chieh, J.J.; Yang, C.C. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Yang, S.Y. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); MagQu Co., Ltd., Sindian Dist., New Taipei City 231, Taiwan (China); Chen, Po-Yu; Huang, Yu-Hao [Actherm Inc., Hsinchu 200, Taiwan (China); Hong, Y.W. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Horng, H.E., E-mail: phyfv001@ntnu.edu.tw [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China)

    2013-01-15

    Reagents for magnetically labeled immunoassay on human Hb and human HbA1c have been synthesized. The reagents consist of Fe{sub 3}O{sub 4} magnetic particles biofunctionalized with antibodies against Hb and HbA1c. It has been demonstrated that the reagents can be applied to quantitatively detect Hb and HbA1c by using immunomagnetic reduction assay. In addition to characterizing the assay properties, such as the standard curve and the low-detection limit, the stability of reagents is investigated. To do this, the temporal dependence of particle sizes and the bio-activity of reagents are monitored. The results show that the reagents are highly stable when stored at 2-8 Degree-Sign C. This means that the reagents synthesized in this work are promising for practical applications. - Highlights: Black-Right-Pointing-Pointer The properties of assaying Hb and HbA1c using immunomagnetic reduction are studied. Black-Right-Pointing-Pointer The magnetic nanoparticles with antibodies are highly stable in solutions. Black-Right-Pointing-Pointer No significant mutual interference between Hb and HbA1c in assays is observed. Black-Right-Pointing-Pointer High-sensitivity assays on Hb and HbA1c using immunomagnetic reduction are achieved.

  2. Extreme disorder in an ultrahigh-affinity protein complex

    Science.gov (United States)

    Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

    2018-03-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

  3. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  4. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    Directory of Open Access Journals (Sweden)

    Renata Angeli

    2009-01-01

    Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

  5. Mapping Affinities in Academic Organizations

    Directory of Open Access Journals (Sweden)

    Dario Rodighiero

    2018-02-01

    Full Text Available Scholarly affinities are one of the most fundamental hidden dynamics that drive scientific development. Some affinities are actual, and consequently can be measured through classical academic metrics such as co-authoring. Other affinities are potential, and therefore do not leave visible traces in information systems; for instance, some peers may share interests without actually knowing it. This article illustrates the development of a map of affinities for academic collectives, designed to be relevant to three audiences: the management, the scholars themselves, and the external public. Our case study involves the School of Architecture, Civil and Environmental Engineering of EPFL, hereinafter ENAC. The school consists of around 1,000 scholars, 70 laboratories, and 3 institutes. The actual affinities are modeled using the data available from the information systems reporting publications, teaching, and advising scholars, whereas the potential affinities are addressed through text mining of the publications. The major challenge for designing such a map is to represent the multi-dimensionality and multi-scale nature of the information. The affinities are not limited to the computation of heterogeneous sources of information; they also apply at different scales. The map, thus, shows local affinities inside a given laboratory, as well as global affinities among laboratories. This article presents a graphical grammar to represent affinities. Its effectiveness is illustrated by two actualizations of the design proposal: an interactive online system in which the map can be parameterized, and a large-scale carpet of 250 square meters. In both cases, we discuss how the materiality influences the representation of data, in particular the way key questions could be appropriately addressed considering the three target audiences: the insights gained by the management and their consequences in terms of governance, the understanding of the scholars’ own

  6. Reliability of reagent strips for semi-quantitative measurement of glucosuria in a neonatal intensive care setting

    NARCIS (Netherlands)

    Bekhof, Jolita; Kollen, Boudewijn J.; van de Leur, Sjef; Kok, Joke H.; van Straaten, Irma H. L. M.

    2014-01-01

    Glucosuria in preterm infants is often measured using a visually readable reagent strip, e.g., when monitoring total parenteral nutrition or during sepsis or when treating with corticosteroids. However, the specific circumstances in a neonatal intensive care unit (NICU), such as the use of diapers

  7. Reliability of Reagent Strips for Semi-quantitative Measurement of Glucosuria in a Neonatal Intensive Care Setting

    NARCIS (Netherlands)

    Bekhof, Jolita; Kollen, Boudewijn J.; van de Leur, Sjef; Kok, Joke H.; van Straaten, Irma H. L. M.

    2014-01-01

    Background: Glucosuria in preterm infants is often measured using a visually readable reagent strip, e.g., when monitoring total parenteral nutrition or during sepsis or when treating with corticosteroids. However, the specific circumstances in a neonatal intensive care unit (NICU), such as the use

  8. On extraction reagents for hydrometallurgy

    International Nuclear Information System (INIS)

    Zolotov, Yu.A.

    1975-01-01

    Fundamental requirements to the extractants are considered. Ways of obtaining selective extractants are discussed in particular on the basis of coordination chemistry achivements. Attention is drawn to expediency of study (as extractants) of flotation reagents, additions to the oil, pesticides, accelerators of caoutchouc vulcanization

  9. Review Article: Toxic Effects of Some Reagents Used in Electron ...

    African Journals Online (AJOL)

    Ultrathin sections prepared for electron microscopy and histochemistry are indispensable in cytological, histological and histochemical studies. The paper discusses the various reagents used in these fields of study. Unfortunately, these reagents and chemicals are hazardous to health. There is wisdom in informing the ...

  10. Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

    Science.gov (United States)

    Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

    2013-04-01

    Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by

  11. Reagents for the assay of cardenolide glycosides and aglycones

    International Nuclear Information System (INIS)

    Wilkinson, S.

    1976-01-01

    Some novel reagents are described for use in the radioimmunoassay of the 3-glycone derivatives of cardenolides (cardiac glycosides) and more especially digoxin, digitoxin, gitoxin, periplocin and lanatosides. Using these reagents these cardenolides and their derivatives may be assayed both in aqueous solution and in urine. A method is also described for performing such assays, including a suitable kit. (U.K.)

  12. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  13. The HLA-DP polymorphism in Denmark investigated by local and international PLT reagents. Definition of two "new" DP antigens

    DEFF Research Database (Denmark)

    Ødum, Niels; Hartzman, R; Jakobsen, B K

    1986-01-01

    Lymphocytes from highly selected donors were primed for 10 days and subsequently bulk-expanded in IL 2 (TCGF) containing cultures. Two well-discriminatory PLT (CDP = Copenhagen DP) reagents against each of the DPw1-w6 specificities and one against each of the two "new" specificities, CDP4s...

  14. Investigation of cleaning reagents for calcium chromate spills

    International Nuclear Information System (INIS)

    Dillard, B.M.

    1979-01-01

    Cleaning of calcium chromate spills can be a problem due to the insolubility of the material and the corrosiveness of several possible cleaning agents on the stainless steel equipment. Because of OSHA Standards for Cr(VI) exposure, it is necessary to remove spills as efficiently as possible in order to prevent the contaminant from becoming airborne. This study involved the comparison of several possible cleaning agents by studying the solubility of calcium chromate in each reagent. Two general types of reagents for dissolution of calcium chromate were investigated; those which act by conversion of the insoluble calcium chromate to a more soluble salt and to H 2 CrO 4 , and those which appear to act as complexing agents and thereby dissolve the calcium chromate. The most efficient of the reagents investigated was hydrochloric acid. However, even dilute solutions of halide acids destroy passivity of stainless steel causing pitting and stress-corrosion. Acetic acid and nitric acid were somewhat less efficient than hydrochloric acid in dissolving calcium chromate. However, both reagents are noncorrosive with stainless steel, nitric acid tending to favor passivity of the materials. Therefore, it is recommended that dilute solutions of either of these two acids be used for removal of calcium chromate spills in conjunction with mechanical methods that might be necessary, depending on the magnitude of the spill

  15. Use of Competition Kinetics with Fast Reactions of Grignard Reagents

    DEFF Research Database (Denmark)

    Holm, Torkil

    2000-01-01

    small.This is concluded from experiments in which results obtained by competition kinetics are compared with results obtained directly by flow stream procedures. A clearer picture of the reactivity ratios is obtained when the highly reactive reagent is highly diluted with its competitor. A fast reagent...

  16. Size and shape dependent deprotonation potential and proton affinity of nanodiamond

    International Nuclear Information System (INIS)

    Barnard, Amanda S; Per, Manolo C

    2014-01-01

    Many important reactions in biology and medicine involve proton abstraction and transfer, and it is integral to applications such as drug delivery. Unlike electrons, which are quantum mechanically delocalized, protons are instantaneously localized on specific residues in these reactions, which can be a distinct advantage. However, the introduction of nanoparticles, such as non-toxic nanodiamonds, to this field complicates matters, as the number of possible sites increases as the inverse radius of the particle. In this paper we present >10 4 simulations that map the size- and shape-dependence of the deprotonation potential and proton affinity of nanodiamonds in the range 1.8–2.7 nm in average diameter. We find that while the average deprotonation potential and proton affinities decrease with size, the site-specific values are inhomogeneous over the surface of the particles, exhibiting strong shape-dependence. The proton affinity is strongly facet-dependent, whereas the deprotonation potential is edge/corner-dependent, which creates a type of spatial hysteresis in the transfer of protons to and from the nanodiamond, and provides new opportunities for selective functionalization. (paper)

  17. Size and shape dependent deprotonation potential and proton affinity of nanodiamond

    Science.gov (United States)

    Barnard, Amanda S.; Per, Manolo C.

    2014-11-01

    Many important reactions in biology and medicine involve proton abstraction and transfer, and it is integral to applications such as drug delivery. Unlike electrons, which are quantum mechanically delocalized, protons are instantaneously localized on specific residues in these reactions, which can be a distinct advantage. However, the introduction of nanoparticles, such as non-toxic nanodiamonds, to this field complicates matters, as the number of possible sites increases as the inverse radius of the particle. In this paper we present \\gt {{10}4} simulations that map the size- and shape-dependence of the deprotonation potential and proton affinity of nanodiamonds in the range 1.8-2.7 nm in average diameter. We find that while the average deprotonation potential and proton affinities decrease with size, the site-specific values are inhomogeneous over the surface of the particles, exhibiting strong shape-dependence. The proton affinity is strongly facet-dependent, whereas the deprotonation potential is edge/corner-dependent, which creates a type of spatial hysteresis in the transfer of protons to and from the nanodiamond, and provides new opportunities for selective functionalization.

  18. Fundamentals of affinity cell separations.

    Science.gov (United States)

    Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

    2018-03-01

    Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  20. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    Miles, L.A.; Plow, E.F.

    1986-01-01

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  1. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  2. Affine field theories

    International Nuclear Information System (INIS)

    Cadavid, A.C.

    1989-01-01

    The author constructs a non-Abelian field theory by gauging a Kac-Moody algebra, obtaining an infinite tower of interacting vector fields and associated ghosts, that obey slightly modified Feynman rules. She discusses the spontaneous symmetry breaking of such theory via the Higgs mechanism. If the Higgs particle lies in the Cartan subalgebra of the Kac-Moody algebra, the previously massless vectors acquire a mass spectrum that is linear in the Kac-Moody index and has additional fine structure depending on the associated Lie algebra. She proceeds to show that there is no obstacle in implementing the affine extension of supersymmetric Yang-Mills theories. The result is valid in four, six and ten space-time dimensions. Then the affine extension of supergravity is investigated. She discusses only the loop algebra since the affine extension of the super-Poincare algebra appears inconsistent. The construction of the affine supergravity theory is carried out by the group manifold method and leads to an action describing infinite towers of spin 2 and spin 3/2 fields that interact subject to the symmetries of the loop algebra. The equations of motion satisfy the usual consistency check. Finally, she postulates a theory in which both the vector and scalar fields lie in the loop algebra of SO(3). This theory has an expanded soliton sector, and corresponding to the original 't Hooft-Polyakov solitonic solutions she now finds an infinite family of exact, special solutions of the new equations. She also proposes a perturbation method for obtaining an arbitrary solution of those equations for each level of the affine index

  3. Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

    Science.gov (United States)

    Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

    2007-01-01

    Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method

  4. Mössbauer spectroscopy research of interaction of alumosilicic reagent and iron dissolved in water

    International Nuclear Information System (INIS)

    Feklistov, D Y; Filippov, V P; Kurchatov, I M; Laguntsov, N I; Salomasov, V A; Permyakov, Yu V

    2016-01-01

    The aim of this work is to reveal the results of alumosilicic reagent interaction with iron compounds contained in the water. This reagent is simultaneously coagulant-flocculant and adsorbent. The iron atoms state is studied in the reagent and in reacted sediment. The valence state of iron atoms are determined in the reagents and sediments. The existence of iron containing superparamagnetic particles in the sediment is shown. (paper)

  5. Electron affinities: theoretical

    International Nuclear Information System (INIS)

    Kaufman, J.J.

    1976-01-01

    A brief description is given of the conceptual background and formalism of the various ab-initio and semi-ab-initio quantum computational techniques for calculating atomic and molecular electron affinities: Hartree--Fock--Roothaan SCF, configuration interaction (CI), multiconfiguration SCF (MC-SCF), Bethe--Goldstone, superposition of configurations (SOC), ab-initio effective core model potentials, Xα-MS, plus other less common methods. Illustrative and comparative examples of electron affinities calculated by these various methods are presented

  6. Lanthanide shift reagents, binding, shift mechanisms and exchange

    International Nuclear Information System (INIS)

    Boer, J.W.M. de

    1977-01-01

    Paramagnetic lanthanide shift reagents, when added to a solution of a substrate, induce shifts in the nuclear magnetic resonance (NMR) spectrum of the substrate molecules. The induced shifts contain information about the structure of the shift reagent substrate complex. The structural information, however, may be difficult to extract because of the following effects: (1) different complexes between shift reagent and substrate may be present in solution, e.g. 1:1 and 1:2 complexes, and the shift observed is a weighed average of the shifts of the substrate nuclei in the different complexes; (2) the Fermi contact interaction, arising from the spin density at the nucleus, contributes to the induced shift; (3) chemical exchange effects may complicate the NMR spectrum. In this thesis, the results of an investigation into the influence of these effects on the NMR spectra of solutions containing a substrate and LSR are presented. The equations describing the pseudo contact and the Fermi contact shift are derived. In addition, it is shown how the modified Bloch equations describing the effect of the chemical exchange processes occurring in the systems studied can be reduced to the familiar equations for a two-site exchange case. The binding of mono- and bifunctional ethers to the shift reagent are reported. An analysis of the induced shifts is given. Finally, the results of the experiments performed to study the exchange behavior of dimethoxyethane and heptafluorodimethyloctanedionato ligands are presented

  7. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  8. Hemoglobin affinity in Andean rodents

    Directory of Open Access Journals (Sweden)

    HRVOJ OSTOJIC

    2002-01-01

    Full Text Available Blood hemoglobin oxygen affinity (P50 was measured in three Andean species and in the laboratory rat (control, all raised near sea level. Chinchilla lanigera (Molina, 1792 has an altitudinal habitat range from low Andean slopes up to 3000 m., while Chinchilla brevicaudata (Waterhouse, 1848 has an altitudinal range from 3000 to 5000 m. The laboratory type guinea pig, wild type guinea pig (Cavia porcellus, (Waterhouse, 1748, and laboratory rat (Rattus norvegicus were also raised at sea level. The Andean species had high hemoglobin oxygen affinities (low P50 compared with the rat. Chinchilla brevicaudata had a higher affinity than Chinchilla lanigera. The wild type guinea pig had a higher affinity than the laboratory type. As has been shown in other species, this is another example of an inverse correlation between the altitude level and the P50 values. This is the first hemoglobin oxygen affinity study in Chinchilla brevicaudata.

  9. Organic-soluble lanthanide nuclear magnetic resonance shift reagents for sulfonium and isothiouronium salts

    International Nuclear Information System (INIS)

    Wenzel, T.J.; Zaia, J.

    1987-01-01

    Lanthanide complexes of the formula [Ln(fod) 4 ] - (FOD, 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedione) are effective organic-soluble nuclear magnetic resonance shift reagents for sulfonium and isothiouronium salts. The shift reagent is formed in solution from Ln(fod) 3 and Ag(fod) or K(fod). The selection of Ag(fod) or K(fod) in forming the shift reagent is dependent on the anion of the organic salt. Ag(fod) is more effective with halide salts, whereas K(fod) is preferred with tetrafluoroborate salts. Resolution of diastereotopic hydrogen atoms was observed in the shifted spectra of certain substrates. Enantiomeric resolution was obtained in the spectrum of sec-butylisothiouronium chloride with a chiral shift reagent. The reagents can be employed in solvents such as chloroform and benzene

  10. New sorption-reagent materials for decontamination of liquid radioactive waste

    International Nuclear Information System (INIS)

    Avramenko, V.A.; Golikov, A.P.; Zheleznov, V.V.; Kaplun, E.V.; Marinin, D.V.; Sokolnitskaya, T.A.

    2001-01-01

    Full text: Use of selective sorbents in liquid radioactive waste (LRW) management is widely spread in the field of nuclear power objects liquid waste decontamination, since the main objective there is to remove long-lived radionuclides of the nuclear cycle. The latter include, first of all, cesium-137, strontium-90, cobalt-60 and a number of α-irradiators. In this case LRW composition for most of the nuclear power objects is rather simple, except acidic deactivation solutions. At the same time, liquid radioactive wastes of different research centers have a variable chemical and radiochemical composition depending on objectives and tasks of a given center research activities. As a result, application of sorption technologies in such waste decontamination determines special requirements to these sorbents selectivity: a wide spectrum of radionuclides that can be removed and fairly high selectivity enabling to remove radionuclides from solutions of complex chemical composition (containing surfactants, complexing agents etc.). This paper is concerned with studying properties of new materials selective to different radionuclides. These materials are capable to interact with solution components whether already contained in the waste or deliberately added into resulting solution. Such sorption-reagent materials combine universal character of co-precipitation methods with simplicity of sorption methods. In this work we studied sorption-reagent inorganic ion-exchange materials interacting with sulfate-, carbonate-, oxalate-, sulfide-, and permanganate-ions. Insoluble compounds formed as a result of this interaction increase tens- and hundreds-fold the sorption selectivity of different radionuclides - strontium, cobalt, mercury, iron, and manganese as compared to conventional ion-exchange system. By means of X-ray phase analysis, IR-spectroscopy, chemical and radiochemical analysis, we have studied the mechanism of radionuclide sorption on different sorption-reagent

  11. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    Science.gov (United States)

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. First two-reagent vitamin D assay for general clinical chemistry.

    Science.gov (United States)

    Saida, Fakhri B; Padilla-Chee, Mario; Dou, Chao; Yuan, Chong

    2018-05-01

    Vitamin D is a lipid-soluble molecule that plays key physiological roles in the metabolism of calcium, phosphate and magnesium. Recent studies show that deficiency in vitamin D is linked to cardiovascular diseases, autoimmune diseases and cancer. As a result, regular monitoring of 25-OH vitamin D (the main circulating form of vitamin D) is becoming essential. Current 25-OH vitamin D testing methodologies are cumbersome (too many reagents, long incubation times, phase separation) and are not compatible with general clinical chemistry platforms. Here, we report on a novel method to detect 25-OH vitamin D that is fast (results in 10 min or less), simple (two reagents) and compatible with virtually all general clinical chemistry analyzers. An immunoturbidimetric assay for 25-OH vitamin D (the Diazyme EZ Vitamin D Assay) has been developed using nanoparticles and vitamin D-specific antibodies. The performance of the assay kit, which consists of two reagents and five calibrators, was tested on the Beckman AU680 analyzer (AU680). The new assay was precise, sensitive (LOD = 7.2 nmol/L), linear (up to 390.1 nmol/L) and correlated strongly (R 2  > 0.95) with major commercial 25-OH vitamin D assays. Additionally, the assay was found to be the fastest to date, with the first results obtained within 10 min. Throughput on the AU680 was estimated at over 300 tests per hour. The newly developed 25-OH vitamin D assay is fast, precise and accurate. It can be run on most general chemistry analyzers. This assay aims at providing vitamin D-testing capabilities to all clinical chemistry laboratories. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  13. Reagent conditions of the flotation of copper, copper - molybdenum and copper -zinc ores in foreing countries

    International Nuclear Information System (INIS)

    Nevaeva, L.M.

    1983-01-01

    Reagents-collectors and frothers, used abroad in reagent regimes of flotation of copper, copper-molybdenum and copper zinc ores, have been considered. Xanthogenates, aerofloats, xanthogenformiates, thionocarbamates are mainly used as reagents-collectors. Methylizobutylcarbinol and Daufros are used as reagents-frothers

  14. Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents.

    Science.gov (United States)

    Yamagata, Masahito; Sanes, Joshua R

    2018-02-27

    Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.

  15. Affinity of antibody secreted by a single cell

    International Nuclear Information System (INIS)

    Doran, D.M.

    1978-01-01

    It was the intention of this research to measure the affinity of antibody secreted by a single cell, and to describe the spectrum of affinities displayed in response to antigenic stimulation. The single cell secreting specific antibody was isolated by means of the hemolytic plaque assay. The amount of antibody secreted by the cell was to be measured through the use of a solid phase radioimmunoassay. The affinity of the antibody would be estimated by comparing the diameter of the plaque, and the amount of antibody secreted, with a mathematical theory of the formation of a plaque in agar. As a test system, a solid phase radioimmunoassay was developed for human serum albumin using antibody coupled to Sephadex. A sensitivity of 1 nanogram was attained with this assay. A solid phase radioimmunoassay for mouse immunoglobulin M was developed, using antibody coupled to Sepharose. The sensitivity attained with this assay was only on the order of 10 micrograms. The mouse immunoglobulin M radioimmunoassay was not sensitive enough to measure the amount of antibody secreted by a single cell. From a theoretical equation, the relationship between antibody affinity, plaque diameter and antibody secretion rate was calculated for the experimental conditions used in this research. By assuming a constant antibody secretion rate, an effective binding constant for the antibody was estimated from the average plaque diameters. This effective binding constant was observed to increase during the immune response

  16. Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

    Science.gov (United States)

    Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

    2015-01-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

  17. Novel trends in affinity biosensors: current challenges and perspectives

    International Nuclear Information System (INIS)

    Arugula, Mary A; Simonian, Aleksandr

    2014-01-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives. (topical review)

  18. 21 CFR 866.3120 - Chlamydia serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3120 Chlamydia... and antisera used in serological tests to identify antibodies to chlamydia in serum. Additionally...

  19. 21 CFR 866.3490 - Rhinovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3490 Rhinovirus... and antisera used in serological tests to identify antibodies to rhinovirus in serum. The...

  20. 21 CFR 866.3020 - Adenovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020 Adenovirus... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease...

  1. 21 CFR 866.3205 - Echovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3205 Echovirus... echoviruses from clinical specimens or from tissue culture isolates derived from clinical specimens. The...

  2. 125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage

    International Nuclear Information System (INIS)

    Denny, J.B.; Blobel, G.

    1984-01-01

    A radioactive crosslinking reagent, N-[4-(p-azido-m-[ 125 I]iodophenylazo)benzoyl]-3-aminopropyl-N'-oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with 125 I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an 125 I-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366-nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid using carrier-free Na 125 I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lower by a factor of 3.6 than that of the heavy chain. 36 references, 3 figures

  3. Reagent precipitation of copper ions from wastewater of machine-building factories

    Science.gov (United States)

    Porozhnyuk, L. A.; Lupandina, N. S.; Porozhnyuk, E. V.

    2018-03-01

    The article presents the results of reagent removal of copper ions from wastewater of machine-building factories. The urgency of the study is conditioned by the widening of the range of effective reagents through the implementation of industrial waste. The investigation covers mineralogical and fractional composition of chalk enrichment waste. In the work, the conditions of thermal activation of chalk enrichment waste used for reagent removal of copper ions from wastewater were elaborated. It was shown that the thermal activation of waste facilitates the increased treatment efficacy up to the set sanitation, hygiene and technological standards.

  4. A importância da qualidade da água reagente no laboratório clínico The importance of water quality in clinical laboratory reagent

    Directory of Open Access Journals (Sweden)

    Maria Elizabete Mendes

    2011-06-01

    Full Text Available A água é um reagente utilizado na maioria dos testes laboratoriais e por isso deve seguir um padrão de controle de qualidade rigoroso. O fornecimento urbano de água apresenta moléculas orgânicas, íons inorgânicos, partículas, coloides, gases, bactérias e seus produtos, que podem alterar os resultados dos exames laboratoriais e causar eventuais erros e falhas mecânicas em equipamentos analíticos. Para remover essas impurezas, é necessário recorrer a uma combinação de tecnologias de purificação. Há várias organizações que especificam normas sobre a água reagente, a fim de minimizar sua interferência nos ensaios laboratoriais. A maioria dos laboratórios utiliza as normas estabelecidas pelo Clinical and Laboratory Standards Institute (CLSI que classifica a água em: clinical laboratory reagent water (CLRW, special reagent water (SRW e instrumental feed water (IFW. O monitoramento da qualidade é realizado pela determinação de resistividade, condutividade, carbono orgânico total (TOC, controle microbiológico e endotoxinas. Os parâmetros são avaliados de acordo com a periodicidade estabelecida pela norma utilizada. Neste artigo, discutem-se a importância da água utilizada nos procedimentos laboratoriais, o controle da qualidade e as interferências nos ensaios laboratoriais.Water is a reagent used in most laboratory tests and, therefore, must follow stringent quality control standards. The urban water supply has organic molecules, inorganic ions, particles, colloids, gases, bacteria and their products, which may alter laboratory test results and cause occasional errors and mechanical failures in diagnostic equipment. To remove these impurities, it is necessary to use a combination of purification technologies. There are several organizations that specify reagent water standards to minimize its interference in laboratory assays. Most laboratories set standards established by the Clinical and Laboratory Standards

  5. 21 CFR 866.3470 - Reovirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3470 Reovirus... and antisera used in serological tests to identify antibodies to reovirus in serum. The identification...

  6. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  7. Extreme disorder in an ultrahigh-affinity protein complex

    DEFF Research Database (Denmark)

    Borgia, Alessandro; Borgia, Madeleine B; Bugge, Katrine

    2018-01-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions...... with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring...... or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex...

  8. Field-Portable Immunoassay Instruments and Reagents to Measure Chelators and Mobile Forms of Uranium

    International Nuclear Information System (INIS)

    Blake, Diane A.

    2003-01-01

    The goals for the 3-year project period are (1) to test and validate the present uranium sensor and develop protocols for its use at the NABIR Field Research Center; (2) to develop new reagents that will provide superior performance for the present hand-held immunosensor; and (3) to develop new antibodies that will permit this sensor to also measure other environmental contaminants (chromium, mercury, and/or DTPA). Sensor design modifications are underway via international collaborations. New reagents that will provide superior performance for the present hand-held immunosensor are being prepared and tested. New methods have been developed, to produce recombinant forms of metal-specific monoclonal antibodies for use with the sensor. Site-directed mutagenesis experiments are underway to determine the mechanisms of binding. Immunization experiments with sheep and rabbits to develop new recombinant forms of antibodies to metal-chelate complexes (chromium, mercury, and/or DTPA) have been initiated

  9. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  10. The application of magnetic force differentiation for the measurement of the affinity of peptide libraries

    International Nuclear Information System (INIS)

    Shang Hao; Kirkham, Perry M.; Myers, Tina M.; Cassell, Gail H.; Lee, Gil U.

    2005-01-01

    A new method has been developed for measuring the binding affinity of phage displayed peptides and a target protein using magnetic particles. The specific interaction between the phage displayed peptides and the target protein was subject to a force generated by the magnetic particle. The binding affinity was obtained by analyzing the force-bond lifetime

  11. 2017 Guralp Affinity Digitizer Evaluation.

    Energy Technology Data Exchange (ETDEWEB)

    Merchant, Bion J.

    2018-03-01

    Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  12. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko

    2014-04-01

    Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

  13. Two-parameter quantum affine algebra Ur,s(sln-circumflex), Drinfeld realization and quantum affine Lyndon basis

    International Nuclear Information System (INIS)

    Hu Naihong; Rosso, M.; Zhang Honglian

    2006-12-01

    We further find the defining structure of a two-parameter quantum affine algebra U r,s (sl n -circumflex) (n > 2) in the sense of Benkart-Witherspoon [BW1] after the work of [BGH1], [HS] and [BH], which turns out to be a Drinfeld double. Of more importance for the 'affine' cases is that we work out the compatible two-parameter version of the Drinfeld realization as a quantum affinization of U r,s (sl n ) and establish the Drinfeld isomorphism Theorem in the two-parameter setting via developing a new remarkable combinatorial approach - quantum 'affine' Lyndon basis with an explicit valid algorithm, based on the Drinfeld realization. (author)

  14. 21 CFR 864.9650 - Quality control kit for blood banking reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control kit for blood banking reagents... SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9650 Quality control kit for blood banking reagents. (a...

  15. Performing in-situ chemical oxidation with Fenton's Reagent at a former gas work in Rotterdam

    Energy Technology Data Exchange (ETDEWEB)

    Plaisier, W.; Pancras, T. [In-situ Technieken BV, Wageningen (Netherlands); Bennen, M.; Bouwhuis, E. [Public Works Rotterdam (Netherlands)

    2003-07-01

    In-Situ Technieken BV is the first company in the Netherlands to apply full-scale in-situ chemical oxidation (ISCO) by means of Fenton's reagent or permanganates. The use of ISCO for soil remediation purposes originated in the USA. After three years of practical experience in the Netherlands it can be stated that ISCO has become a proven technology for source area treatment in the Netherlands as well. We present the results of a pilot ISCO treatment program with Fenton's reagent that was performed at the former gas works facility 'Feyenoord'. The site is one of the largest gas works facilities built in Rotterdam. The former gas production activities have lead to severe pollution of soil and groundwater over an area of approximately 3 hectares. Both laboratory and field observations showed that it would be very difficult to execute an efficient Fenton's reagent ISCO treatment program at this specific site for the following reasons. First of all, the contamination consisting of tar and tar-related compounds is found in an anthropogenic (man-made) soil of 9 meter in thickness. Secondly, high contents of carbonates and organic material were found in the soils, which act as a scavenger for the hydroxyl free radical in a Fenton's reagent ISCO system. The pilot test was performed in two phases with an intermediate period of 2 months. The injection of totally 24 tonnes hydrogen peroxide (50%) was anticipated. Reactions of the reagents in the soil were very strong. The presence of old (forgotten) boreholes and an intensive drainage system, in combination with the aggressive reaction resulted in short-circuiting of reagents to the surface. Out of the proposed 24 tonnes of hydrogen peroxide (50%) only 14.5 tonnes were injected. This would have effect on the amount of contamination oxidized. Nevertheless, the oxidation had a significant effect on the soil contamination. A reduction of approximately 90% for PAH was obtained. Reduction was also

  16. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    Science.gov (United States)

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

    Science.gov (United States)

    Artigas, Pablo; Gadsby, David C

    2006-08-15

    The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na/K pump. We used outside-out patch recording of Xenopus alpha1beta3 Na/K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus alpha1beta3 Na/K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the alpha subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pump-channels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na/K pump.

  18. Rapid heteroatom transfer to arylmetals utilizing multifunctional reagent scaffolds

    Science.gov (United States)

    Gao, Hongyin; Zhou, Zhe; Kwon, Doo-Hyun; Coombs, James; Jones, Steven; Behnke, Nicole Erin; Ess, Daniel H.; Kürti, László

    2017-07-01

    Arylmetals are highly valuable carbon nucleophiles that are readily and inexpensively prepared from aryl halides or arenes and widely used on both laboratory and industrial scales to react directly with a wide range of electrophiles. Although C-C bond formation has been a staple of organic synthesis, the direct transfer of primary amino (-NH2) and hydroxyl (-OH) groups to arylmetals in a scalable and environmentally friendly fashion remains a formidable synthetic challenge because of the absence of suitable heteroatom-transfer reagents. Here, we demonstrate the use of bench-stable N-H and N-alkyl oxaziridines derived from readily available terpenoid scaffolds as efficient multifunctional reagents for the direct primary amination and hydroxylation of structurally diverse aryl- and heteroarylmetals. This practical and scalable method provides one-step synthetic access to primary anilines and phenols at low temperature and avoids the use of transition-metal catalysts, ligands and additives, nitrogen-protecting groups, excess reagents and harsh workup conditions.

  19. Representations of affine Hecke algebras

    CERN Document Server

    Xi, Nanhua

    1994-01-01

    Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

  20. Decolorizing textile wastewater with Fenton's reagent electrogenerated with a solar photovoltaic cell.

    Science.gov (United States)

    Figueroa, Sandra; Vázquez, Leticia; Alvarez-Gallegos, A

    2009-02-01

    In this work it is demonstrated that Fenton's reagent can be electroproduced with abundant and cheap feedstock: oxygen saturated wastewater and solar energy. Experiments were carried out in a divided electrochemical flow cell using two electrodes: a three dimensional reticulated vitreous carbon cathode and stainless steel anode. Fenton's reagent is produced by oxygen reduction on the cathode in the presence of 1mM Fe(2+). The influence of electrolyte nature and its concentration and potential difference on the current efficiency, as well as the rate of Fenton's reagent electroproduction is discussed and it is concluded that over this extended range of conditions the current efficiency, for Fenton's reagent production, fell within the range 50-70%. It is possible to electroproduce a stoichiometric amount of Fenton reagent for the oxidation of 0.061mM Reactive Black 5 (in tap water+0.05M Na(2)SO(4), approximately pH 2.8). Similar results were obtained for solutions containing 0.1mM Acid Green 25. Some practical applications in the field of water treatment are included. The energy required for drive electrochemical reaction is supplied to the flow cell by means of a commercial solar panel.

  1. Dewaterability of five sewage sludges in Guangzhou conditioned with Fenton's reagent/lime and pilot-scale experiments using ultrahigh pressure filtration system.

    Science.gov (United States)

    Liang, Jialin; Huang, Shaosong; Dai, Yongkang; Li, Lei; Sun, Shuiyu

    2015-11-01

    Sludge conditioning with Fenton's reagent and lime is a valid method for sludge dewatering. This study investigated the influence of different organic matter content sludge on sludge dewatering and discussed the main mechanism of sludge conditioning by combined Fenton's reagent and lime. The results indicated that the specific resistance to filterability (SRF) of sludge was reduced efficiently by approximately 90%, when conditioned with Fenton's reagent and lime. Through single factor experiments, the optimal conditioning combinations were found. In addition, the relationship between VSS% and consumption of the reagents was detected. Furthermore, it was also demonstrated that the SRF and filtrate TOC values had a significant correlation with VSS% of sludge (including raw and conditioned). The main mechanism of sludge dewatering was also investigated. Firstly, it revealed that the dewaterability of sludge was closely correlated to extracellular polymeric substances (EPS) and bound water contents. Secondly, the results of scanning electron microscopy (SEM) stated that sludge particles were to be smaller and thinner after conditioning. And this structure could easily form outflow channels for releasing free water. Additionally, with the ultrahigh pressure filtration system, the water content of sludge cake conditioned with Fenton's reagent and lime could be reduced to below 50%. Moreover, the economic assessment shows that Fenton's reagent and lime combined with ultrahigh pressure filtration system can be an economical and viable technology for sewage sludge dewatering. Finally, three types of sludge were classified: (1) Fast to dewater; (2) Moderately fast to dewater; (3) Slow to dewater sludge. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Improvements to parallel plate flow chambers to reduce reagent and cellular requirements

    Directory of Open Access Journals (Sweden)

    Larson Richard S

    2001-09-01

    Full Text Available Abstract Background The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents. Results In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements. Conclusions These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur.

  3. A Piecewise Affine Hybrid Systems Approach to Fault Tolerant Satellite Formation Control

    DEFF Research Database (Denmark)

    Grunnet, Jacob Deleuran; Larsen, Jesper Abildgaard; Bak, Thomas

    2008-01-01

    In this paper a procedure for modelling satellite formations   including failure dynamics as a piecewise-affine hybrid system is   shown. The formulation enables recently developed methods and tools   for control and analysis of piecewise-affine systems to be applied   leading to synthesis of fault...... tolerant controllers and analysis of   the system behaviour given possible faults.  The method is   illustrated using a simple example involving two satellites trying   to reach a specific formation despite of actuator faults occurring....

  4. Antisymmetric tensor generalizations of affine vector fields.

    Science.gov (United States)

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  5. Manifolds with integrable affine shape operator

    Directory of Open Access Journals (Sweden)

    Daniel A. Joaquín

    2005-05-01

    Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

  6. Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

    Science.gov (United States)

    Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

    2017-12-01

    Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Testing Affine Term Structure Models in Case of Transaction Costs

    NARCIS (Netherlands)

    Driessen, J.J.A.G.; Melenberg, B.; Nijman, T.E.

    1999-01-01

    In this paper we empirically analyze the impact of transaction costs on the performance of affine interest rate models. We test the implied (no arbitrage) Euler restrictions, and we calculate the specification error bound of Hansen and Jagannathan to measure the extent to which a model is

  8. Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guifang [Department of Chemistry, Jinan University, Guangzhou 510632 (China); Lu, Gang [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Yin, Pinghe, E-mail: tyinph@jnu.edu.cn [Research Center of Analysis and Test, Jinan University, Guangzhou 510632 (China); Zhao, Ling, E-mail: zhaoling@jnu.edu.cn [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Jimmy Yu, Qiming [Griffith School of Engineering, Griffith University, Nathan Campus, Brisbane, Queensland 4111 (Australia)

    2016-04-15

    Highlights: • Membrane concentrates have a threat to human health and environment. • Untreated membrane concentrates induces cytotoxic and genotoxic to HepG2 cells. • Both methods were effective method for degradation of BPA and NP in concentrates. • Both methods were efficient in reducing genotoxic effects of concentrates. • UV-Fenton reagent had higher removal efficiency and provides toxicological safety. - Abstract: Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24 h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

  9. Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

    International Nuclear Information System (INIS)

    Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Jimmy Yu, Qiming

    2016-01-01

    Highlights: • Membrane concentrates have a threat to human health and environment. • Untreated membrane concentrates induces cytotoxic and genotoxic to HepG2 cells. • Both methods were effective method for degradation of BPA and NP in concentrates. • Both methods were efficient in reducing genotoxic effects of concentrates. • UV-Fenton reagent had higher removal efficiency and provides toxicological safety. - Abstract: Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24 h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

  10. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... used to identify Mycobacterium tuberculosis directly from clinical specimens. The identification aids...

  11. The affine quantum gravity programme

    International Nuclear Information System (INIS)

    Klauder, John R

    2002-01-01

    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix { g-hat ab (x)} composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that still retain some basic characteristics of gravity, specifically a partial second-class constraint operator structure. Although perturbatively nonrenormalizable, gravity may possibly be understood nonperturbatively from a hard-core perspective that has proved valuable for specialized models. Finally, developing a procedure to pass to the genuine physical Hilbert space involves several interconnected steps that require careful coordination

  12. Projections of climate-driven changes in tuna vertical habitat based on species-specific differences in blood oxygen affinity.

    Science.gov (United States)

    Mislan, K A S; Deutsch, Curtis A; Brill, Richard W; Dunne, John P; Sarmiento, Jorge L

    2017-10-01

    Oxygen concentrations are hypothesized to decrease in many areas of the ocean as a result of anthropogenically driven climate change, resulting in habitat compression for pelagic animals. The oxygen partial pressure, pO 2 , at which blood is 50% saturated (P 50 ) is a measure of blood oxygen affinity and a gauge of the tolerance of animals for low ambient oxygen. Tuna species display a wide range of blood oxygen affinities (i.e., P 50 values) and therefore may be differentially impacted by habitat compression as they make extensive vertical movements to forage on subdaily time scales. To project the effects of end-of-the-century climate change on tuna habitat, we calculate tuna P 50 depths (i.e., the vertical position in the water column at which ambient pO 2 is equal to species-specific blood P 50 values) from 21st century Earth System Model (ESM) projections included in the fifth phase of the Climate Model Intercomparison Project (CMIP5). Overall, we project P 50 depths to shoal, indicating likely habitat compression for tuna species due to climate change. Tunas that will be most impacted by shoaling are Pacific and southern bluefin tunas-habitat compression is projected for the entire geographic range of Pacific bluefin tuna and for the spawning region of southern bluefin tuna. Vertical shifts in P 50 depths will potentially influence resource partitioning among Pacific bluefin, bigeye, yellowfin, and skipjack tunas in the northern subtropical and eastern tropical Pacific Ocean, the Arabian Sea, and the Bay of Bengal. By establishing linkages between tuna physiology and environmental conditions, we provide a mechanistic basis to project the effects of anthropogenic climate change on tuna habitats. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  13. High-throughput fragment screening by affinity LC-MS.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  14. Decontaminating reagents for radioactive systems

    International Nuclear Information System (INIS)

    Seddon, W.A.

    1982-01-01

    A decontaminating reagent composition has been developed comprising EDTA, citric acid, oxalic acid, and formic acid. Formic acid inhibits the decomposition of both EDTA and citric acid, and yields oxalic acid as a result of its own radiolysis. The invention includes the improvement of initially incorporating formic acid in the mixture and maintaining the presence of formic acid by at least one further addition

  15. Affinity Programs and the Real Estate Brokerage Industry

    OpenAIRE

    G Stacy Sirmans; David A. Macpherson

    2001-01-01

    This study surveys active real estate brokers obtaining information on involvement in affinity programs and referral/relocation networks. Some results regarding affinity involvement are: (a) 13% of respondents reported affinity affilliations, 75% reported no affiliations, and 12% indicated plans to become involved within the next year; (b) about half having affinity affiliations were involved with 2-4 groups; (c) affinity relationships were most often with membership organizations, corporatio...

  16. Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations

    Directory of Open Access Journals (Sweden)

    Franklin C. Wong

    2013-01-01

    Full Text Available Photoaffinity labeling, a useful in vivo biochemical tool, is limited when applied in vivo because of the poor tissue penetration by ultraviolet (UV photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp at low doses (<50 cGy or Rad, specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp, RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105 and to the dopamine (D2 membrane receptors (by azidoclebopride, respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R, and peripheral benzodiazepine receptor (PBDZR. It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites.

  17. Inactivation of viable Ascaris eggs by reagents during enumeration.

    Science.gov (United States)

    Nelson, K L; Darby, J L

    2001-12-01

    Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.

  18. Remotely controlled reagent feed system for mixed waste treatment Tank Farm

    International Nuclear Information System (INIS)

    Dennison, D.K.; Bowers, J.S.; Reed, R.K.

    1995-02-01

    LLNL has developed and installed a large-scale. remotely controlled, reagent feed system for use at its existing aqueous low-level radioactive and mixed waste treatment facility (Tank Farm). LLNL's Tank Farm is used to treat aqueous low-level and mixed wastes prior to vacuum filtration and to remove the hazardous and radioactive components before it is discharged to the City of Livermore Water Reclamation Plant (LWRP) via the sanitary sewer in accordance with established limits. This reagent feed system was installed to improve operational safety and process efficiency by eliminating the need for manual handling of various reagents used in the aqueous waste treatment processes. This was done by installing a delivery system that is controlled either remotely or locally via a programmable logic controller (PLC). The system consists of a pumping station, four sets of piping to each of six 6,800-L (1,800-gal) treatment tanks, air-actuated discharge valves at each tank, a pH/temperature probe at each tank, and the PLC-based control and monitoring system. During operation, the reagents are slowly added to the tanks in a preprogrammed and controlled manner while the pH, temperature, and liquid level are continuously monitored by the PLC. This paper presents the purpose of this reagent feed system, provides background related to LLNL's low-level/mixed waste treatment processes, describes the major system components, outlines system operation, and discusses current status and plans

  19. In situ generation of the Ohira-Bestmann Reagent from stable sulfonyl azide

    DEFF Research Database (Denmark)

    Jepsen, Tue Heesgaard; Kristensen, Jesper Langgaard

    2014-01-01

    We report an improved method for in situ generation of the Ohira-Bestmann reagent. Using the recently reported bench stable imidazole-1-sulfonyl azide as diazotransfer reagent, this new method represents a safe and scalable approach for the transformation of aldehydes into terminal alkynes...

  20. Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

    Science.gov (United States)

    Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

    2015-03-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

    Science.gov (United States)

    Szewczyk, A; Wójcik, G; Lobanov, N A; Nalecz, M J

    1999-08-19

    The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor. Copyright 1999 Academic Press.

  3. 21 CFR 866.3250 - Erysipelothrix rhusiopathiae serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3250... Erysipelothrix rhusiopathiae from cultured isolates derived from clinical specimens. The identification aids in...

  4. 21 CFR 866.3270 - Flavobacterium spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3270.... from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of...

  5. 21 CFR 866.3320 - Histoplasma capsulatum serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3320... capsulatum from clinical specimens or cultured isolates derived from clinical specimens. The identification...

  6. Using Affinity Diagrams to Evaluate Interactive Prototypes

    DEFF Research Database (Denmark)

    Lucero, Andrés

    2015-01-01

    our particular use of affinity diagramming in prototype evaluations. We reflect on a decade’s experience using affinity diagramming across a number of projects, both in industry and academia. Our affinity diagramming process in interaction design has been tailored and consists of four stages: creating...

  7. Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease

    DEFF Research Database (Denmark)

    Jørgensen, C S; Hansen, K B; Jacobsen, Søren

    2005-01-01

    agent. Using these conditions we found a relatively high level of non-specific binding in many sera. Antibodies to proteins that are used as blocking reagents in ELISA (bovine serum albumin (BSA), ovalbumin, skimmed milk powder) are frequently present in sera, and these may cause false-positive results...

  8. Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

    Science.gov (United States)

    Horejsí, V; Tichá, M; Kocourek, J

    1977-09-29

    Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

  9. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  10. Single-step affinity purification for fungal proteomics.

    Science.gov (United States)

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  11. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3165... clinical specimens or from cultured isolates derived from clinical specimens. The identification aids in...

  12. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3140.... from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria...

  13. 21 CFR 866.3110 - Campylobacter fetus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3110 Campylobacter... clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the...

  14. 21 CFR 866.3340 - Klebsiella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3340 Klebsiella... from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria...

  15. 21 CFR 866.3930 - Vibrio cholerae serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3930 Vibrio... from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of...

  16. 21 CFR 866.3010 - Acinetobacter calcoaceticus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3010... this bacterium from cultured isolates derived from clinical specimens. The identification aids in the...

  17. A Snippet of Grignard Reagent's History

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 18; Issue 8. A Snippet of Grignard Reagent's History. Sujan Singh Dua. Classroom Volume 18 Issue 8 August 2013 pp 777-780. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/018/08/0777-0780. Keywords.

  18. [Affine transformation-based automatic registration for peripheral digital subtraction angiography (DSA)].

    Science.gov (United States)

    Kong, Gang; Dai, Dao-Qing; Zou, Lu-Min

    2008-07-01

    In order to remove the artifacts of peripheral digital subtraction angiography (DSA), an affine transformation-based automatic image registration algorithm is introduced here. The whole process is described as follows: First, rectangle feature templates are constructed with their centers of the extracted Harris corners in the mask, and motion vectors of the central feature points are estimated using template matching technology with the similarity measure of maximum histogram energy. And then the optimal parameters of the affine transformation are calculated with the matrix singular value decomposition (SVD) method. Finally, bilinear intensity interpolation is taken to the mask according to the specific affine transformation. More than 30 peripheral DSA registrations are performed with the presented algorithm, and as the result, moving artifacts of the images are removed with sub-pixel precision, and the time consumption is less enough to satisfy the clinical requirements. Experimental results show the efficiency and robustness of the algorithm.

  19. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

    Directory of Open Access Journals (Sweden)

    Fabian König

    Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

  20. Connections between quantized affine algebras and superalgebras

    International Nuclear Information System (INIS)

    Zhang, R.B.

    1992-08-01

    Every affine superalgebra with a symmetrizable Cartan matrix is closely related to an ordinary affine algebra with the same Cartan matrix. It is shown that the quantum supergroup associated with the former is essentially isomorphic to the quantum group associated with the latter in an appropriate class of representations. At the classical level, each integrable irreducible highest weight representation of the affine superalgebra has a corresponding irreducible representation of the affine algebra, which has the same weight space decomposition. (author). 5 refs, 3 tabs

  1. 21 CFR 866.3850 - Trichinella spiralis serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3850... devices that consist of antigens and antisera used in serological tests to identify antibodies to...

  2. 21 CFR 866.3680 - Sporothrix schenckii serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3680... devices that consist of antigens and antisera used in serological tests to identify antibodies to...

  3. 21 CFR 866.3400 - Parainfluenza virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3400 Parainfluenza... that consist of antigens and antisera used in serological tests to identify antibodies to parainfluenza...

  4. 21 CFR 866.3040 - Aspergillus spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3040 Aspergillus... consist of antigens and antisera used in various serological tests to identify antibodies to Aspergillus...

  5. 21 CFR 866.3125 - Citrobacter spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3125 Citrobacter... isolates derived from clinical specimens. The identification aids in the diagnosis of disease caused by...

  6. 21 CFR 866.3740 - Streptococcus spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3740 Streptococcus... derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria...

  7. 21 CFR 866.3065 - Bordetella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3065 Bordetella... serological tests to identify Bordetella spp. from cultured isolates or directly from clinical specimens. The...

  8. Mobile Technology Affinity in Renal Transplant Recipients.

    Science.gov (United States)

    Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

    Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 21 CFR 866.3145 - Coxsackievirus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145... fluorescent dye that are used to identify coxsackievirus from clinical specimens or from tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of coxsackievirus...

  10. USE OF FENTON'S REAGENT AS A DISINFECTANT

    Science.gov (United States)

    Combined sewage samples obtained from a wastewater treatment facility were disinfected by the Fenton's Reagent of several different compositions. The pre-settled samples contained both suspended solids (SS) and dissolved organic carbon (DOC) at concentrations of 28 and 290 mg/L,...

  11. Selective Flotation of Calcite from Fluorite: A Novel Reagent Schedule

    Directory of Open Access Journals (Sweden)

    Zhiyong Gao

    2016-10-01

    Full Text Available Fluorite is an important strategic mineral. In general, fluorite ores will contain a certain amount of calcite gangue mineral. Thus, they need to be separated from each other. For an economic separation, a reverse flotation process is used to float calcite gangue from fluorite. However, little information on the separation is available. In this study, a novel reagent schedule using citric acid (CA as the depressant, sodium fluoride (NaF as the regulator and sulfoleic acid (SOA as the collector, was developed to separate calcite from fluorite. The results demonstrated a high selectivity for the flotation of calcite from fluorite using this new reagent schedule. The best selective separation for a single mineral and mixed binary minerals was obtained when 200 mg/L of NaF, 50 mg/L of CA, and 6 mg/L of SOA were used at pH 9. In addition, a batch flotation experiment was carried out using a run-of-mine feed material. Selective separation was achieved with 85.18% calcite removal while only 11.2% of fluorite was lost. An attempt was made to understand the effect of the new reagent schedule on the flotation of calcite. The results from both microflotation and bench scale flotation demonstrated a great potential for industrial application using this novel reagent schedule to upgrade fluorite ore.

  12. Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line.

    Science.gov (United States)

    Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Yu, Qiming Jimmy

    2016-04-15

    Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. POLYNUCLEAR AROMATIC HYDROCARBON (PAH) RELEASE FROM SOIL DURING TREATMENT WITH FENTON'S REAGENT

    Science.gov (United States)

    Fenton's Reagent was used to treat soil from a wood-treating site in southeastern Ohio which had been contaminated with creosote. Slurries, consisting of 10 µg of contaminated soil and 30 mL water were treated with 40 mL of Fenton's Reagent (1:1 of 30% H2O2 ...

  14. 21 CFR 866.3870 - Trypanosoma spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3870 Trypanosoma... consist of antigens and antisera used in serological tests to identify antibodies to Trypanosoma spp. in...

  15. 21 CFR 866.3630 - Serratia spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3630 Serratia spp... antigens and antisera used in serological tests to identify Serratia spp. from cultured isolates. The...

  16. 21 CFR 866.3660 - Shigella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3660 Shigella spp...), used in serological tests to identify Shigella spp. from cultured isolates. The identification aids in...

  17. 21 CFR 866.3330 - Influenza virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3330 Influenza... consist of antigens and antisera used in serological tests to identify antibodies to influenza in serum...

  18. 21 CFR 866.3035 - Arizona spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3035 Arizona spp... antisera and antigens used to identify Arizona spp. in cultured isolates derived from clinical specimens...

  19. 21 CFR 866.3380 - Mumps virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3380 Mumps virus... serological tests to identify mumps viruses from tissue culture isolates derived from clinical specimens. The...

  20. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  1. Final Report for research grant entitled 'Development of Reagents for Application of At-211 and Bi-213 to Targeted Radiotherapy of Cancer'

    International Nuclear Information System (INIS)

    Wilbur, D. Scott

    2011-01-01

    This grant was a one-year extension of another grant with the same title (DE-FG03-98ER62572). The objective of the studies was to continue in vivo evaluation of reagents to determine which changes in structure were most favorable for in vivo use. The focus of our studies was development and optimization of reagents for pretargeting alpha-emitting radionuclides At-211 or Bi-213 to cancer cells. Testing of the reagents was conducted in vitro and in animal model systems. During the funding period, all three specific aims set out in the proposed studies were worked on, and some additional studies directed at development of a method for direct labeling of proteins with At-211 were investigated. We evaluated reagents in two different approaches in 'two step' pretargeting protocols. These approaches are: (1) delivery of the radionuclide on recombinant streptavidin to bind with pretargeted biotinylated monoclonal antibody (mAb), and alternatively, (2) delivery of the radionuclide on a biotin derivative to bind with pretargeted antibody-streptavidin conjugates. The two approaches were investigated as it was unclear which will be superior for the short half-lived alpha-emitting radionuclides.

  2. Final Report for research grant entitled "Development of Reagents for Application of At-211 and Bi-213 to Targeted Radiotherapy of Cancer"

    Energy Technology Data Exchange (ETDEWEB)

    Wilbur, D. Scott

    2011-12-23

    This grant was a one-year extension of another grant with the same title (DE-FG03-98ER62572). The objective of the studies was to continue in vivo evaluation of reagents to determine which changes in structure were most favorable for in vivo use. The focus of our studies was development and optimization of reagents for pretargeting alpha-emitting radionuclides At-211 or Bi-213 to cancer cells. Testing of the reagents was conducted in vitro and in animal model systems. During the funding period, all three specific aims set out in the proposed studies were worked on, and some additional studies directed at development of a method for direct labeling of proteins with At-211 were investigated. We evaluated reagents in two different approaches in 'two step' pretargeting protocols. These approaches are: (1) delivery of the radionuclide on recombinant streptavidin to bind with pretargeted biotinylated monoclonal antibody (mAb), and alternatively, (2) delivery of the radionuclide on a biotin derivative to bind with pretargeted antibody-streptavidin conjugates. The two approaches were investigated as it was unclear which will be superior for the short half-lived alpha-emitting radionuclides.

  3. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  4. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    Science.gov (United States)

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  5. Affine Fullerene C60 in a GS-Quasigroup

    Directory of Open Access Journals (Sweden)

    Vladimir Volenec

    2014-01-01

    Full Text Available It will be shown that the affine fullerene C60, which is defined as an affine image of buckminsterfullerene C60, can be obtained only by means of the golden section. The concept of the affine fullerene C60 will be constructed in a general GS-quasigroup using the statements about the relationships between affine regular pentagons and affine regular hexagons. The geometrical interpretation of all discovered relations in a general GS-quasigroup will be given in the GS-quasigroup C(1/2(1+5.

  6. From affine Hecke algebras to boundary symmetries

    International Nuclear Information System (INIS)

    Doikou, Anastasia

    2005-01-01

    Motivated by earlier works we employ appropriate realizations of the affine Hecke algebra and we recover previously known non-diagonal solutions of the reflection equation for the U q (gl n -bar ) case. The corresponding N site spin chain with open boundary conditions is then constructed and boundary non-local charges associated to the non-diagonal solutions of the reflection equation are derived, as coproduct realizations of the reflection algebra. With the help of linear intertwining relations involving the aforementioned solutions of the reflection equation, the symmetry of the open spin chain with the corresponding boundary conditions is exhibited, being essentially a remnant of the U q (gl n -bar ) algebra. More specifically, we show that representations of certain boundary non-local charges commute with the generators of the affine Hecke algebra and with the local Hamiltonian of the open spin chain for a particular choice of boundary conditions. Furthermore, we are able to show that the transfer matrix of the open spin chain commutes with a certain number of boundary non-local charges, depending on the choice of boundary conditions

  7. Copper-Catalyzed Oxy-Alkynylation of Diazo Compounds with Hypervalent Iodine Reagents.

    Science.gov (United States)

    Hari, Durga Prasad; Waser, Jerome

    2016-02-24

    Alkynes have found widespread applications in synthetic chemistry, biology, and materials sciences. In recent years, methods based on electrophilic alkynylation with hypervalent iodine reagents have made acetylene synthesis more flexible and efficient, but they lead to the formation of one equivalent of an iodoarene as side-product. Herein, a more efficient strategy involving a copper-catalyzed oxy-alkynylation of diazo compounds with ethynylbenziodoxol(on)e (EBX) reagents is described, which proceeds with generation of nitrogen gas as the only waste. This reaction is remarkable for its broad scope in both EBX reagents and diazo compounds. In addition, vinyl diazo compounds gave enynes selectively as single geometric isomers. The functional groups introduced during the transformation served as easy handles to access useful building blocks for synthetic and medicinal chemistry.

  8. Affinity Spaces and 21st Century Learning

    Science.gov (United States)

    Gee, James Paul

    2017-01-01

    This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

  9. A new achiral reagent for the incorporation of multiple amino groups into oligonucleotides

    DEFF Research Database (Denmark)

    Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael

    1995-01-01

    The synthesis of a new functionalized achiral linker reagent (10) for the incorporation of multiple primary amino groups into oligonucleotides is described. The linker reagent is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and the linker can be incorporated...

  10. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  11. On the structure of self-affine convex bodies

    Energy Technology Data Exchange (ETDEWEB)

    Voynov, A S [M. V. Lomonosov Moscow State University, Faculty of Mechanics and Mathematics, Moscow (Russian Federation)

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  12. Organophosphorus reagents in actinide separations: Unique tools for production, cleanup and disposal

    International Nuclear Information System (INIS)

    Nash, K. L.

    2000-01-01

    Interactions of actinide ions with phosphate and organophosphorus reagents have figured prominently in nuclear science and technology, particularly in the hydrometallurgical processing of irradiated nuclear fuel. Actinide interactions with phosphorus-containing species impact all aspects from the stability of naturally occurring actinides in phosphate mineral phases through the application of the bismuth phosphate and PUREX processes for large-scale production of transuranic elements to the development of analytical separation and environment restoration processes based on new organophosphorus reagents. In this report, an overview of the unique role of organophosphorus compounds in actinide production, disposal, and environment restoration is presented. The broad utility of these reagents and their unique chemical properties is emphasized

  13. Sequence2Vec: A novel embedding approach for modeling transcription factor binding affinity landscape

    KAUST Repository

    Dai, Hanjun

    2017-07-26

    Motivation: An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Results: Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model (HMM) which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these HMMs into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA data sets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods.

  14. The adsorption of chelating reagents on oxide minerals

    International Nuclear Information System (INIS)

    Bryson, M.A.W.

    1984-06-01

    This work constitutes a fundamental study of the interaction between chelating reagents and oxide minerals. The adsorption mechanisms have been elucidated for most of the systems generated by the oxides of copper(II) or iron(III) and chelating reagents octyl hydroxamate, N-phenylbenzohydroxamate, salicylaldoxime, 5-nitro-salicylaldoxime or 8-hydroxyquinoline. In order to better understand the adsorption process associated with copper(II) oxide, the oxide was recrystallized to produce a coarser material with a more uniform surface. This allowed the oxide surface to be viewed under the scanning electron microscope. A detailed investigation of the effect of the system variables; pH, conditioning period, concentration, temperature, surface area and dispersing reagent on the rate of precipitation of the copper chelate species of general form, Cu(chel) 2 , was made. In addition the chemical nature of the adsorbed species and the structural form of the precipitates were determined with the aid of infra-red spectroscopy and the scanning electron microscope. On the basis of these results a model has been formulated for the adsorption processes. The precipitation process was examined in more detail by the study of the adsorption of chelate on copper metal. Contact angle measurements of air bubbles on copper metal conditioned with chelate were related to the adsorption results in an attempt to isolate the optimum conditions for flotation of oxide minerals

  15. Application of flotational reagents obtained from coke-industry byproducts

    Energy Technology Data Exchange (ETDEWEB)

    N.I. Nikitin; I.N. Nikitin; N.I. Toporkova [Khar' kov Polytechnic Institute (Ukraine)

    2007-06-15

    Today, the operational efficiency of coal-preparation shops at coke plants largely depends on the flotation process, since flotation is the basic method of regenerating the slurry water in the water-slurry systems and the basic enrichment process for small-grain coal slurries. At The Coal-Chemistry Institute, attempts have been made to address the growing demand for readily available and relatively inexpensive flotational reagents. In particular, a list of promising coke-industry byproducts for use as flotational reagents has been compiled, and the possibility of reducing their toxicity has been established. In addition, various industrial byproducts and wastes have been investigated in terms of flotational activity.

  16. Green Imidazolium Ionics-From Truly Sustainable Reagents to Highly Functional Ionic Liquids.

    Science.gov (United States)

    Tröger-Müller, Steffen; Brandt, Jessica; Antonietti, Markus; Liedel, Clemens

    2017-09-04

    We report the synthesis of task-specific imidazolium ionic compounds and ionic liquids with key functionalities of organic molecules from electro-, polymer-, and coordination chemistry. Such products are highly functional and potentially suitable for technology applications even though they are formed without elaborate reactions and from cheap and potentially green reagents. We further demonstrate the versatility of the used synthetic approach by introducing different functional and green counterions to the formed ionic liquids directly during the synthesis or after metathesis reactions. The influence of different cation structures and different anions on the thermal and electrochemical properties of the resulting ionic liquids is discussed. Our goal is to make progress towards economically competitive and sustainable task-specific ionic liquids. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: Structure–Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations

    Science.gov (United States)

    2013-01-01

    Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure–activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6–10-fold over commercial reagents under their respective optimal conditions. Emerging structure–activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity. PMID:23682947

  18. Quality control of radioimmunoassay reagents for T4

    International Nuclear Information System (INIS)

    Wayan Radiatning, S.

    1987-01-01

    Quality control of radioimmunoassay reagents for T4. A program of quality control testing has been carried out for 125 I-T4, T4 standards and T4 antisera. 125 I-labelled T4 has been tested for its specific activity, radiochemical purity using a Sephadex G-25 column, immunological activity, based on the immunological reaction between labelled antigen and excess T4 antibody, and non-specific binding. The useful shelf-life of the labelled compound was determined by monitoring the decrease in radiochemical purity and immunological activity, and the increase in non-specific binding. T4 standards were calibrated by means of T4 RIA kit manufactured by DPC (Diagnostic Products Corporation). A test on parallelism was also performed using hyperthyroid sera. T4 antisera were evaluated with respect to titre, avidity and specifity. The test results on 125 I-T4 show a specific activity varying between 1830-2020 uCi/ug, a radiochemical purity above 90%, an immunological more than 80% and a non-specific binding of less than 5%. The standard curve for T4 was found to coincide well with the standard curve of the DPC kit and parallel with the curve for hyperthyroid sera. The titre of T4 antisera obtained was 1:300, the avidity was about 4.8 x 10 7 and the cross-reaction for T3 was 1.6%. It can be concluded from the experimental results, that the 125 I-T4, T4 standards and T4 antisera prepared meet the requirements for the manufacture of T4 kits. (author). 5 refs.; 14 figs

  19. Polynomials associated with equilibria of affine Toda-Sutherland systems

    International Nuclear Information System (INIS)

    Odake, S; Sasaki, R

    2004-01-01

    An affine Toda-Sutherland system is a quasi-exactly solvable multi-particle dynamics based on an affine simple root system. It is a 'cross' between two well-known integrable multi-particle dynamics, an affine Toda molecule (exponential potential, periodic nearest-neighbour interaction) and a Sutherland system (inverse sine-square interaction). Polynomials describing the equilibrium positions of affine Toda-Sutherland systems are determined for all affine simple root systems

  20. Reagents Activity in a Copper Droplets / Post-Processing Slag Suspension

    OpenAIRE

    Wołczyński W.; Karwan-Baczewska J.; Najman K.; Bydałek A.W.

    2016-01-01

    The suspension of the copper droplets in the post-processing slag taken directly from the KGHM-Polska Miedź S.A. Factory (from the direct-to-blister technology as performed in the flash furnace) was subjected to the special treatment with the use of the one of the typical industrial reagent and with the complex reagent newly patented by the authors. This treatment was performed in the BOLMET S.A. Company in the semi-industrial conditions. The result of the CaCO3, and Na2CO3 chemicals influenc...

  1. Risk-based Strategy to Determine Testing Requirement for the Removal of Residual Process Reagents as Process-related Impurities in Bioprocesses.

    Science.gov (United States)

    Qiu, Jinshu; Li, Kim; Miller, Karen; Raghani, Anil

    2015-01-01

    The purpose of this article is to recommend a risk-based strategy for determining clearance testing requirements of the process reagents used in manufacturing biopharmaceutical products. The strategy takes account of four risk factors. Firstly, the process reagents are classified into two categories according to their safety profile and history of use: generally recognized as safe (GRAS) and potential safety concern (PSC) reagents. The clearance testing of GRAS reagents can be eliminated because of their safe use historically and process capability to remove these reagents. An estimated safety margin (Se) value, a ratio of the exposure limit to the estimated maximum reagent amount, is then used to evaluate the necessity for testing the PSC reagents at an early development stage. The Se value is calculated from two risk factors, the starting PSC reagent amount per maximum product dose (Me), and the exposure limit (Le). A worst-case scenario is assumed to estimate the Me value, that is common. The PSC reagent of interest is co-purified with the product and no clearance occurs throughout the entire purification process. No clearance testing is required for this PSC reagent if its Se value is ≥1; otherwise clearance testing is needed. Finally, the point of the process reagent introduction to the process is also considered in determining the necessity of the clearance testing for process reagents. How to use the measured safety margin as a criterion for determining PSC reagent testing at process characterization, process validation, and commercial production stages are also described. A large number of process reagents are used in the biopharmaceutical manufacturing to control the process performance. Clearance testing for all of the process reagents will be an enormous analytical task. In this article, a risk-based strategy is described to eliminate unnecessary clearance testing for majority of the process reagents using four risk factors. The risk factors included

  2. Different endothelin receptor affinities in dog tissues

    International Nuclear Information System (INIS)

    Loeffler, B.M.L.; Loehrer, W.

    1991-01-01

    Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

  3. Duals of Affine Grassmann Codes and Their Relatives

    DEFF Research Database (Denmark)

    Beelen, P.; Ghorpade, S. R.; Hoholdt, T.

    2012-01-01

    Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine...... Grassmann code of any level and compute its minimum distance. Further, we ameliorate the results by Beelen concerning the automorphism group of affine Grassmann codes. Finally, we prove that affine Grassmann codes and their duals have the property that they are linear codes generated by their minimum......-weight codewords. This provides a clean analogue of a corresponding result for generalized Reed-Muller codes....

  4. A Novel Vertex Affinity for Community Detection

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  5. Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization

    Science.gov (United States)

    Leelawattanachai, Jeerapond; Kwon, Keon-Woo; Michael, Praveesuda; Ting, Richard; Kim, Ju-Young; Jin, Moonsoo M.

    2015-01-01

    The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization. PMID:25901755

  6. Tetrameric DABCO™-Bromine: an Efficient and Versatile Reagent ...

    African Journals Online (AJOL)

    NJD

    Reagent for Bromination of Various Organic Compounds. Majid M. Heravi,a* ... aDepartment of Chemistry, School of Sciences, Azzahra University, Vanak, Tehran, Iran. bChemistry ... Synthesis of "-bromo ketones and nitriles has also been ...

  7. On affine non-negative matrix factorization

    DEFF Research Database (Denmark)

    Laurberg, Hans; Hansen, Lars Kai

    2007-01-01

    We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate id...

  8. Effect of some colloid surfactants on spectrophotometric characteristics of metal chelates with chromophore organic reagents

    International Nuclear Information System (INIS)

    Chernova, R.K.

    1977-01-01

    Theoretical regularities and prospects of using surface active substances (SAS) in spectrophotometric determination of metal ions (including ions of rare-earth elements, transition metals, Be(3)) with chromophore chelating reagents were investigated. The chromophore reagents investigated were pyrocatechol violet, phenolcarboxylic acids of the triarylmethane series, fluorones, phthalexones and azo-compounds. As SAS certain long-chain quaternary ammonium and pyridinium salts (LQAS) were employed. From the results reported it follows that the introduction of LQAS in the system of Mesup(n+)-chromophore reagent is a rather effective method of enhancing the contrast rendition and, in some cases, the sensitivity and selectivity of the reagents. Explanations are suggested as to the factors which cause the changes observed in the contrast of the reactions in the presence of SAS; the underlying phenomena are the ligand-ligand interactions between the organic reagents and SAS and solubilization processes of the reaction products by the micelles of SAS

  9. Improving image segmentation by learning region affinities

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  10. Selection of imprinted nanoparticles by affinity chromatography.

    Science.gov (United States)

    Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

    2009-04-15

    Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

  11. Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3

    International Nuclear Information System (INIS)

    Hong Chenglin; Yuan Ruo; Chai Yaqin; Zhuo Ying

    2009-01-01

    The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 U mL -1 with a coefficient 0.9986 and a detection limit of 0.64 U mL -1 at S/N = 3

  12. Ferrocenyl-doped silica nanoparticles as an immobilized affinity support for electrochemical immunoassay of cancer antigen 15-3.

    Science.gov (United States)

    Hong, Chenglin; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying

    2009-02-09

    The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0-240 UmL(-1) with a coefficient 0.9986 and a detection limit of 0.64 UmL(-1) at S/N=3.

  13. 21 CFR 866.3240 - Equine encephalomyelitis virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... these viruses. Equine encephalomyelitis viruses are transmitted to humans by the bite of insects, such...

  14. Contractions of affine spherical varieties

    International Nuclear Information System (INIS)

    Arzhantsev, I V

    1999-01-01

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL 2 -varieties are considered

  15. 21 CFR 866.3480 - Respiratory syncytial virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3480... respiratory syncytial viruses from clinical specimens or from tissue culture isolates derived from clinical...

  16. Kalman filtering applied to a reagent feed system

    International Nuclear Information System (INIS)

    Griffin, C.D.; Croson, D.V.; Feeley, J.J.

    1988-01-01

    Using a Kalman filter solves a troublesome measurement noise problem and, at the same time, improves nuclear safety by detecting leaks to the process' feed tanks. To demonstrate how this technology of optimal estimation can be exploited, this article presents a systematic plan and example of how a Kalman filter was proven in industrial use on a reagent analyzer. A process to recycle uranium from spent fuel elements uses a reagent stream containing boron to dissolve the fuel. The boron is the neutron poison that prevents a nuclear chain reaction during the uranium dissolution. The purpose of the Kalman filter for this system is to reduce the uncertainty in the boron concentration measurement. The filter also provides incipient fault detection by estimating the unmeasured state of any unpoisoned solution, which would dilute the boron solution, entering the feed vessel

  17. Estimation of 239Pu in urine, influence of Sulkowich reagent

    International Nuclear Information System (INIS)

    Kalaiselvan, S.; Prasad, M.V.R.; Jeevanram, R.K.

    1988-01-01

    Plutonium is known to be co-precipitated with Sulkowich reagent as calcium ammonium oxalate. In adopting this technique for bio-assay of plutonium, its accuracy depends on the self-absorption of the resulting precipitate in each urine sample. Pu recovery experiments were carried out with varying concentration of Ca and Mg, using different volumes of Sulkowich reagent. When the sample volume is 500 ml, Pu in urine can be estimated with an accuracy and precision of 74.38%+-7.4%, with a detection limit of 0.06 Bq (1.6 pCi) per dm 3 . (author) 3 refs.; 2 figs.; 2 tabs

  18. The use of permanganate as a sequencing reagent for identification of 5-methylcytosine residues in DNA.

    OpenAIRE

    Fritzsche, E; Hayatsu, H; Igloi, G L; Iida, S; Kössel, H

    1987-01-01

    The use of permanganate as a reagent for DNA sequencing by chemical degradation has been studied with respect to its specificity for 5-methylcytosine residues. At weakly acidic pH and room temperature, 0.2 mM potassium permanganate reacts preferentially with thymine, 5-methylcytosine, and to a lesser extent with purine residues, while cytosine remains essentially intact. Permanganate oxidation is, therefore, a suitable DNA sequencing reaction for positive discrimination between 5-methylcytosi...

  19. Facile synthesis of aliphatic isothiocyanates and thioureas on solid phase using peptide coupling reagents

    DEFF Research Database (Denmark)

    Boas, Ulrik; Andersen, Heidi Gertz; Christensen, Jørn B.

    2004-01-01

    Peptide coupling reagents can be used as versatile reagents for the formation of aliphatic isothiocyanates and thioureas on solid phase from the corresponding solid-phase anchored aliphatic primary amines. The formation of the thioureas is fast and highly chemoselective, and proceeds via formatio...

  20. 21 CFR 866.3940 - West Nile virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3940 West Nile... detection aids in the clinical laboratory diagnosis of viral meningitis/encephalitis caused by West Nile...

  1. Microwave-induced electrostatic etching: generation of highly reactive magnesium for application in Grignard reagent formation.

    Science.gov (United States)

    van de Kruijs, Bastiaan H P; Dressen, Mark H C L; Meuldijk, Jan; Vekemans, Jef A J M; Hulshof, Lumbertus A

    2010-04-07

    A detailed study regarding the influence of microwave irradiation on the formation of a series of Grignard reagents in terms of rates and selectivities has revealed that these heterogeneous reactions may display a beneficial microwave effect. The interaction between microwaves and magnesium turnings generates violent electrostatic discharges. These discharges on magnesium lead to melting of the magnesium surface, thus generating highly active magnesium particles. As compared to conventional operation the microwave-induced discharges on the magnesium surface lead to considerably shorter initiation times for the insertion of magnesium in selected substrates (i.e. halothiophenes, halopyridines, octyl halides, and halobenzenes). Thermographic imaging and surface characterization by scanning electron microscopy showed that neither selective heating nor a "specific" microwave effect was causing the reduction in initiation times. This novel and straightforward initiation method eliminates the use of toxic and environmentally adverse initiators. Thus, this initiation method limits the formation of by-products. We clearly demonstrated that microwave irradiation enables fast Grignard reagent formation. Therefore, microwave technology is promising for process intensification of Grignard based coupling reactions.

  2. Radioimmunoassay reagent and its use in a radioimmunoassay

    International Nuclear Information System (INIS)

    Polito, A.J.; Knight, W.S.

    1977-01-01

    A radioimmunoassay to detect or determine a steroid of the cortisone and aldosterone group has been developed. The invention particularly concerns a steroid derivative containing imidazole group which is radioactively labelled. The invented reagents are labelled with iodine 125. (VJ) [de

  3. Fenton's Reagent. Innovative Technology Summary Report

    International Nuclear Information System (INIS)

    None

    1999-01-01

    The Fenton's Reagent DNAPL treatment process is an in situ oxidation method to destroy DNAPLs in groundwater. Residual industrial solvents, primarily Dense Non-Aqueous Phase Liquids (DNAPLs), are currently the most significant barrier to successful completion of most large groundwater and soil cleanup efforts. DNAPL pools and residues slowly dissolve into surrounding groundwater to create large plumes of organic solvent contamination with concentration levels far above regulatory limits

  4. Reagents and fractions impact on sulphide ore heap bioleaching at Smolnik mine

    Science.gov (United States)

    Oros, L. M.; Zavada, J.

    2017-10-01

    Mine Smolnik is one of the oldest sulphide ore mines in Europe and it is also an important part of bioleaching development. This paper follows previous attempts to extract residual metals from nearby heaps via variations in bioleaching reagents with regard to recent findings and needs in the related industry. Furthermore, economic and process relations between reagents and chosen heap fractions were also investigated in this case study.

  5. Oxidative Degradation of Phenol containing Wastewater using Fenton Reagent, Permanganate and Ultraviolet Radiation

    International Nuclear Information System (INIS)

    Abd El-Rahman, N.M.; Talaat, H.A.; Sorour, M.H.

    1999-01-01

    Phenol containing wastewaters are generated by numerous industrial units including integrated steel mills, textile mills, plastic production, etc. The present work is targeted to explore the viable oxidation techniques for degradation of phenolic wastewater. Three modes of treatment have been adopted in this study, namely, sole oxidant mode using Fenton reagent or permanganate, UV-assisted oxidation and two consequent chemical oxidation steps. Results indicated the superiority of fenton reagent over KMnO 4 oxidation in the sole oxidant mode. On the other hand, UV-assisted KMnO 4 oxidation enables almost complete COD reduction. Dual chemical oxidation mode employing KMnO 4 oxidation followed by Fenton reagent is also an efficient oxidative degradation system

  6. Conversion of coal mine drainage ochre to water treatment reagent: Production, characterisation and application for P and Zn removal.

    Science.gov (United States)

    Sapsford, Devin; Santonastaso, Marco; Thorn, Peter; Kershaw, Steven

    2015-09-01

    Coal mine drainage ochre is a ferruginous precipitate that forms from mine water in impacted watercourses and during treatment. With thousands of tonnes per annum of such ochre arising from mine water treatment in the UK alone, management of these wastes is a substantive issue. This paper demonstrates that the ochre from both active and passive treatment of coal mine drainage can be transformed into an effective water treatment reagent by simple acid dissolution and that the reagent can be used for the removal of dissolved phosphorous from municipal wastewater and zinc from non-coal mine waters. Ochre is readily soluble in H2SO4 and HCl. Ochre is more soluble in HCl with solubilities of up to 100 g/L in 20% (w/w) HCl and 68 g/L in 10% (w/w) H2SO4. For four of the eight tested ochres solubility decreased in higher concentrations of H2SO4. Ochre compositional data demonstrate that the coal mine ochres tested are relatively free from problematic levels of elements seen by other authors from acid mine drainage-derived ochre. Comparison to British Standards for use of iron-based coagulants in drinking water treatment was used as an indicator of the acceptability of use of the ochre-derived reagents in terms of potentially problematic elements. The ochre-derived reagents were found to meet the 'Grade 3' specification, except for arsenic. Thus, for application in municipal wastewater and mine water treatment additional processing may not be required. There was little observed compositional difference between solutions prepared using H2SO4 or HCl. Ochre-derived reagents showed applicability for the removal of P and Zn with removals of up to 99% and 97% respectively measured for final pH 7-8, likely due to sorption/coprecipitation. Furthermore, the results demonstrate that applying a Fe dose in the form of liquid reagent leads to a better Fe:P and Fe:Zn removal ratio compared to ochre-based sorption media tested in the literature. Copyright © 2015 The Authors. Published by

  7. Repertoire Analysis of Antibody CDR-H3 Loops Suggests Affinity Maturation Does Not Typically Result in Rigidification

    Directory of Open Access Journals (Sweden)

    Jeliazko R. Jeliazkov

    2018-03-01

    Full Text Available Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody’s antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity.

  8. Chemical cleaning specification: few tube test model

    International Nuclear Information System (INIS)

    Hampton, L.V.; Simpson, J.L.

    1979-09-01

    The specification is for the waterside chemical cleaning of the 2 1/4 Cr - 1 Mo steel steam generator tubes. It describes the reagents and conditions for post-chemical cleaning passivation of the evaporator tubes

  9. Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

    Science.gov (United States)

    Borjigin, Jimo; Nathans, Jeremy

    1993-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

  10. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    International Nuclear Information System (INIS)

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

    2011-01-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  11. Mechanism of red mud combined with Fenton's reagent in sewage sludge conditioning.

    Science.gov (United States)

    Zhang, Hao; Yang, Jiakuan; Yu, Wenbo; Luo, Sen; Peng, Li; Shen, Xingxing; Shi, Yafei; Zhang, Shinan; Song, Jian; Ye, Nan; Li, Ye; Yang, Changzhu; Liang, Sha

    2014-08-01

    Red mud was evaluated as an alternative skeleton builder combined with Fenton's reagent in sewage sludge conditioning. The results show that red mud combined with Fenton's reagent showed good conditioning capability with the pH of the filtrate close to neutrality, indicating that red mud acted as a neutralizer as well as a skeleton builder when jointly used with Fenton's reagent. Through response surface methodology (RSM), the optimal dosages of Fe(2+), H2O2 and red mud were proposed as 31.9, 33.7 and 275.1 mg/g DS (dry solids), respectively. The mechanism of the composite conditioner could be illuminated as follows: (1) extracellular polymeric substances (EPS), including loosely bound EPS and tightly bound EPS, were degraded into dissolved organics, e.g., proteins and polysaccharides; (2) bound water was released and converted into free water due to the degradation of EPS; and (3) morphology of the conditioned sludge exhibited a porous structure in contrast with the compact structure of raw sludge, and the addition of red mud formed new mineral phases and a rigid lattice structure in sludge, allowing the outflow of free water. Thus, sludge dewatering performance was effectively improved. The economic assessment for a wastewater treatment plant of 370,000 equivalent inhabitants confirms that using red mud conditioning, combined with Fenton's reagent, leads to a saving of approximately 411,000 USD/y or 50.8 USD/t DS comparing with using lime and ordinary Portland cement combined with Fenton's reagent, and approximately 612,000 USD/y or 75.5 USD/t DS comparing with the traditional treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Alkylation of pyridines at their 4-positions with styrenes plus yttrium reagent or benzyl Grignard reagents.

    Science.gov (United States)

    Mizumori, Tomoya; Hata, Takeshi; Urabe, Hirokazu

    2015-01-02

    A new regioselective alkylation of pyridines at their 4-position was achieved with styrenes in the presence of yttrium trichloride, BuLi, and diisobutylaluminium hydride (DIBAL-H) in THF. Alternatively, similar products were more simply prepared from pyridines and benzyl Grignard reagents. These reactions are not only a useful preparation of 4-substituted pyridines but are also complementary to other relevant reactions usually giving 2-substituted pyridines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 21 CFR 866.3085 - Brucella spp. serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp... from clinical specimens or to identify antibodies to Brucella spp. in serum. Additionally, some of... to identify Brucella spp. directly from clinical specimens or cultured isolates derived from clinical...

  14. 21 CFR 866.3235 - Epstein-Barr virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3235 Epstein-Barr... consist of antigens and antisera used in serological tests to identify antibodies to Epstein-Barr virus in...

  15. Global affine differential geometry of hypersurfaces

    CERN Document Server

    Li, An-Min; Zhao, Guosong; Hu, Zejun

    2015-01-01

    This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

  16. High throughput screening method for identification of new lipofection reagents.

    Science.gov (United States)

    Regelin, A E; Fernholz, E; Krug, H F; Massing, U

    2001-08-01

    Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

  17. Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, N O; Tok, J B; Tarasow, T M

    2008-02-08

    Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. Methodology/Principal Findings. High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and interchip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  18. Massively parallel interrogation of aptamer sequence, structure and function.

    Directory of Open Access Journals (Sweden)

    Nicholas O Fischer

    Full Text Available BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

  19. Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans.

    Science.gov (United States)

    Díaz-Sánchez, Ángel Gabriel; Alvarez-Parrilla, Emilio; Martínez-Martínez, Alejandro; Aguirre-Reyes, Luis; Orozpe-Olvera, Jesica Aline; Ramos-Soto, Miguel Armando; Núñez-Gastélum, José Alberto; Alvarado-Tenorio, Bonifacio; de la Rosa, Laura Alejandra

    2016-11-26

    Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2'-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a "hinge" located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

  20. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    Science.gov (United States)

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Affinity monolith chromatography: A review of principles and recent analytical applications

    Science.gov (United States)

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  2. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  3. New unitary affine-Virasoro constructions

    International Nuclear Information System (INIS)

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M.; Yamron, J.P.

    1990-01-01

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g

  4. Stability of Loop-Mediated Isothermal Amplification (LAMP) Reagents and its Amplification Efficiency on Crude Trypanosome DNA Templates

    OpenAIRE

    Thekisoe, Oriel M. M; Bazie, Raoul S. B; Coronel-Servian, Andrea M; Sugimoto, Chihiro; Kawazu, Shin-ichiro; Inoue, Noboru

    2009-01-01

    This study evaluated the stability of LAMP reagents when stored at 25C and 37C, and also assessed its detection efficiency on different DNA template preparations. Accordingly, LAMP using reagents stored at 25C and 37C amplified DNA of in vitro cultured T. b. brucei (GUTat 3.1) from day 1 to day 15 of reagent storage. There were no significant differences (P>0.05) in detection sensitivity of LAMP among the reagents stored at 25C, 37C and –20C (recommended storage temperature). LAMP usin...

  5. Development of a nonlinear model for the prediction of response times of glucose affinity sensors using concanavalin A and dextran and the development of a differential osmotic glucose affinity sensor

    Science.gov (United States)

    Reis, Louis G.

    With the increasing prevalence of diabetes in the United States and worldwide, blood glucose monitoring must be accurate and reliable. Current enzymatic sensors have numerous disadvantages that make them unreliable and unfavorable among patients. Recent research in glucose affinity sensors correct some of the problems that enzymatic sensors experience. Dextran and concanavalin A are two of the more common components used in glucose affinity sensors. When these sensors were first explored, a model was derived to predict the response time of a glucose affinity sensor using concanavalin A and dextran. However, the model assumed the system was linear and fell short of calculating times representative of the response times determined through experimental tests with the sensors. In this work, a new model that uses the Stokes-Einstein Equation to demonstrate the nonlinear behavior of the glucose affinity assay was developed to predict the response times of similar glucose affinity sensors. In addition to the device tested by the original linear model, additional devices were identified and tested with the proposed model. The nonlinear model was designed to accommodate the many different variations between systems. The proposed model was able to accurately calculate response times for sensors using the concanavalin A-dextran affinity assay with respect to the experimentally reported times by the independent research groups. Parameter studies using the nonlinear model were able to identify possible setbacks that could compromise the response of thesystem. Specifically, the model showed that the improper use of asymmetrical membranes could increase the response time by as little as 20% or more as the device is miniaturized. The model also demonstrated that systems using the concanavalin Adextran assay would experience higher response times in the hypoglycemic range. This work attempted to replicate and improve an osmotic glucose affinity sensor. The system was designed to

  6. Interaction of Celestine Concentrate and Reagent Grade SrSO4 with Oxalate Solutions

    Directory of Open Access Journals (Sweden)

    Abdullah Obut

    2012-12-01

    Full Text Available The interaction of reagent grade strontium sulphate and celestine concentrate with aqueous solutions of oxalic acid, sodiumoxalate and ammonium oxalate for the production of strontium carbonate were investigated for different oxalate compound:SrSO4 moleratios and reaction times using x-ray diffraction analysis and dissolution tests. Under the same experimental conditions, it was foundthat aqueous oxalic acid and sodium oxalate solutions had no or little effect on reagent grade strontium sulphate or celestineconcentrate, but aqueous ammonium oxalate solution converted them into strontium oxalate hydrate. Strontium carbonate was obtainedat conversion ratios of 74.7% for the celestine concentrate and 84.6 % for the reagent grade strontium sulphate by the decompositionof the obtained strontium oxalate hydrate at 600 °C under air atmosphere.

  7. Learning a peptide-protein binding affinity predictor with kernel ridge regression

    Science.gov (United States)

    2013-01-01

    Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting

  8. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  9. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  10. Affine stochastic mortality

    NARCIS (Netherlands)

    Schrager, D.F.

    2006-01-01

    We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing

  11. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    Science.gov (United States)

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Rank Two Affine Manifolds in Genus 3

    OpenAIRE

    Aulicino, David; Nguyen, Duc-Manh

    2016-01-01

    We complete the classification of rank two affine manifolds in the moduli space of translation surfaces in genus three. Combined with a recent result of Mirzakhani and Wright, this completes the classification of higher rank affine manifolds in genus three.

  13. 21 CFR 864.1860 - Immunohistochemistry reagents and kits.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunohistochemistry reagents and kits. 864.1860 Section 864.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1860...

  14. Chromosome-specific staining to detect genetic rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Joe W.; Pinkel, Daniel; Tkachuk, Douglas; Westbrook, Carol

    2013-04-09

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyzes. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  15. The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

    Science.gov (United States)

    Giannone, Richard J.; Liu, Yie; Wang, Yisong

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  16. Statistical validation of reagent lot change in the clinical chemistry laboratory can confer insights on good clinical laboratory practice.

    Science.gov (United States)

    Cho, Min-Chul; Kim, So Young; Jeong, Tae-Dong; Lee, Woochang; Chun, Sail; Min, Won-Ki

    2014-11-01

    Verification of new lot reagent's suitability is necessary to ensure that results for patients' samples are consistent before and after reagent lot changes. A typical procedure is to measure results of some patients' samples along with quality control (QC) materials. In this study, the results of patients' samples and QC materials in reagent lot changes were analysed. In addition, the opinion regarding QC target range adjustment along with reagent lot changes was proposed. Patients' sample and QC material results of 360 reagent lot change events involving 61 analytes and eight instrument platforms were analysed. The between-lot differences for the patients' samples (ΔP) and the QC materials (ΔQC) were tested by Mann-Whitney U tests. The size of the between-lot differences in the QC data was calculated as multiples of standard deviation (SD). The ΔP and ΔQC values only differed significantly in 7.8% of the reagent lot change events. This frequency was not affected by the assay principle or the QC material source. One SD was proposed for the cutoff for maintaining pre-existing target range after reagent lot change. While non-commutable QC material results were infrequent in the present study, our data confirmed that QC materials have limited usefulness when assessing new reagent lots. Also a 1 SD standard for establishing a new QC target range after reagent lot change event was proposed. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  17. Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates.

    Science.gov (United States)

    Thekisoe, Oriel M M; Bazie, Raoul S B; Coronel-Servian, Andrea M; Sugimoto, Chihiro; Kawazu, Shin-Ichiro; Inoue, Noboru

    2009-04-01

    This study evaluated the stability of LAMP reagents when stored at 25 degrees C and 37 degrees C, and also assessed its detection efficiency on different DNA template preparations. Accordingly, LAMP using reagents stored at 25 degrees C and 37 degrees C amplified DNA of in vitro cultured T. b. brucei (GUTat 3.1) from day 1 to day 15 of reagent storage. There were no significant differences (P>0.05) in detection sensitivity of LAMP among the reagents stored at 25 degrees C, 37 degrees C and -20 degrees C (recommended storage temperature). LAMP using the reagents stored at above-mentioned temperatures amplified serially diluted DNAs (genomic DNA extracted by phenol-chloroform method, FTA card and hemolysed blood) of T. b. gambiense (IL2343) with high sensitivity. Reactions were conducted on the reagents stored from 1 day to 30 days. LAMP detection sensitivity was poor when fresh blood as DNA template was added directly into reactive solution. Results of this study demonstrated that LAMP has the potential to be used in field conditions for diagnosis of trypanosome infections without being affected by ambient temperatures of tropical and sub-tropical countries where trypanosomosis is endemic.

  18. Dynamics of Open Systems with Affine Maps

    International Nuclear Information System (INIS)

    Zhang Da-Jian; Liu Chong-Long; Tong Dian-Min

    2015-01-01

    Many quantum systems of interest are initially correlated with their environments and the reduced dynamics of open systems are an interesting while challenging topic. Affine maps, as an extension of completely positive maps, are a useful tool to describe the reduced dynamics of open systems with initial correlations. However, it is unclear what kind of initial state shares an affine map. In this study, we give a sufficient condition of initial states, in which the reduced dynamics can always be described by an affine map. Our result shows that if the initial states of the combined system constitute a convex set, and if the correspondence between the initial states of the open system and those of the combined system, defined by taking the partial trace, is a bijection, then the reduced dynamics of the open system can be described by an affine map. (paper)

  19. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  20. USING MICROSCALE THERMOPHORESIS TO EASILY MEASURE BINDING AFFINITY

    Directory of Open Access Journals (Sweden)

    Dennis Breitsprecher*

    2018-03-01

    Full Text Available While it’s very common for biologists and chemists to test whether or not two molecules interact with each other, it’s much more useful to gather information on the nature of that interaction. How strong is it? How long will it last? What does that mean for its biological function? One way to answer these questions is to study affinity. Binding affinity is defined as the strength of the binding interaction between a single biomolecule to its binding partner, or ligand, and it can be quantifiably measured, providing information on whether or not molecules are interacting, as well as assigning a value to the affinity. When measuring binding affinity, there are several parameters to look at, but the dissociation constant (Kd, which defines the likelihood that an interaction between two molecules will break, is a very common measurement. The smaller the dissociation constant, the more tightly bound the ligand is, and the higher the affinity is between the two molecules.

  1. Trifluoromethanesulfonic Anhydride as a Low-Cost and Versatile Trifluoromethylation Reagent.

    Science.gov (United States)

    Ouyang, Yao; Xu, Xiu-Hua; Qing, Feng-Ling

    2018-04-19

    A large number of reagents have been developed for the synthesis of trifluoromethylated compounds. However, an ongoing challenge in trifluoromethylation reaction is the use of less expensive and practical trifluoromethyl sources. We report herein the unprecedented direct trifluoromethylation of (hetero)arenes using trifluoromethanesulfonic anhydride as a radical trifluoromethylation reagent by merging photoredox catalysis and pyridine activation. Furthermore, introduction of both the CF 3 and OTf groups of the trifluoromethanesulfonic anhydride into internal alkynes to access tetrasubstituted trifluoromethylated alkenes was achieved. Since trifluoromethanesulfonic anhydride is a low-cost and abundant chemical, this method provides a cost-efficient and practical route to trifluoromethylated compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

    Science.gov (United States)

    Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

    2018-02-23

    Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. An affine model of the dynamics of astrophysical discs

    Science.gov (United States)

    Ogilvie, Gordon I.

    2018-06-01

    Thin astrophysical discs are very often modelled using the equations of 2D hydrodynamics. We derive an extension of this model that describes more accurately the behaviour of a thin disc in the absence of self-gravity, magnetic fields, and complex internal motions. The ideal fluid theory is derived directly from Hamilton's Principle for a 3D fluid after making a specific approximation to the deformation gradient tensor. We express the equations in Eulerian form after projection on to a reference plane. The disc is thought of as a set of fluid columns, each of which is capable of a time-dependent affine transformation, consisting of a translation together with a linear transformation in three dimensions. Therefore, in addition to the usual 2D hydrodynamics in the reference plane, the theory allows for a deformation of the mid-plane (as occurs in warped discs) and for the internal shearing motions that accompany such deformations. It also allows for the vertical expansions driven in non-circular discs by a variation of the vertical gravitational field around the horizontal streamlines, or by a divergence of the horizontal velocity. The equations of the affine model embody conservation laws for energy and potential vorticity, even for non-planar discs. We verify that they reproduce exactly the linear theories of 3D warped and eccentric discs in a secular approximation. However, the affine model does not rely on any secular or small-amplitude assumptions and should be useful in more general circumstances.

  4. Agonist-induced affinity alterations of a central nervous system. cap alpha. -bungarotoxin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lukas, R.J.; Bennett, E.L.

    1979-01-01

    The ability of cholinergic agonists to block the specific interaction of ..cap alpha..-bungarotoxin (..cap alpha..-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of ..cap alpha..-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of ..cap alpha..-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.

  5. Single-Step Affinity Purification for Fungal Proteomics ▿ †

    OpenAIRE

    Liu, Hui-Lin; Osmani, Aysha H.; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B.; De Souza, Colin P.; Osmani, Stephen A.

    2010-01-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  6. Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities.

    Science.gov (United States)

    Bhosle, Amrisha; Chandra, Nagasuma

    2016-03-01

    Antifolates are competitive inhibitors of dihydrofolate reductase (DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the 'supersite' and classified supersites of DHFRs from 56 species into 16 'site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates. © 2016 Federation of European Biochemical Societies.

  7. Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

    Science.gov (United States)

    Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

    2014-07-11

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. 21 CFR 864.9225 - Cell-freezing apparatus and reagents for in vitro diagnostic use.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell-freezing apparatus and reagents for in vitro diagnostic use. 864.9225 Section 864.9225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH... Establishments That Manufacture Blood and Blood Products § 864.9225 Cell-freezing apparatus and reagents for in...

  9. The Structure of Affine Buildings

    CERN Document Server

    Weiss, Richard M

    2009-01-01

    In The Structure of Affine Buildings, Richard Weiss gives a detailed presentation of the complete proof of the classification of Bruhat-Tits buildings first completed by Jacques Tits in 1986. The book includes numerous results about automorphisms, completions, and residues of these buildings. It also includes tables correlating the results in the locally finite case with the results of Tits's classification of absolutely simple algebraic groups defined over a local field. A companion to Weiss's The Structure of Spherical Buildings, The Structure of Affine Buildings is organized around the clas

  10. The dynamics of metric-affine gravity

    International Nuclear Information System (INIS)

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-01-01

    Highlights: → The role and the dynamics of the connection in metric-affine theories is explored. → The most general second order action does not lead to a dynamical connection. → Including higher order invariants excites new degrees of freedom in the connection. → f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy

  11. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

    International Nuclear Information System (INIS)

    Zhou, Ruokun; Huan, Tao; Li, Liang

    2015-01-01

    Highlights: • Two new reagents were developed for chemical isotope labeling mass spectrometry (MS). • They could be used to label amine-containing metabolites in a metabolomic sample. • The labeled metabolites could be detected with much improved sensitivity in MS. • One of the reagents could also help generate useful MS/MS spectra for structural analysis. • These reagents should be useful for quantitative metabolomics. - Abstract: Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented

  13. Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Ruokun; Huan, Tao; Li, Liang, E-mail: Liang.Li@ualberta.ca

    2015-06-30

    Highlights: • Two new reagents were developed for chemical isotope labeling mass spectrometry (MS). • They could be used to label amine-containing metabolites in a metabolomic sample. • The labeled metabolites could be detected with much improved sensitivity in MS. • One of the reagents could also help generate useful MS/MS spectra for structural analysis. • These reagents should be useful for quantitative metabolomics. - Abstract: Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented

  14. Spectral affinity in protein networks.

    Science.gov (United States)

    Voevodski, Konstantin; Teng, Shang-Hua; Xia, Yu

    2009-11-29

    Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to quickly find nodes closest to a queried vertex in any protein

  15. Competitive roles of reagent vibration and translation in the exothermic proton transfer reaction H+2+Ar→HAr++H

    International Nuclear Information System (INIS)

    Bilotta, R.M.; Farrar, J.M.

    1981-01-01

    We present a crossed beam study of the title reaction at fixed collision energies of 1.2 and 2.3 eV with reagent H + 2 average vibrational energies of 0.44 and 0.89 eV; we also present data at fixed total energies with variable proportions of reagent vibrational and translational energy. At fixed collision energy, reagent vibrational excitation is found to have negligible effect on the total cross section for proton transfer. At fixed total energy, a decrease in reagent vibrational excitation with a corresponding increase in reagent translation leads to partial disposal of the incremental translation in product translation: At a total energy of 3.5 eV, 50% of this incremental reagent translation appears as product translation. At a total energy of 4.6 eV, 78% of the incremental translation appears in product translation. The experimental data are discussed in terms of induced attractive and repulsive energy release on an attractive potential surface. The role of noncollinear geometries and compressed reactant configurations is judged to be of substantial importance in assessing product rotational excitation and dissociation

  16. Multiprocessor Real-Time Scheduling with Hierarchical Processor Affinities

    OpenAIRE

    Bonifaci , Vincenzo; Brandenburg , Björn; D'Angelo , Gianlorenzo; Marchetti-Spaccamela , Alberto

    2016-01-01

    International audience; Many multiprocessor real-time operating systems offer the possibility to restrict the migrations of any task to a specified subset of processors by setting affinity masks. A notion of " strong arbitrary processor affinity scheduling " (strong APA scheduling) has been proposed; this notion avoids schedulability losses due to overly simple implementations of processor affinities. Due to potential overheads, strong APA has not been implemented so far in a real-time operat...

  17. Method of Generating Hydrocarbon Reagents from Diesel, Natural Gas and Other Logistical Fuels

    Science.gov (United States)

    Herling, Darrell R [Richland, WA; Aardahl, Chris L [Richland, WA; Rozmiarek, Robert T [Middleton, WI; Rappe, Kenneth G [Richland, WA; Wang, Yong [Richland, WA; Holladay, Jamelyn D [Kennewick, WA

    2008-10-14

    The present invention provides a process for producing reagents for a chemical reaction by introducing a fuel containing hydrocarbons into a flash distillation process wherein the fuel is separated into a first component having a lower average molecular weight and a second component having a higher average molecular weight. The first component is then reformed to produce synthesis gas wherein the synthesis gas is reacted catalytically to produce the desire reagent.

  18. Monoclonal TCR-redirected tumor cell killing.

    Science.gov (United States)

    Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

    2012-06-01

    T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

  19. Sorption-reagent treatment of brines produced by reverse osmosis unit for liquid radioactive waste management

    International Nuclear Information System (INIS)

    Avramenko, V. A.; Zheleznov, V. V.; Sergienko, V. I.; Chizhevsky, I. Yu

    2003-01-01

    The results of the pilot plant tests (2002-2003) of the sorption-reagent decontamination of high salinity radioactive waste (brines) remaining after the low-salinity liquid radioactive waste (LRW) treatment in the reverse-osmosis unit from long-lived radionuclides are presented. The sorption-reagent materials used in this work were developed in the Institute of Chemistry FEDRAS. They enable one to decontaminate brines with total salt content up to 50 g/l from long-lived radionuclides of Cs, Sr and Co. At joint application of the reverse-osmosis and sorption-reagent technologies total volume of solid radioactive waste (SRW) decreases up to 100-fold as compared to the technology of cementation of reverse osmosis brines. In this case total cost of LRW treatment and SRW disposal decreases more than 10-fold. Brines decontaminated from radionuclides are then diluted down to the ecologically safe total salts content in water to be disposed of. Tests were performed to compare the efficiency of technologies including evaporation of brines remaining after reverse osmosis process and their decontamination by means of the sorption-reagent method. It was shown that, as compared to evaporation, the sorption-reagent technology provides substantial advantages as in regard to radioactive waste total volume reduction as in view of total cost of the waste management

  20. 21 CFR 866.3390 - Neisseria spp. direct serological test reagents.

    Science.gov (United States)

    2010-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3390... Neisseria spp. directly from clinical specimens. The identification aids in the diagnosis of disease caused...

  1. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Calligaro, D.; Eldefrawi, M.

    1986-01-01

    Binding of [ 3 H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [ 3 H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95 0 C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [ 3 H]cocaine (15 nM) was inhibited by increasing concentrations of Na + ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [ 3 H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC 50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [ 3 H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC 50 values below μM concentrations

  2. Protein-Protein Interaction Reagents | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu

  3. [Thermodynamic forecasting of reagents composition for soils decontamination].

    Science.gov (United States)

    Nikolaev, V P; Nikolaevskiĭ, V B; Chirkina, I V; Shcheglov, M Iu

    2009-01-01

    Based on thermodynamic studies, the authors conducted laboratory experiments on searching optimal composition of leaching reagents solution for soils decontamination, when contaminated with Cs-137, of activity coefficient for caesium sulfate microquantities in macrocomponents solutions. The method could be used for modelling the radionuclides phase equillibrium and relocations in soils.

  4. Affine fractal functions as bases of continuous funtions | Navascues ...

    African Journals Online (AJOL)

    The objective of the present paper is the study of affine transformations of the plane, which provide self-affine curves as attractors. The properties of these curves depend decisively of the coefficients of the system of affinities involved. The corresponding functions are continuous on a compact interval. If the scale factors are ...

  5. Antibody Affinity Maturation in Fishes—Our Current Understanding

    Directory of Open Access Journals (Sweden)

    Brad G. Magor

    2015-07-01

    Full Text Available It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig mutator enzyme activation-induced cytidine deaminase (AID. We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

  6. Small-scale extraction and radioiodination of human hormones for the substitution of imported radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Gimbo, E.K.; Ribela, M.T.C.P.; Borghi, V.C.; Schwarz, I.; Morganti, L.; Araujo, E.A.; Bartolini, P.

    1988-01-01

    The methods for national production of radioimmunoassay reagents to substitute imported kits of: highly purified unlabelled hormones for radioiodination; 125 I-labelled hormones; and specific high titre antisera are presented. The extraction and purification of human growth hormone (hGH) and human luteinizing hormone (hGH) were done from human pituitaries. The 125 I-labelled hormones are obtained by stoichiometric methods. The 125 I-hGH, 125 I-hLH, I-hTSH and 125 I- h calcitonin were prepared and tested in internal and external quality control, in comparison with imported products. The parameters such as: maximum binding to specific antiserum (Bo), nonspecific binding (NSB), mean effective dose (ED 50), sensitivity and accuracy were evaluated. (M.C.K.) [pt

  7. Evaluation of Questionnaire, Reagent Strip and Egg Count as ...

    African Journals Online (AJOL)

    A longitudinal study covering 55 months evaluated the three diagnostic tools used for confirmation of prevalence of urinary schistosomiasis among 1151 consented primary school pupils in 13 communities of Edo State, Nigeria. Questionnaire, reagent strip method and parasitological examination were employed.

  8. Thermokinetic model of borosilicate glass dissolution: contextual affinity

    International Nuclear Information System (INIS)

    Advocat, T.; Vernaz, E.; Crovisier, J.L.; Fritz, B.

    1989-01-01

    Short and long-term geochemical interactions of R7T7 nuclear glass with water at 100 0 C were simulated with the DISSOL thermokinetic computer code. Both the dissolved glass quantity and the resulting water composition, saturation states and mineral quantities produced were calculated as a function of time. The rate equation used in the simulation was first proposed by Aagaard and Helgeson. It simulates a gradually diminishing dissolution rate as the reaction affinity diminishes. The best agreement with 1-year experimental data was obtained with a reaction affinity calculated from silica activity (Grambow's hypothesis) rather than taking into account the activity of all the glass components as proposed by Jantzen and Plodinec. The concept of residual affinity was introduced by Grambow to express the fact that the glass dissolution rate does not cease. We prefer to replace the term residual affinity by contextual affinity, which expresses the influence on the dissolution rate of three factors: the solution chemistry, the metastability of SiO 2 (m), and the possible precipitation of certain aluminosilicates such as zeolites. 19 refs

  9. Manganese-Catalyzed Aerobic Heterocoupling of Aryl Grignard Reagents

    DEFF Research Database (Denmark)

    Ghaleshahi, Hajar Golshahi; Antonacci, Giuseppe; Madsen, Robert

    2017-01-01

    An improved protocol has been developed for the MnCl2-catalyzed cross-coupling reaction of two arylmagnesium bromides under dioxygen. The reaction was achieved by using the Grignard reagents in a 2:1 ratio and 20 % of MnCl2. Very good yields of the heterocoupling product were obtained when the li...

  10. Affinity Strings: Enterprise Data for Resource Recommendations

    Directory of Open Access Journals (Sweden)

    Shane Nackerud

    2008-12-01

    Full Text Available The University of Minnesota Libraries have created a MyLibrary portal, with databases and e-journals targeted to users, based on their affiliations. The University's enterprise authentication system provides an "affinity string", now used to personalize the MyLibrary portal. This affinity string automates discovery of a user's relationship to the University--describing a user's academic department and degree program or position at the University. Affinity strings also provide the Libraries with an anonymized view of resource usage, allowing data collection that respects users' privacy and lays the groundwork for automated recommendation of relevant resources based on the practices and habits of their peers.

  11. Factors involved in the selection of limestone reagents for use in wet FGD systems

    International Nuclear Information System (INIS)

    Jarvis, J.B.; Roothaan, E.S.; Meserole, F.B.; Owens, D.R.

    1992-01-01

    With recent activity in the design and construction of retrofit flue gas desulfurization (FGD) systems, many utilities are faced with the task of selecting limestones which will allow FGD systems to function as designed, and at the same time, provide cost-effective operation. The Electric Power Research Institute (EPRI) has sponsored research to identify factors which should be considered in the reagent selection process. A set of capabilities has been developed which is currently being employed to assist six utilities in selecting cost-effective reagent sources. The major elements in the selection package consist of an analytical characterization of candidate limestones; grindability, reactivity, and magnesium availability testing; and performance modeling utilizing EPRI's FGD PRocess Integration and Simulation Model (FGDPRISM). The results from these measurements are used to perform a site-specific economic analysis which can be used to rank the candidate limestones and quantify the impact of various limestone properties on plant operating costs. This paper includes a description of each element in the selection package along with a review of current research activities aimed at improving predictions of limestone reactivity and magnesium availability. An example is presented which illustrates how reactivity and magnesium availability affect both the performance of an FGD system and plant operating costs

  12. Recyclable bio-reagent for rapid and selective extraction of contaminants from soil

    International Nuclear Information System (INIS)

    Lomasney, H.L.

    1997-01-01

    This Phase I Small Business Innovation Research program is confirming the effectiveness of a bio-reagent to cost-effectively and selectively extract a wide range of heavy metals and radionuclide contaminants from soil. This bioreagent solution, developed by ISOTRON reg-sign Corporation (New Orleans, LA), is flushed through the soil and recycled after flowing through an electrokinetic separation module, also developed by ISOTRON reg-sign. The process is ex situ, and the soil remains in its transport container through the decontamination process. The transport container can be a fiberglass box, or a bulk bag or open-quotes super sack.close quotes Rocks, vegetation, roots, etc. need not be removed. High clay content soils are accommodated. The process provides rapid injection of reagent solution, and when needed, sand is introduced to speed up the heap leach step. The concentrated waste form is eventually solidified. The bio-reagent is essentially a natural product, therefore any solubizer residual in soil is not expected to cause regulatory concern. The Phase I work will confirm the effectiveness of this bio-reagent on a wide range of contaminants, and the engineering parameters that are needed to carry out a full-scale demonstration of the process. ISOTRON reg-sign scientists will work with contaminated soil from Los Alamos National Laboratory. LANL is in the process of decontaminating and decommissioning more than 300 sites within its complex, many of which contain heavy metals or radionuclides; some are mixed wastes containing TCE, PCB, and metals

  13. Recyclable bio-reagent for rapid and selective extraction of contaminants from soil

    Energy Technology Data Exchange (ETDEWEB)

    Lomasney, H.L. [ISOTRON Corp., New Orleans, LA (United States)

    1997-10-01

    This Phase I Small Business Innovation Research program is confirming the effectiveness of a bio-reagent to cost-effectively and selectively extract a wide range of heavy metals and radionuclide contaminants from soil. This bioreagent solution, developed by ISOTRON{reg_sign} Corporation (New Orleans, LA), is flushed through the soil and recycled after flowing through an electrokinetic separation module, also developed by ISOTRON{reg_sign}. The process is ex situ, and the soil remains in its transport container through the decontamination process. The transport container can be a fiberglass box, or a bulk bag or {open_quotes}super sack.{close_quotes} Rocks, vegetation, roots, etc. need not be removed. High clay content soils are accommodated. The process provides rapid injection of reagent solution, and when needed, sand is introduced to speed up the heap leach step. The concentrated waste form is eventually solidified. The bio-reagent is essentially a natural product, therefore any solubizer residual in soil is not expected to cause regulatory concern. The Phase I work will confirm the effectiveness of this bio-reagent on a wide range of contaminants, and the engineering parameters that are needed to carry out a full-scale demonstration of the process. ISOTRON{reg_sign} scientists will work with contaminated soil from Los Alamos National Laboratory. LANL is in the process of decontaminating and decommissioning more than 300 sites within its complex, many of which contain heavy metals or radionuclides; some are mixed wastes containing TCE, PCB, and metals.

  14. 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

    Science.gov (United States)

    Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

    2007-01-01

    Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

  15. Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

    Science.gov (United States)

    Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

    2009-08-17

    Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

  16. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    Science.gov (United States)

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley

  17. [A stable reagent for the-single stage determination of inorganic phosphate].

    Science.gov (United States)

    Pupyshev, A B

    1991-01-01

    A recipe of a simple reagent for phosphorus detection has been developed, consisting of ammonium molybdate (4 mM), sulfuric acid (0.2 N), and Tween-80 (0.2%). The developing phosphate staining may be registered in 15 min at a wavelength of 350 nm. The product molar extinction is equal to 1.20.10(4) M-1.cm-1, this being close to that of molybdic blue. Phosphate staining is characterized by the stability of results and insensitivity to the presence of a number of substances used in enzymology. The prepared reagent is fit for experiments within a fortnight if stored in the cold.

  18. Affinity between information retrieval system and search topic

    International Nuclear Information System (INIS)

    Ebinuma, Yukio

    1979-01-01

    Ten search profiles are tested on the INIS system at the Japan Atomic Energy Research Institute. The results are plotted on recall-precision chart ranging from 100% recall to 100% precision. The curves are not purely systems-dependent nor search-dependent, and are determined substantially by the ''affinity'' between the system and the search topic. The curves are named ''Affinity curves of search topics with information retrieval systems'', and hence retrieval affinity factors are derived. They are obtained not only for individual search topics but also for averages in the system. By such a quantitative examination, the difference of affinity among search topics in a given system, that of the same search topic among various systems, and that of systems to the same group of search topics can be compared reasonably. (author)

  19. Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

    Science.gov (United States)

    Shukla, Girja S.; Krag, David N.

    2010-01-01

    Novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries constructed in fusion to the amino-terminus of P99 β-lactamase molecules were used for identifying β-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified severalβ-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of ~40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific β-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer. PMID:19751096

  20. Affine group formulation of the Standard Model coupled to gravity

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Ching-Yi, E-mail: l2897107@mail.ncku.edu.tw [Department of Physics, National Cheng Kung University, Taiwan (China); Ita, Eyo, E-mail: ita@usna.edu [Department of Physics, US Naval Academy, Annapolis, MD (United States); Soo, Chopin, E-mail: cpsoo@mail.ncku.edu.tw [Department of Physics, National Cheng Kung University, Taiwan (China)

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  1. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    Science.gov (United States)

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  2. A Figment of Their Imagination: Adolescent Poetic Literacy in an Online Affinity Space

    Science.gov (United States)

    Padgett, Elizabeth R.; Curwood, Jen Scott

    2016-01-01

    Drawing on sociocultural perspectives, this case study investigated ways that youths write, read, and critique poetry in an online affinity space. Specifically, it used a thematic analysis of interviews, a linguistic analysis of online feedback mechanisms, and poetic analysis to gain insight into how four teens engaged with poetry on the Figment…

  3. Specific chelating reagent to extract uranium from the sea water

    International Nuclear Information System (INIS)

    Tabushi, Iwao; Kobuke, Yoshiaki

    1982-01-01

    The behavior of uranyl ions as seen from the chemistry of complex as the object of molecule recognition was examined on the basis of the research of structure and equilibrium in solution, in an attempt for the search of specific ligands and the molecular design of adsorbents. What is the most essential for the extraction of uranyl ions from sea water is the development of an excellent adsorbent with rapid adsorption, large equilibrium adsorption and high physical-chemical stability. Based on this appreciation, attempt has been made to find out the molecular design of an excellent adsorbent, starting with the chemistry of complex of uranyl ions. For the purpose, the following matters are reviewed: uranium in nature, the chemistry of complex of uranyl ions, specific ligands for the uranyl ions in sea water, the adsorbent resins for sea-water uranium, the proposals for an adsorbent system. (Mori, K.)

  4. Peptide bond-forming reagents HOAt and HATU are not mutagenic in the bacterial reverse mutation test.

    Science.gov (United States)

    Nicolette, John; Neft, Robin E; Vanosdol, Jessica; Murray, Joel

    2016-04-01

    The peptide bond-forming reagents 1-hydroxy-7-azabenzotriazole (HOAt, CAS 39968-33-7) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS 148893-10-1) either have structural alerts, unclassified features or are considered out of domain when evaluated for potential mutagenicity with in silico programs DEREK and CaseUltra. Since they are commonly used reagents in pharmaceutical drug syntheses, they may become drug substance or drug product impurities and would need to be either controlled to appropriately safe levels or tested for mutagenicity. Both reagents were tested in the bacterial reverse mutation (Ames) test at Covance, under GLP conditions, following the OECD test guideline and ICH S2(R1) recommendations and found to be negative. Our data show that HOAt and HATU-common pharmaceutical synthesis reagents-are not mutagenic, and can be treated as ordinary drug impurities. © 2016 Wiley Periodicals, Inc.

  5. Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

    International Nuclear Information System (INIS)

    Haber, R.S.; Loeb, J.N.

    1988-01-01

    The administration of thyroid hormone is known to result in an induction of the Na + -K + -adenosinetriphosphatase (Na + -K + -ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na + -K + -ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3',5-triiodo-L-thyronine (T 3 ) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na + -K + -ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K I s) for ouabain of ∼10 -7 and 10 -4 M, respectively. Measurement of the specific binding of [ 3 H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K d ) of slightly less than 10 -7 M, whose maximal binding capacity was increased by T 3 treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T 3 treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na + -K + -ATPase by ouabain-dependent phosphorylation from [ 32 P]orthophosphate confirmed that T 3 treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na + -K + -ATPase are subject to selective hormonal induction

  6. Palladium-catalysed direct cross-coupling of secondary alkyllithium reagents

    NARCIS (Netherlands)

    Vila, Carlos; Giannerini, Massimo; Hornillos, Valentin; Fananas-Mastral, Martin; Feringa, Ben L.

    2014-01-01

    Palladium-catalysed cross-coupling of secondary C(sp(3)) organometallic reagents has been a long-standing challenge in organic synthesis, due to the problems associated with undesired isomerisation or the formation of reduction products. Based on our recently developed catalytic C-C bond formation

  7. SATL Based Lesson for Teaching Grignard Reagents in Synthetic ...

    African Journals Online (AJOL)

    Synthesizing new products from raw materials has been very popular aspects of research in organic chemistry. Traditionally, Grignard reagent has been very vital component of such synthetic procedures. Hence learning of various issues concerning with applications of Grignard reactions in synthetic organic chemistry is ...

  8. Affine.m—Mathematica package for computations in representation theory of finite-dimensional and affine Lie algebras

    Science.gov (United States)

    Nazarov, Anton

    2012-11-01

    In this paper we present Affine.m-a program for computations in representation theory of finite-dimensional and affine Lie algebras and describe implemented algorithms. The algorithms are based on the properties of weights and Weyl symmetry. Computation of weight multiplicities in irreducible and Verma modules, branching of representations and tensor product decomposition are the most important problems for us. These problems have numerous applications in physics and we provide some examples of these applications. The program is implemented in the popular computer algebra system Mathematica and works with finite-dimensional and affine Lie algebras. Catalogue identifier: AENA_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENB_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, UK Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 24 844 No. of bytes in distributed program, including test data, etc.: 1 045 908 Distribution format: tar.gz Programming language: Mathematica. Computer: i386-i686, x86_64. Operating system: Linux, Windows, Mac OS, Solaris. RAM: 5-500 Mb Classification: 4.2, 5. Nature of problem: Representation theory of finite-dimensional Lie algebras has many applications in different branches of physics, including elementary particle physics, molecular physics, nuclear physics. Representations of affine Lie algebras appear in string theories and two-dimensional conformal field theory used for the description of critical phenomena in two-dimensional systems. Also Lie symmetries play a major role in a study of quantum integrable systems. Solution method: We work with weights and roots of finite-dimensional and affine Lie algebras and use Weyl symmetry extensively. Central problems which are the computations of weight multiplicities, branching and fusion coefficients are solved using one general recurrent

  9. Spectral affinity in protein networks

    Directory of Open Access Journals (Sweden)

    Teng Shang-Hua

    2009-11-01

    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to

  10. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

    Science.gov (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2018-01-01

    Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

  11. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2018-01-01

    " impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication......Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien......-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types....

  12. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.

    1987-01-01

    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  13. Thermokinetic model of borosilicate glass dissolution: Contextual affinity

    International Nuclear Information System (INIS)

    Advocat, T.; Vernaz, E.; Crovisier, J.L.; Fritz, B.

    1990-01-01

    Short and long-term geochemical interactions of R7T7 nuclear glass with water at 100C were simulated with the DISSOL thermokinetic computer code. Both the dissolved glass quantity and the resulting water composition, saturation states and mineral quantities produced were calculated as a function of time. The rate equation used in the simulation was first proposed by Aagaard and Hegelson: v = k + · S · a( H + ) -n · (1 - e -(A/RT) ). It simulates a gradually diminishing dissolution rate as the reaction affinity diminishes. The best agreement with 1-year experimental data was obtained with a reaction affinity calculated from silica activity (Grambow's hypothesis) rather than taking into account the activity of all the glass components as proposed by Jantzen and Plodinec. The concept of residual affinity was introduced by Grambow to express the fact that the glass dissolution rate does not cease. The authors prefer to replace the term residual affinity by contextual affinity, which expresses the influence on the dissolution rate of three factors: the solution chemistry, the metastability of SiO 2 (m), and the possible precipitation of certain aluminosilicates such as zeolites

  14. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    Science.gov (United States)

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Exploring the Potential for Using Inexpensive Natural Reagents Extracted from Plants to Teach Chemical Analysis

    Science.gov (United States)

    Hartwell, Supaporn Kradtap

    2012-01-01

    A number of scientific articles report on the use of natural extracts from plants as chemical reagents, where the main objective is to present the scientific applications of those natural plant extracts. The author suggests that natural reagents extracted from plants can be used as alternative low cost tools in teaching chemical analysis,…

  16. A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of ß-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Willats, William George Tycho; Knox, J.P.

    1996-01-01

    Seedlings of Arabidopsis thaliana were germinated and grown in medium containing ß-glucosyl Yariv reagent (ßGlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of ßGlcY on the seedlings...

  17. Reliability of Reagent Strips for Semi-quantitative Measurement of Glucosuria in a Neonatal Intensive Care Setting

    Directory of Open Access Journals (Sweden)

    Jolita Bekhof

    2014-12-01

    Conclusion: The reliability of the semi-quantitative measurement of glucosuria in newborn infants using reagent strips is good, even under the conditions of a NICU. Changes in the rating of reagent strips of more than one category are most likely to be beyond measurement error.

  18. A highly efficient dual-diazonium reagent for protein crosslinking and construction of a virus-based gel.

    Science.gov (United States)

    Ma, Dejun; Zhang, Jie; Zhang, Changyu; Men, Yuwen; Sun, Hongyan; Li, Lu-Yuan; Yi, Long; Xi, Zhen

    2018-05-09

    A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

  19. APPLICATION OF FENTON’S REAGENT ON REMEDIATION OF POLYCYCLIC AROMATIC HYDROCARBONs (PAHs IN SPIKED SOIL

    Directory of Open Access Journals (Sweden)

    Nursiah La Nafie

    2010-06-01

    Full Text Available Problem associated with Polycyclic Aromatic Hydrocarbons (PAHs contaminated site in environmental media have received increasing attention. To resolve such problems, innovative in situ methods are urgently required. This work investigated the feasibility of using Fenton's Reagent to remediate PAHs in spiked soil. PAHs were spiked into soil to simulate contaminated soil. Fenton's Reagent (H2O2 + Fe2+ and surfactant were very efficient in destruction of PAHs including naphthalene, anthracene, fluoranthene, pyrene, and benzo(apyrene from spiked soil. It was indicated by the fact that more than 96% of PAHs were degraded in the solution and the spiked soil.   Keywords: Environmental, Fenton's Reagent, Polycyclic Aromatic Hydrocarbons, and Spiked soil.

  20. Site-specifically {sup 89}Zr-labeled monoclonal antibodies for ImmunoPET

    Energy Technology Data Exchange (ETDEWEB)

    Tinianow, Jeff N.; Gill, Herman S.; Ogasawara, Annie; Flores, Judith E.; Vanderbilt, Alexander N.; Luis, Elizabeth; Vandlen, Richard; Darwish, Martine; Junutula, Jagath R.; Williams, Simon-P. [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States); Marik, Jan [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States)], E-mail: marik.jan@gene.com

    2010-04-15

    Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled {sup 89}Zr-Df-thio-trastuzumab conjugates were investigated. Methods: The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with {sup 89}Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. Results: The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with {sup 89}Zr in yields exceeding 75%. {sup 89}Zr-Df-Ac-thio-trastuzumab and {sup 89}Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20-25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1-7.1. Conclusions: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with {sup 89}Zr. The site-specifically {sup 89}Zr-labeled thio

  1. Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer reagents.

    Directory of Open Access Journals (Sweden)

    Noh Jin Park

    Full Text Available Response to cetuximab (Erbitux® and panitumumab (Vectibix® varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™ reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR.This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery of the assay were assessed using normal sera and purified EGFR ECD.This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA.This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing.

  2. Selenium and tellurium reagents in organic synthesis

    International Nuclear Information System (INIS)

    Comasseto, J.V.

    1984-01-01

    A review of the contribution of the University of Sao Paulo (SP, Brazil) to the organic synthesis of selenium and tellurium reagents is made. Major reactions amoung selenium compounds and insaturated substrates, phosphorus, ester enolates as well as the use of phase transference catalysed reactions to produce arylselenolate are described. For tellurium, interactions of its compounds with organic substrates and reactive intermediates (e.g. benzino diazomethane) are reported. (C.L.B.) [pt

  3. Structural insights into a high affinity nanobody:antigen complex by homology modelling

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2017-01-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby...... simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for Rgp...

  4. Design, synthesis, and validation of an in vitro platform peptide-whole cell screening assay using MTT reagent

    Directory of Open Access Journals (Sweden)

    Sahar Ahmed

    2017-05-01

    Full Text Available An in vitro platform to perform peptide screening against different cancer cell lines was designed. The strategy for this screening relied on the design and detection of high-affinity cancer-targeting peptides based on the sequences of NGR and P160. Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager. For proof of concept, a peptide library (132 spots, and 66 different peptides was designed, synthesized, and screened against different cancer cell lines. The current strategy assists in the identification of positive and negative peptides as well as the relative binding between positive ones. Better binding peptide sequences of the NGR motif were demonstrated to show up to a 2.6-fold increase in CD13+ cell lines with insignificant binding to CD13− ones. Comparable results were observed for P160 peptide sequences, to which different peptides had increased binding, with an up to 3-fold increase relative to the native P160 peptide. Based on our results, new peptide sequences for cancer targeting were identified, and the developed strategy was applied to two different peptide libraries.

  5. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    Science.gov (United States)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  6. The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

    International Nuclear Information System (INIS)

    Nicolau, L.A.D.; Silva, R.O.; Damasceno, S.R.B.; Carvalho, N.S.; Costa, N.R.D.; Aragão, K.S.; Barbosa, A.L.R.; Soares, P.M.G.; Souza, M.H.L.P.; Medeiros, J.V.R.

    2013-01-01

    Our objective was to investigate the protective effect of Lawesson's reagent, an H 2 S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm 2 ); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm 2 ); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (K ATP ) channels

  7. Functions of chalcogenide electrodes in solutions of complexing reagents and interfering ions

    International Nuclear Information System (INIS)

    Kiyanskij, V.V.

    1990-01-01

    The possibility to modify chalcogenide electrodes and their behaviour in solutions of complexing reagents for the development of new methods of potentiometric titration has been studied. It is shown that complexing reagents (EDTA, cupferron, 8-hydroxyquinoline, sodium dithiocarbaminate) and Cu(2), Hg(2) produce a strong effect on the functions of Ag, Cu, Cd, Pb - selective electrodes, which is used for titration of potential-determining and non-potential-determining ions ions (Sr 2+ , La 3+ etc.) and also for modification of sulfide-selecting electrode. A method of potentiometric titration of sulfates and chlorides with modified Cd- and Ag-selective electrodes is suggested

  8. Oxytetracycline as a new analytical reagent for the spectrophotometric determination of boron

    Energy Technology Data Exchange (ETDEWEB)

    Narayana, G L

    1984-05-01

    Oxytetracycline hydrochloride, Terramycin, is introduced as a new reagent for the spectrophotometric determination of trace quantities of boron in concentrated sulphuric acid medium. The reagent has an absorption maximum at 430 nm, and that of the boron complex at 520 nm. The colored system conformed to Beer's law between 2 and 10 ..mu..g of boron at 520 nm. The molar absorptivity calculated on the basis of boron is 10,800 1 mol/sup -1/ cm/sup -1/. The composition of the complex has been shown to be 1:1 both by the slope ratio and molar ratio methods. 16 references.

  9. Oxytetracycline as a new analytical reagent for the spectrophotometric determination of boron

    International Nuclear Information System (INIS)

    Narayana, G.L.

    1984-01-01

    Oxytetracycline hydrochloride, Terramycin, is introduced as a new reagent for the spectrophotometric determination of trace quantities of boron in concentrated sulphuric acid medium. The reagent has an absorption maximum at 430 nm, and that of the boron complex at 520 nm. The coloured system conformed to Beer's law between 2 and 10 μg of boron at 520 nm. The molar absorptivity calculated on the basis of boron is 10,800 1 mol -1 cm -1 . The composition of the complex has been shown to be 1:1 both by the slope ratio and molar ratio methods. (author)

  10. THE USE OF GRIGNARD REAGENT IN PHEROMONE SYNTHESIS FOR PALM WEEVIL (Rhynchorus, Sp

    Directory of Open Access Journals (Sweden)

    Warsito Warsito

    2010-06-01

    Full Text Available In an integrated controlling system of palm weevil, using of synthetic feromoid is strickly needed. The research is aimed to synthesize pheromone which secreted by the weevil, e.g. 4-methyl-5-nonanol (R. ferrugineus and 3-methyl-4-octanol (R. schach through Grignard reagent which formed in situ. The synthesis was proceded by retrosynthesis to determine the precursor, valeraldehyde. The precursor was reacted with Grignard reagent of sec-amyl magnesium bromide (R. ferrugenieus and sec-butyl magnesium bromide (R. shach which made in situ. Characterization of the synthetic molecular pheromone was performed by Gas Chromatography-mass spectroscopy and Fourier Transformed Infra Red. The bioassay of the molecule was carried out by olfactometer. The result showed that the conversion of the reactions were 51.28% (4-methyl-5-nonanol and 85.90% (3-methyl-4-octanol. The character of physico-chemical and bioactivity of the synthetic pheromone are identic with natural pheromones.   Keywords: palm weevil, pheromone, grignard reagent

  11. Characterization of high-affinity (/sup 3/H)ouabain binding in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Hauger, R.; Luu, H.M.; Meyer, D.K.; Goodwin, F.K.; Paul, S.M.

    1985-06-01

    The characteristics of (/sup 3/H)ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. (/sup 3/H)Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific (/sup 3/H)ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for (/sup 3/H)ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal (/sup 3/H)ouabain binding was examined. Kainic acid lesions of the striatum reduced (/sup 3/H)ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the high-affinity (/sup 3/H)ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of (/sup 3/H)ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

  12. ASIFT: An Algorithm for Fully Affine Invariant Comparison

    Directory of Open Access Journals (Sweden)

    Guoshen Yu

    2011-02-01

    Full Text Available If a physical object has a smooth or piecewise smooth boundary, its images obtained by cameras in varying positions undergo smooth apparent deformations. These deformations are locally well approximated by affine transforms of the image plane. In consequence the solid object recognition problem has often been led back to the computation of affine invariant image local features. The similarity invariance (invariance to translation, rotation, and zoom is dealt with rigorously by the SIFT method The method illustrated and demonstrated in this work, Affine-SIFT (ASIFT, simulates a set of sample views of the initial images, obtainable by varying the two camera axis orientation parameters, namely the latitude and the longitude angles, which are not treated by the SIFT method. Then it applies the SIFT method itself to all images thus generated. Thus, ASIFT covers effectively all six parameters of the affine transform.

  13. The behavior of thiourea and flotation reagents in zinc electrowinning circuits

    Science.gov (United States)

    MacKinnon, D. J.; Dutrizac, J. E.; Brannen, J. M.; Hardy, D. J.

    1988-04-01

    The effect of thiourea and flotation reagents on the electrowinning of zinc from industrial electrolytes was studied, and all the compounds were found to reduce the zinc deposition current efficiency and to change the properties of the zinc deposits. The effectiveness of activated carbon, two-stage cementation, and hot acid leaching on the destruction/removal of the organic compounds also was addressed. Activated carbon pretreatment of thiourea-containing electrolytes restored the current efficiency for 1-hour zinc deposits to values comparable to those obtained for thiourea-free electrolytes. The activated carbon pretreatment, however, altered the deposit morphology and orientation, but produced a cyclic voltammogram similar to that of the thiourea-free solution. Two-stage cementation did not counteract the harmful effects of thiourea. Hot acid leaching destroyed the thiourea but generated large concentrations of ferrous ion that reduced the current efficiency. The ferrous concentrations, however, were readily controlled by KMnO4 or MnO2 oxidation. None of the treatment options (activated carbon, two-stage cementation, or hot acid leaching) was effective in controlling the flotation reagents, and their moderately harmful effect on zinc electrowinning persisted. Even low concentrations of these reagents polarized zinc deposition, and this resulted in a “glue-type” zinc deposit.

  14. Extraction of uranium(6), transuranium elements and europium by bidentate neutral phosphorus- and phosphorus-nitrogen-containing reagents with substituent in methylene bridge

    International Nuclear Information System (INIS)

    Kochetkova, N.E.; Kojro, O.Eh.; Nesterova, N.P.; Medved', T.Ya.; Chmutova, M.K.; Myasoedov, B.F.; Kabachnik, M.I.

    1986-01-01

    The influence of substituents in methylene bridge on solubility, extractivity and selectivity of bidentate neutral phosphorus- and phosphorus-nitrogen-containing reagents in the process of U(6), TUE, Eu extraction has been studied. It is ascertained that hydrogen substitution in the bridge of tetraphenylmethylenediphosphine dioxide (1) causes a decrease in the extractivity of reagent as to TPE, uranium (6) and europium. There is no visible regular relation between basicity and extractivity of substituted reagents. Hydrogen substitution in the bridge of diphenyl[diethylcarbamoylmethyl]phosphine oxide (2) causes a decrease in extractivity of the reagent as to TPE, uranium (6) and europium. In contrast to monodentate neutral reagents, when bidentate neutral reagents are used, sometimes no increase in the reagent extractivity with an increase in its basicity is observed. When fragments restricting the conformation mobility of bidentate reagent molecule are introduced in it (here substituents in methylene bridge), it may result in the violation of the regularity, since of all the factors affecting the reagent extractivity the spatial factor may become the prevailing one. On hydrogen substitution in the bridge of 1 separation factors of practically all (with few exceptions) studied pairs of elements increase. Hydrogen substitution in the bridge of 2 causes an increase in separation factor of U (6) /Am pair and it does not affect the separation factor of Am/Eu pair. Hydrogen substitution in the bridge of 1 and 2 does not result in the preparation of more efficient and considerably more selective reagents for extractive isolation and separation of the elements, but some of the substituted reagents (Cl-substituted 1, for instance) may turn out useful for the element separation

  15. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  16. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  17. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  18. Applications of Affine and Weyl geometry

    CERN Document Server

    García-Río, Eduardo; Nikcevic, Stana

    2013-01-01

    Pseudo-Riemannian geometry is, to a large extent, the study of the Levi-Civita connection, which is the unique torsion-free connection compatible with the metric structure. There are, however, other affine connections which arise in different contexts, such as conformal geometry, contact structures, Weyl structures, and almost Hermitian geometry. In this book, we reverse this point of view and instead associate an auxiliary pseudo-Riemannian structure of neutral signature to certain affine connections and use this correspondence to study both geometries. We examine Walker structures, Riemannia

  19. Reaction of lupane and oleanane triterpenoids with Lawesson's reagent

    Czech Academy of Sciences Publication Activity Database

    Kvasnica, Miroslav; Rudovská, I.; Císařová, I.; Šarek, J.

    2008-01-01

    Roč. 64, č. 17 (2008), s. 3736-3743 ISSN 0040-4020 Grant - others:GA ČR(CZ) GP203/05/P025 Institutional research plan: CEZ:AV0Z40550506 Keywords : terpenoids * Lawesson's reagent * ketones * sulfur Subject RIV: CC - Organic Chemistry Impact factor: 2.897, year: 2008

  20. Whole transcriptome analysis for T cell receptor-affinity and IRF4-regulated clonal expansion of T cells

    Directory of Open Access Journals (Sweden)

    Wei Shi

    2014-12-01

    Full Text Available Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8+ T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq to interrogate global expression changes when IRF4 was deleted in CD8+ T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929.

  1. Theoretical determination of proton affinity differences in zeolites

    NARCIS (Netherlands)

    Kramer, G.J.; Santen, van R.A.

    1993-01-01

    An important factor in zeolite catalysis is the proton affinity, i.e., the energy required to remove a proton from the zeolite lattice. Differences in proton affinity are expected to influence the catalytic activity of acid sites, making the catalytically active sites inhomogeneous (within one

  2. Pseudo-affinity chromatography of rumen microbial cellulase on ...

    African Journals Online (AJOL)

    Pseudo-affinity chromatography of rumen microbial cellulase on Sepharose- Cibacron Blue F3GA. ... African Journal of Biotechnology ... Pseudo affinity adsorption of bioproducts on Sepharose-cibacron blue F3-GA was subjected to rumen microbial enzyme evaluation through batch binding and column chromatography of ...

  3. Volatility Components, Affine Restrictions and Non-Normal Innovations

    DEFF Research Database (Denmark)

    Christoffersen, Peter; Jacobs, Kris; Dorian, Christian

    Recent work by Engle and Lee (1999) shows that allowing for long-run and short-run components greatly enhances a GARCH model's ability fit daily equity return dynamics. Using the risk-neutralization in Duan (1995), we assess the option valuation performance of the Engle-Lee model and compare...... models to four conditionally non-normal versions. As in Hsieh and Ritchken (2005), we find that non-affine models dominate affine models both in terms of fitting return and in terms of option valuation. For the affine models we find strong evidence in favor of the component structure for both returns...

  4. Realization of Robertson-Walker spacetimes as affine hypersurfaces

    International Nuclear Information System (INIS)

    Chen Bangyen

    2007-01-01

    Due to the growing interest in embeddings of spacetimes in higher dimensional spaces, we consider a special type of embedding. We prove that Robertson-Walker spacetimes can be embedded as centroaffine hypersurfaces and graph hypersurfaces in some affine spaces in such a way that the induced relative metrics are exactly the Lorentzian metrics on the Robertson-Walker spacetimes. Such realizations allow us to view Robertson-Walker spacetimes and their submanifolds as affine submanifolds in a natural way. Consequently, our realizations make it possible to apply the tools of affine differential geometry to study Robertson-Walker spacetimes and their submanifolds

  5. Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents.

    Science.gov (United States)

    Müller, Torsten; Winter, Dominic

    2017-07-01

    Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  7. Development of reagents for radioimmunoassay of: triiodothyronine, thyroxine and thyrotrophin; Desarrollo de reactivos para el radioinmunoanalisis de: triyodotironina, tiroxina y tirotrofina

    Energy Technology Data Exchange (ETDEWEB)

    Delgado S, B.; Lavalley E, C.; Ruiz J, A.; Garcia F, C.; Zamorano A, F

    1991-12-15

    The radioimmunoassay (RIA) of thyroid hormones it is the but it frequents of all the studies carried out by RIA in the laboratories of Nuclear Medicine, these essays are carried out with imported reagents. In the ININ the reagents and the necessary methodology have been developed for the triiodothyronine (T3), thyroxine (T4) and thyrotrophin (TSH). The good titles of the antibodies (Ac) primary for each hormone were of 1:4,000; 1:750 and 1:1,500. The used separation system was of double Ac with PEG to 10%, with titles of 1:10 for the second Ac of lamb. The specific activity for 125-I-T3 and 125-I-T4 oscillate between 850 at 900 {mu}Ci / {mu} g: being this of 90 {mu} Ci /{mu}g for TSH. To the first two hormones they were added 1-8 aniline naftalen sulfonic acid (ANS) to concentrations of 3 and 2 mg/ml respectively. As buffer for T3 and T4 it was used Tris-HCl pH 8.6 and PBS with normal serum of rabbit (SNC) for TSH. The standards got ready in buffer or free serum of thyroid hormones. The slope of the standard curves varied between -2.3 to -2.7 and the variation intra and inter assay among 4 to 10%. It is had at the moment in the ININ with standardized reagents for the RIA of T3, T4 and TSH, it is hoped to carry out tests in other laboratories and to establish the conditions of stability more appropriate to begin the preparation of pilot reagents. (Author)

  8. Methyl Cation Affinities of Neutral and Anionic Maingroup-Element Hydrides: Trends Across the Periodic Table and Correlation with Proton Affinities

    NARCIS (Netherlands)

    Mulder, R. Joshua; Guerra, Celia Fonseca; Bickelhaupt, F. Matthias

    2010-01-01

    We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and

  9. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  10. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Evans, D.M.; Williams, K.P.; McGuinness, B. [PerSeptive Biosystems, Framingham, MA (United States)] [and others

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  11. Generalized Warburg impedance on realistic self-affine fractals ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals.

  12. Cross-reactivity of amphetamine analogues with Roche Abuscreen radioimmunoassay reagents

    International Nuclear Information System (INIS)

    Cody, J.T.

    1990-01-01

    Cross-reactivity of amphetamine analogues with the Abuscreen amphetamine radioimmunoassay reagents was determined for both the standard and high specificity antibody systems. Compounds tested included 2-methoxyamphetamine, 4-hydroxymethamphetamine, 2,5-dimethoxyamphetamine (DMA), 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxy-beta-phenethylamine (BDMPEA), 3,4,5-trimethoxyamphetamine (TMA), 3,4-methylenedioxyamphetamine (MDA), N,N-dimethyl-3,4-methylenedioxyamphetamine and N-hydroxy-3,4-methylenedioxyamphetamine (N-OH MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), 2,5-dimethoxy-4-ethylamphetamine, 2,5-dimethoxy-4-methylamphetamine (DOM), and 3,4,5-trimethoxyphenethylamine (mescaline). Blank negative reference material was spiked with 1,000 to 100,000 ng/mL of the amphetamine analogue and used as sample in the assays. MDA was the only analogue that showed cross reactivity equal to or greater than that of amphetamine. None of the other analogue compounds demonstrated a positive result at even the highest concentration; however several showed depressed counts at various concentration levels

  13. Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

    2013-07-01

    In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

  14. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    Science.gov (United States)

    Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

    2008-01-01

    The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

  15. Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

    2014-04-01

    The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

  16. Non-affine fields in solid-solid transformations: the structure and stability of a product droplet.

    Science.gov (United States)

    Paul, Arya; Sengupta, Surajit; Rao, Madan

    2014-01-08

    We describe the microstructure, morphology, and dynamics of growth of a droplet of martensite nucleating in a parent austenite during a solid-solid transformation, using a Landau theory written in terms of both conventional affine elastic deformations and non-affine deformations. Non-affineness, φ, serves as a source of strain incompatibility and screens long-ranged elastic interactions. It is produced wherever the local stress exceeds a threshold and anneals diffusively thereafter. Using a variational calculation, we find three types of stable solution (labeled I, II, and III) for the structure of the product droplet, depending on the stress threshold and the scaled mobilities of φ parallel and perpendicular to the parent-product interface. The profile of the non-affine field φ is different in these three solutions: I is characterized by a vanishingly small φ, II admits large values of φ localized in regions of high stress within the parent-product interface, and III is a structure in which φ completely wets the parent-product interface. The width l and size W of the twins follow the relation l is proportional to √W in solution I; this relation does not hold for II or III. We obtain a dynamical phase diagram featuring these solutions, and argue that they represent specific solid-state microstructures.

  17. Radiation chemical technology of industrial polymer reagents development

    International Nuclear Information System (INIS)

    Kudaibergenov, S.; Nurkeeva, Z.; Mun, G.; Sigitov, V.; Maltzeva, R.; Petukhov, V.; Tchekushin, A.

    1996-01-01

    The goal of this project is to develop the technology of producing of polymeric reagents from the raw materials of Kazakstan for application in medicine, agriculture, enhanced oil recovery and ecology. To achieve the objectives the next technological lines or operations (Blocks) should be realized: 1. Rectification column and distilling apparatus for purification of monomers and solvents including analytical equipment to control the quality of the final product; 2. Irradiation of reaction mixture by either gamma-irradiation source Co-60; 3. Purification of polymer reagents; 4. Producing of commercial products. It is supposed that the power irradiation devices for producing of hydrogels will be mounted on the research atomic reactor of the Almaty Branch of the Institute of Atomic Energy of the National Nuclear Center. There are high qualification personal which has much experience in radioactive materials operating. Irradiation technologies will provide the low cost of hydrogels, approximately 250-300 US$ per 1 ton. Expected results. One can expect that the realization of this project allows to produce hydrogels in industrial scale to cover partly the requirements of medicine, agriculture, oil industry and ecology

  18. Poly(4-vinylpyridine) as a reagent with silanol-masking effect for silica and its specific selectivity for PAHs and dinitropyrenes in a reversed phase

    International Nuclear Information System (INIS)

    Ihara, Hirotaka; Fukui, Megumi; Mimaki, Takamasa; Shundo, Atsuomi; Dong, Wei; Derakhshan, Mahnaz; Sakurai, Toshihiko; Takafuji, Makoto; Nagaoka, Shoji

    2005-01-01

    This paper demonstrates that poly(4-vinylpyridine) is applicable as an effective masking reagent for silica to reduce undesirable side effects due to silanol groups. It also shows that this chemical modification brings about unique retention behaviors absolutely different from conventional ODS, which appear in molecular-shape selectivity for polycyclic aromatic hydrocarbons and in selectivity for position isomerism, especially for electron-withdrawing substitution compounds. Separation of 1,6- and 1,8-dinirtopyrenes as carcinogens is also described

  19. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    Science.gov (United States)

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Generalized Warburg impedance on realistic self-affine fractals

    Indian Academy of Sciences (India)

    We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals. The information about the ...