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Epitope mapping

Brian M Chan, Anita Badh, Kelly A Berry, Stephanie A Grauer, Chadwick T King
A key step in the therapeutic antibody drug discovery process is early identification of diverse candidate molecules. Information comparing antibody binding epitopes can be used to classify antibodies within a large panel, guiding rational lead molecule selection. We describe a novel epitope binning method utilizing high-throughput flow cytometry (HTFC) that leverages cellular barcoding or spectrally distinct beads to multiplex samples to characterize antibodies raised against cell membrane receptor or soluble protein targets...
May 1, 2018: SLAS Discovery
Lee I Garner, Andrew Hartland, Johannes Breuning, Marion H Brown
CD6 is a type I T cell surface receptor which modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab™ ) which has reached the clinic for treatment of autoimmune disease. We characterised molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action...
May 17, 2018: Immunology
David F Tucker, Jonathan T Sullivan, Kimberly-Anne Mattia, Christine R Fisher, Trevor Barnes, Manu N Mabila, Rona Wilf, Chidananda Sulli, Meghan Pitts, Riley J Payne, Moniquetta Hall, Duncan Huston-Paterson, Xiaoxiang Deng, Edgar Davidson, Sharon H Willis, Benjamin J Doranz, Ross Chambers, Joseph B Rucker
The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4...
May 16, 2018: Proceedings of the National Academy of Sciences of the United States of America
Jan Poolman
Because of diverse sequences and differential expression of surface structures on individual invasive Neisseria meningitidis serogroup B (MenB) strains, predicting the efficacy of MenB vaccines using traditional human serum bactericidal assays (hSBA) is impractical. The meningococcal antigen surface expression (MEASURE) assay uses flow cytometry to quantitate the expression of factor H binding proteins (fHbp) contained in the bivalent rLP2086 MenB vaccine. To date, experience with MEASURE has been lacking, and in a long-awaited article, McNeil et al...
May 15, 2018: MBio
Piet A van Rijn, Mieke A Maris-Veldhuis, Jan Boonstra, René G P van Gennip
African Horse Sickness Virus (AHSV) (Orbivirus genus, Reoviridae family) causes high mortality in naïve domestic horses with enormous economic and socio-emotional impact. There are nine AHSV serotypes showing limited cross neutralization. AHSV is transmitted by competent species of Culicoides biting midges. AHS is a serious threat beyond the African continent as endemic Culicoides species in moderate climates transmit the closely related prototype bluetongue virus. There is a desperate need for safe and efficacious vaccines, while DIVA (Differentiating Infected from Vaccinated) vaccines would accelerate control of AHS...
May 11, 2018: Vaccine
Abu Z Dayem Ullah, Jorge Oscanoa, Jun Wang, Ai Nagano, Nicholas R Lemoine, Claude Chelala
Broader functional annotation of genetic variation is a valuable means for prioritising phenotypically-important variants in further disease studies and large-scale genotyping projects. We developed SNPnexus to meet this need by assessing the potential significance of known and novel SNPs on the major transcriptome, proteome, regulatory and structural variation models. Since its previous release in 2012, we have made significant improvements to the annotation categories and updated the query and data viewing systems...
May 11, 2018: Nucleic Acids Research
Athéna C Patterson-Orazem, Shannon E Hill, Michael P Fautsch, Raquel L Lieberman
No abstract text is available yet for this article.
May 9, 2018: Experimental Eye Research
Lucas D Caeiro, Catalina D Alba-Soto, Mariana Rizzi, María Elisa Solana, Giselle Rodriguez, Agustina M Chidichimo, Matías E Rodriguez, Daniel O Sánchez, Gabriela V Levy, Valeria Tekiel
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes...
May 4, 2018: PLoS Neglected Tropical Diseases
Beate E Preuß, Christoph P Berg, Christoph Werner, Sandra Plankenhorn, Nisar P Malek, Reinhild Klein
BACKGROUND: In a recent study we had evidence that sulphite oxidase (SO) may be a relevant autoantigen in primary sclerosing cholangitis (PSC). Aim of the present study was, therefore, to analyse humoral and cellular immune-reactivity towards SO in these patients in more detail. METHODS: Sera from 53 patients with PSC (30 untreated and 23 treated with ursodeoxycholic acid [UDCA] at time of analysis), from 422 patients with different hepatic and non-hepatic disorders, and from 50 healthy individuals were tested by ELISA for antibodies against full-length-SO (SO-fl) and its three major domains expressed in E...
May 2, 2018: BMC Gastroenterology
John T Ballew, Jack R Reifert, Patrick S Daugherty
Antigen discovery and mapping strategies that enable the precise identification of serum antibody-binding epitopes in human diseases will be invaluable for translational diagnostics and therapeutic development. Protein and peptide library display screening techniques have shown utility for the discovery of antigens associated with disease onset and progression. Here, we describe a screening methodology using bacterial peptide library display to identify consensus families of disease-specific binding motifs to multiple pools of human serum antibodies...
2018: Methods in Molecular Biology
Burcu Ayoglu, Peter Nilsson, Jochen M Schwenk
With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides...
2018: Methods in Molecular Biology
Anna-Luisa Volk, Johan Rockberg
Knowledge of the exquisite-binding surface of an antibody on its target protein is of great value, in particular for therapeutic antibodies for understanding method of action and for stratification of patients carrying the necessary epitope for desired drug efficacy, but also for capture assays under native conditions. Several epitope mapping methodologies have been described for this purpose, with the laborious X-ray crystallography method being the ideal method for mapping of discontinuous epitopes in antibody-antigen crystal complexes and high-throughput peptide-based methods for mapping of linear epitopes...
2018: Methods in Molecular Biology
Anna-Luisa Volk, Francis Jingxin Hu, Johan Rockberg
The unique property of specific high affinity binding to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe an updated protocol for high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric analysis...
2018: Methods in Molecular Biology
Urpo Lamminmäki, Gaurav Batra, Petri Saviranta
Novel affinity reagents, such as single chain (scFv) antibody fragments, can be generated by isolating them from recombinant protein libraries using phage display selection. A successful selection process against a target protein can produce a number of binder candidates among which the desired binders are identified by screening and characterization of individual clones. Obtaining information on the binding properties, such as the binding epitope, already during the screening step helps to choose the most useful candidates for further development at early phase saving time and resources...
2018: Methods in Molecular Biology
Thiru Vanniasinkam, Mary D Barton, Tongted Phumoonna Das, Michael W Heuzenroeder
This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R...
2018: Methods in Molecular Biology
Thomas Van Blarcom, Andrea Rossi, Davide Foletti, Purnima Sundar, Steven Pitts, Zea Melton, Dilduz Telman, Lora Zhao, Wai Ling Cheung, Jan Berka, Wenwu Zhai, Pavel Strop, Jaume Pons, Arvind Rajpal, Javier Chaparro-Riggers
Monoclonal antibodies are the largest class of therapeutic proteins due in part to their ability to bind an antigen with a high degree of affinity and specificity. A precise determination of their epitope is important for gaining insights into their therapeutic mechanism of action and to help differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of multiple antibodies in parallel over the course of just several weeks. This approach is based on a combination of rational library design, yeast surface display, and next generation DNA sequencing and provides quantitative insights into the epitope residues most critical for the antibody-antigen interaction...
2018: Methods in Molecular Biology
Tariq Ahmad Najar, Shruti Khare, Raghavan Varadarajan
Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described [1] a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption...
2018: Methods in Molecular Biology
Thomas Huber, Thomas P Sakmar
We describe a methodology to map epitopes of monoclonal antibodies that bind to G protein-coupled receptors (GPCRs). The method relies on an amber codon suppression strategy to genetically encode photo-activatable cross-linkers, such as p-azido-L-phenylalanine (azF) or p-benzoly-L-phenylalanine (BzF), in GPCRs expressed in mammalian cells in culture. Individual receptor variants that harbor a site-specific photo-crosslinker residue can be assayed for functional activity in standard cell-based assays. The interaction sites between the receptor variants and an antibody can be mapped by determining which of the azF or BzF residues cross-link to the antibody upon UV irradiation...
2018: Methods in Molecular Biology
Luca Simonelli, Mattia Pedotti, Marco Bardelli, Simon Jurt, Oliver Zerbe, Luca Varani
Identifying an epitope, the region of the antigen in contact with an antibody, is useful in both basic and pharmaceutical research, as well as in vaccine design. Solution NMR spectroscopy is particularly well suited to the residue level characterization of intermolecular interfaces, including antibody-antigen interactions, and thus to epitope identification. Here, we describe the use of NMR for residue level characterization of protein epitopes, focusing on experimental protocols and practical considerations, highlighting advantages and drawbacks of the approach...
2018: Methods in Molecular Biology
Moeko Toride King, Cory L Brooks
Therapeutic antibodies constitute one of the fastest areas of growth in the field of biologic drugs. A molecular understanding of how antibodies interact with their target antigens is known as epitope mapping. The data provided by epitope mapping is extremely valuable in the process of antibody humanization, as well as in vaccine design. In many cases the epitope recognized by the antibody is a complex, discontinuous 3D conformational epitope. Mapping the interactions of an antibody to a conformational epitope is difficult by many standard approaches...
2018: Methods in Molecular Biology
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