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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

Mesalam, Ahmed Atef; Desombere, Isabelle; Farhoudi, Ali; Van Houtte, Freya; Verhoye, Lieven; Ball, Jonathan; Dubuisson, Jean; Foung, Steven K.H.; Patel, Arvind H.; Persson, Mats A.A.; Leroux-Roels, Geert; Meuleman, Philip
Virology.
Jan 2018
10.1016/j.virol.2017.10.019
Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230–239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies: Peptide Arrays for Antibody Selectivity Studies

Vernet, Thierry; Choulier, Laurence; Nominé, Yves; Bellard, Laure; Baltzinger, Mireille; Travé, Gilles; Altschuh, Danièle
J. Mol. Recognit..
Oct 2015
10.1002/jmr.2477
Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP® technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore® technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence 6TAMFQDPQER15) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice

Hamilton, Jennie A.; Li, Jun; Wu, Qi; Yang, PingAr; Luo, Bao; Li, Hao; Bradley, John E.; Taylor, Justin J.; Randall, Troy D.; Mountz, John D.; Hsu, Hui-Chen
J.I..
May 2015
10.4049/jimmunol.1402335
Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2+CD80+ memory B cells, with significantly elevated CD69 and CD86 observed in RBP+ marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.

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