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

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Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms

Smith, Tara C.; Saul, Richard G.; Barton, Elisabeth R.; Luna, Elizabeth J.
PLoS ONE.
Oct 2018
10.1371/journal.pone.0205910
Supervillin isoforms have been implicated in cell proliferation, actin filament-based motile processes, vesicle trafficking, and signal transduction. However, an understanding of the roles of these proteins in cancer metastasis and physiological processes has been limited by the difficulty of obtaining specific antibodies against these highly conserved membrane-associated proteins. To facilitate research into the biological functions of supervillin, monoclonal antibodies were generated against the bacterially expressed human supervillin N-terminus. Two chimeric monoclonal antibodies with rabbit Fc domains (clones 1E2/CPTC-SVIL-1; 4A8/CPTC-SVIL-2) and two mouse monoclonal antibodies (clones 5A8/CPTC-SVIL-3; 5G3/CPTC-SVIL-4) were characterized with respect to their binding sites, affinities, and for efficacy in immunoblotting, immunoprecipitation, immunofluorescence microscopy and immunohistochemical staining. Two antibodies (1E2, 5G3) recognize a sequence found only in primate supervillins, whereas the other two antibodies (4A8, 5A8) are specific for a more broadly conserved conformational epitope(s). All antibodies function in immunoblotting, immunoprecipitation and in immunofluorescence microscopy under the fixation conditions identified here. We also show that the 5A8 antibody works on immunohistological sections. These antibodies should provide useful tools for the study of mammalian supervillins.

The immunome of soy bean allergy: Comprehensive identification and characterization of epitopes

Kern, Karolin; Havenith, Heide; Delaroque, Nicolas; Rautenberger, Paul; Lehmann, Jörg; Fischer, Markus; Spiegel, Holger; Schillberg, Stefan; Ehrentreich-Foerster, Eva; Aurich, Stefanie; Treudler, Regina; Szardenings, Michael
Clin Exp Allergy.
Sep 2018
10.1111/cea.13285
Background The precise mapping of multiple antibody epitopes recognized by patients’ sera allows a more detailed and differentiated understanding of immunological diseases. It may lead to the development of novel therapies and diagnostic tools. Objective Mapping soy bean specific epitopes relevant for soy bean allergy patients and persons sensitized to soy bean, and analysis of their IgE/IgG binding spectrum. Methods Identification of epitopes using sera, applying an optimized peptide phage display library followed by next-generation sequencing, specially designed in silico data analysis and subsequent peptide microarray analysis. Results We were able to identify more than 400 potential epitope motifs in soy bean proteins. More than 60% of them have not yet been described as potential epitopes. Eighty-three peptides, representing the 42 most frequently found epitope candidates, were validated by microarray analysis using 50 sera from people who have been tested positive in skin prick test (SPT). Of these peptides, 56 were bound by antibodies, 55 by serum IgE, 43 by serum IgG and 30 by both. Person-specific epitope patterns were found for each individual and protein. Conclusions For individuals with clinical symptoms, epitope resolved analyses reveal a high prevalence of IgE binding to a few soy bean specific epitopes. Evaluation of individual immune profiles of patients with soy bean sensitization allows the identification of peptides that do facilitate studying individual IgE/IgG epitope binding patterns. This enables discrimination of sensitization from disease, such assay test has the potential to replace SPT assays

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms

Gadermaier, Elisabeth; Tesarz, Manfred; Suciu, Andreea Ana-Maria; Wallwitz, Jacqueline; Berg, Gabriela; Himmler, Gottfried
J Clin Lab Anal.
Feb 2018
10.1002/jcla.22252
Background Periostin (osteoblast-specific factor OSF-2) is a secreted protein occurring in seven known isoforms, and it is involved in a variety of biological processes in osteology, tissue repair, oncology, cardiovascular and respiratory systems or allergic manifestations. To analyze functional aspects of periostin, or the ability of periostin as potential biomarker in physiological and pathological conditions, there is the need for a precise, well-characterized assay that detects periostin in peripheral blood. Methods In this study the development of a sandwich ELISA using monoclonal and affinity-purified polyclonal anti-human periostin antibodies was described. Antibodies were characterized by mapping of linear epitopes with microarray technology, and by analyzing cross-reactive binding to human periostin isoforms with western blot. The assay was validated according to ICH/EMEA guidelines. Results The monoclonal coating antibody binds to a linear epitope conserved between the isoforms. The polyclonal detection antibody recognizes multiple conserved linear epitopes. Therefore, the periostin ELISA detects all known human periostin isoforms. The assay is optimized for human serum and plasma and covers a calibration range between 125 and 4000 pmol/L for isoform 1. Assay characteristics, such as precision (intra-assay: ≤3%, inter-assay: ≤6%), spike-recovery (83%-106%), dilution linearity (95%-126%), as well as sample stability meet the standards of acceptance. Periostin levels of apparently healthy individuals are 864±269 pmol/L (serum) and 817±170 pmol/L (plasma) respectively. Conclusion This ELISA is a reliable and accurate tool for determination of all currently known periostin isoforms in human healthy and diseased samples.

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