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Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry

Pimenova, Tatiana ; Meier, Lukas ; Roschitzki, Bernd ; Paraschiv, Gabriela ; Przybylski, Michael ; Zenobi, Renato

In: Analytical and Bioanalytical Chemistry, 2009, vol. 395, no. 5, p. 1395-1401

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    Titre
    • Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry
    Auteur
    • Pimenova, Tatiana. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093, Zurich, Switzerland
    • Meier, Lukas. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093, Zurich, Switzerland
    • Roschitzki, Bernd. Functional Genomics Center Zurich, UZH/ETH Zurich, 8057, Zurich, Switzerland
    • Paraschiv, Gabriela. Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457, Constance, Germany
    • Przybylski, Michael. Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457, Constance, Germany
    • Zenobi, Renato. Department of Chemistry and Applied Biosciences, ETH Zurich, 8093, Zurich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Analytical and Bioanalytical Chemistry, 2009, vol. 395, no. 5, p. 1395-1401. Springer-Verlag
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:318780
    Summary
    The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrPC) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP binding