Journal
ANALYTICAL BIOCHEMISTRY
Volume 395, Issue 1, Pages 100-107Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.08.018
Keywords
Monoclonal antibody; Antigen-antibody interaction; MS; Epitope mapping; Linear epitopes
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The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide a beta(1-40). In epitope excision, the Fab and a beta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-a beta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, a beta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to a beta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-a beta(1-40) mAbs. (C) 2009 Elsevier Inc. All rights reserved.
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