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Updated: Sep 17, 2025
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Antibody-Antigen Epitope Mapping by X-Ray Crystallography
Andrew Manion1, Teresa M Brooks1, Cory L Brooks2
1Department of Chemistry and Biochemistry, California State University Fresno, Fresno, CA, USA.
Abstract:
Antibodies have emerged as a highly effective class of biologic drugs. A molecular understanding of how antibodies interact with their antigen can be extraordinarily valuable. The data provided by epitope mapping can inform antibody engineering efforts, lead to breakthroughs in dissecting an antibody's mechanism of action, and provide significant insights into vaccine design. While a variety of approaches are available for epitope mapping, X-ray crystallography is considered to be the gold standard as it can provide a near-atomic resolution model of the antibody-antigen interaction. An X-ray structure allows for the inspection of specific antibody-antigen interactions, even in the case of complex conformational epitopes. Here we describe a procedure for epitope mapping using X-ray crystallography. Fab fragments are produced by transient transfection in expiCHO cells and protein antigens produced in E. coli. The Fab-antigen complex is purified by size exclusion chromatography. Crystallization of the Fab-antigen complex and epitope mapping of the resultant high-resolution X-ray structure provides a complete picture of the antibody-antigen interaction. The method described here can be adapted for structure determination and epitope mapping of any antibody fragment to a simple or complex antigen.
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