21. Monoclonal antibody characterization by defining the antigen epitope

GA CANZIANI, S-J WU, J LUO, M KRUSZYNSKI, P TSUI, A DELVECCHIO, AND M BRIGHAM-BURKE.

Boston, MA, USA

Simposio; 19th Annual Protein Society; 2005

International Protein Society

Binding conditions were optimized to explore the kinetic and affinity screening of a set of antigen peptides that overlap with the putative binding epitope of a monoclonal antibody. The 25 amino acid binding epitope of the monoclonal antibody was identified by affinity-based protease digestion followed by mass spectrometry. Peptides with overlapping sequences were synthesized to better define the antigen-antibody interaction. Based on synthetic peptide ELISA data, the antibody-binding region could be reduced to 15 amino acids. Using Biacore SPR technology, the kinetics and affinity of binding of antibody and its Fab fragment to peptides showed the presence of a C-terminal Cys and positively charged residues (Arg and Lys) were critical in the formation and stability of the peptide-antibody complexes.