Epitope Mapping of an Anti-CD20 Monoclonal Antibody (C₂₀Mab-60) Using Enzyme-Linked Immunosorbent Assay

CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies. Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody-antigen binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed an anti-human CD20 monoclonal antibody, C₂₀Mab-60 (IgG_2a, kappa), using the Cell-Based Immunization and Screening (CBIS). C₂₀Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses. In this study, we examined the critical epitope of C₂₀Mab-60 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. We performed ELISA with deletion mutants, and C₂₀Mab-60 reacted to the 160-179 amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C₂₀Mab-60 did not recognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that the binding epitope of C₂₀Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.