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 (IgG2a, 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.
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