Epitope Mapping of an Anti-Human Epidermal Growth Factor Receptor Monoclonal Antibody (EMab-51) Using the RIEDL...

The classic method for identifying the epitope that monoclonal antibodies (mAbs) bind uses deletion mutants and point mutants of the target protein. However, determining the epitope of mAbs-reactive membrane proteins is often challenging. We recently developed the RIEDL insertion for epitope mapping (REMAP) method to identify mAb-binding epitopes. Herein, we first checked the reactivity of an anti-epidermal growth factor receptor (EGFR) mAb (EMab-51) to several EGFR deletion mutants such as EGFR/dN152, EGFR/dN313, EGFR/dN370, EGFR/dN375, EGFR/dN380, and EGFR/dN482. We found the N-terminus of the EMab-51-binding epitope between residues 375 and 380 of EGFR. We next produced EGFR/dN313 mutants with the RIEDL peptide tag inserted at each possible position of ₃₇₅-AFRGDSFTHTPPLDP-₃₈₉. EMab-51 lost its reactivity with the mutants having a RIEDL tag inserted at each position of ₃₇₇-RGDSFTHTPP-₃₈₆, whereas LpMab-7 (an anti-RIEDL mAb) detected every mutant. Thus, using the REMAP method, we identified the EMab-51-binding epitope of EGFR as ₃₇₇-RGDSFTHTPP-₃₈₆.