Background: To clarify the binding region of monoclonal antibodies (mAbs) to target molecules is very critical for understanding the pharmacological function of each mAb. Although deletion mutants and point mutants are usefully utilized for the epitope mapping, we often experience the difficulty of determining the mAb epitope against membrane proteins.
Purpose: We aimed to develop a novel method to determine the binding region of mAbs using the epitope tag system.
Methods: We first checked the reactivity of an anti-CD44 mAb (C₄₄Mab-5) to several deletion mutants of CD44. We then employed the RIEDL tag system ("RIEDL" peptide and LpMab-7 mAb). We inserted the RIEDL peptide into CD44 protein from 22nd to 41st amino acid (aa). Produced transfectants were stained by LpMab-7 and C₄₄Mab-5 in flow cytometry.
Results: C₄₄Mab-5 did not react with the 30-361 aa deletion mutant of CD44. Further, the reaction of C₄₄Mab-5 to RIEDL tag-inserted CD44 from 25 aa to 36 aa was lost although LpMab-7 detected RIEDL tag-inserted CD44 from 22 aa to 41 aa.
Conclusions: The epitope of C₄₄Mab-5 for CD44 was determined to be the peptide from 25-36 aa of CD44 using RIEDL insertion for epitope mapping (REMAP) method. REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.