Epitope Analysis of an Anti-Mouse CCR1 Monoclonal Antibody S15040E Using Flow Cytometry

The C-C motif chemokine receptor 1 (CCR1) is widely expressed in various immune cells and plays crucial roles in the maturation and migration of immune cells. CCR1 has been considered an attractive drug target for treating allergic and autoimmune diseases. An anti-mouse CCR1 (mCCR1) monoclonal antibody (clone S15040E) has been used in various in vivo studies to identify mCCR1-positive cells by flow cytometry. However, the binding epitope has not been determined. This study investigated the binding epitope of S15040E using flow cytometry. The mCCR1 extracellular domain-substituted mutant analysis showed that S15040E recognizes the extracellular loop 2 (ECL2, aa 172–197) of mCCR1. Next, alanine (or glycine) scanning was conducted in the ECL2 region. The results revealed that the Trp176, Phe178, Arg181, and Cys183 are essential amino acids for the recognition by S15040E. These results showed the involvement of the ECL2 of mCCR1 in the recognition by S15040E.