ANALYSIS OF C5A RECEPTOR BY MONOCLONAL-ANTIBODY

We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the huma n C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk(-) cells transfected with cDNA of human C5aR (Ltk(-)/C 5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the Ig M kappa subclass, detected a 43 kDa band on cell lysates of Ltk(-)/C5a R by immunoblotting analysis. Flow cytometry revealed that 4C8 specifi cally bound to Ltk(-)/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracell ular Ca2+ increase in PMN stimulated by C5a and I-125-C5a binding to C 5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibi ted the binding of C5a to C5aR on PMN and Ltk(-)/C5aR. Therefore, it s eems likely that this mAb does not recognize the C5aR active site but sterically inhibits the binding of C5a to its receptor. Using this mAb , we detected a 50 kDa band of C5aR on cell lysates of PMN, monocytes and platelets by immunoblotting. C5aR was expressed on PMN and monocyt es as determined by flow cytometry, whereas it was not demonstrated on the surface of platelets. Based on these results, this mAb should be useful for analysis of C5aR expression in various immunological condit ions and inflammatory diseases.