Analysis of C5a receptor by monoclonal antibody.
We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the human C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk- cells transfected with cDNA of human C5aR (Ltk-/C5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the IgM kappa subclass, detected a 43 kDa band on cell lysates of Ltk-/C5aR by immunoblotting analysis. Flow cytometry revealed that 4C8 specifically bound to Ltk-/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracellular Ca2+ increase in PMN stimulated by C5a and 125I-C5a binding to C5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibited the binding of C5a to C5aR on PMN and Ltk-/C5aR. Therefore, it seems 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 monocytes 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 conditions and inflammatory diseases.
Watanabe
H
H
Department of Biochemistry, Fukushima Medical College, Japan.
Kuraya
M
M
Kasukawa
R
R
Yanagisawa
H
H
Yanagisawa
M
M
Fujita
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015703:Antigens, CD / Q000032:analysis* / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000032:analysis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; D002118:Calcium