TY - JOUR
T1 - Analysis of C5a receptor by monoclonal antibody
AU - Watanabe, Hiroshi
AU - Kuraya, Mikio
AU - Kasukawa, Reiji
AU - Yanagisawa, Hiromi
AU - Yanagisawa, Masashi
AU - Fujita, Teizo
PY - 1995/9/11
Y1 - 1995/9/11
N2 - 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 κ 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.
AB - 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 κ 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.
KW - C5a receptor
KW - Flow cytometry
KW - Immunoblotting
KW - Monoclonal antibody
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U2 - 10.1016/0022-1759(95)00101-F
DO - 10.1016/0022-1759(95)00101-F
M3 - Article
C2 - 7665898
AN - SCOPUS:0029023019
SN - 0022-1759
VL - 185
SP - 19
EP - 29
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1
ER -