Watanabe H, Kuraya M, Kasukawa R, Yanagisawa H, Yanagisawa M, Fujita T
Department of Biochemistry, Fukushima Medical College, Japan.
J Immunol Methods. 1995 Sep 11;185(1):19-29. doi: 10.1016/0022-1759(95)00101-f.
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.
我们通过将用转染了人C5a受体（C5aR，CD88）cDNA的小鼠Ltk-细胞（Ltk-/C5aR）免疫的小鼠脾细胞与小鼠骨髓瘤细胞系P3U1融合，制备了一种针对人C5a受体的小鼠单克隆抗体（mAb），命名为4C8。这种属于IgM κ亚类的单克隆抗体，通过免疫印迹分析在Ltk-/C5aR的细胞裂解物中检测到一条43 kDa的条带。流式细胞术显示4C8特异性结合Ltk-/C5aR，表明该抗体对C5aR具有特异性。此外，发现4C8可部分阻断C5a刺激的PMN细胞内Ca2+增加以及125I-C5a与PMN上C5aR的结合。当用抗小鼠IgM交联时，4C8完全抑制C5a与PMN和Ltk-/C5aR上C5aR的结合。因此，这种单克隆抗体似乎不识别C5aR活性位点，而是在空间上抑制C5a与其受体的结合。使用这种单克隆抗体，我们通过免疫印迹在PMN、单核细胞和血小板的细胞裂解物中检测到一条50 kDa的C5aR条带。通过流式细胞术确定C5aR在PMN和单核细胞上表达，而在血小板表面未检测到。基于这些结果，这种单克隆抗体应该有助于分析各种免疫条件和炎症性疾病中C5aR的表达。
