Analysis of C5a receptor by monoclonal antibody

Hiroshi Watanabe, Mikio Kuraya, Reiji Kasukawa, Hiromi Yanagisawa, Masashi Yanagisawa, Teizo Fujita

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19-29
Number of pages11
JournalJournal of Immunological Methods
Volume185
Issue number1
DOIs
StatePublished - Sep 11 1995

Keywords

  • C5a receptor
  • Flow cytometry
  • Immunoblotting
  • Monoclonal antibody

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Watanabe, H., Kuraya, M., Kasukawa, R., Yanagisawa, H., Yanagisawa, M., & Fujita, T. (1995). Analysis of C5a receptor by monoclonal antibody. Journal of Immunological Methods, 185(1), 19-29. https://doi.org/10.1016/0022-1759(95)00101-F