Preparation And Characterization Of Monoclonal Antibodies Against Human Proteinase-activated Receptor-3

Background and objective: Proteinase-activated receptor-3(PAR3) is the member of the protease-activated receptor(PAR) family, which belongs to a new subfamily of G protein-coupled receptors(GPCRs) with seven transmembrane domains activated via proteolytic cleavage by serine proteinases. PAR3 is locally produced by monocytes, macrophages, dendritic cell and is widely distributed at heart, liver, pancreas, gastrointestinal tract and marrow. PAR3 is participating in the disease, such as inflammation and tumor. The monoclonal antibodies(mAbs) against human proteinase activated receptor-3 help us to measure the expression and understand it more. Accordingly, it is meaningful to prepare and identify mAb against hPAR3.Methods: BALB/c mice were immunized with hPAR3, and hybridoma cell lines were obtained by fusing mouse spleen cells with myeloma NS-1 cells. The specificity of mAbs was characterized by enzyme-linked immunosorbent assay(ELISA), flow cytometry(FCM), confocal laser scanning microscopy(CLSM), immunohistochemical staining and Dot blot.Results: 2 hybridoma cells that secreted the mAbs to PAR3 were obtained. 2 mAbs were IgM. Immunohistochemical staining revealed that the reactivities of 2 mAbs to the macrophage cells in lung, mast cell-like cells in colon, lymphocyte cells in tonsil and prepuce tissues were positive. The result of FCM and CLSM showed that the 2 mAbs recognized PAR3 expressed on the A549 cells. Dot blot analysis showed that the mAbs could recognize the PAR3 antigen that absorbed on the NC membrane.Conclusion: The mAbs against hPAR3 are prepared successfully, which provides valuable tool for studies on expression and related diseases.