Preparation And Characterization Of Monoclonal Antibody Against Protease Activated Receptor-2

Objective: To prepare and identify the monoclonal antibody (mAb) against proteaseactivated receptor-2 (PAR-2).Methods: BLAB/c mice were immunized with specific PAR-2 polypeptide bymultiple-site hypodermic injection. The spleen cells of immunized mice were fused with NS-1myeloma cells. Indirect ELISA was used to screen the hybridoma culture supernants. Thespecificity of mAb were characterized by ELISA, Dot blot, immunohistochemistry, flowcytometry and confocal laser scanning microscopy (CLSM).Results: One hybridoma cell line against PAR-2 was successfully obtained, this mAb wasIgM isotype and the titer of antibody in supernants was 1:16. Dot blot analysis showed that themAb could specifically bind to PAR-2. Immunohistochemical staining revealed that thereactivity of mAb to epithelial cells and smooth muscle cells in lung, epithelial cells andlymphocyte-like cells in colon, lymphocytes in tonsil and squamous epithelial cells in skintissue. The result of flow cytometry showed that the mAb recognized PAR-2 in A549 cells.CLSM examination confirmed that the fluorescent markers were localized in bothcytomembrane and cytoplasm of A549 cells.Conclusion: The mAb against PAR-2 is a useful tool for investigating PAR-2 relateddisease.