Preparation and characterization of monoclonal antibody against human carboxylesterasesª²¢ò

    WU Zhengª²hua, TANG Xiaoª²bo, WANG Zhiª²gang, JI Hongª²yu, YU Lei, LIU Huiª²wen, ZHU Daª²ling*

    Biopharmacy Department of Pharmacy College; Pharmacy Department of the Second Affiliated Hospital; Key Laboratory of Biopharmaceutical Engineering of Heilongjiang Province; Histology and Embryology Department of Basic Medicine College, Harbin Medical University, Harbin 150086, China

    [Abstract]AIM: Topreparemonoclonalantibody(mAb) against human carboxylesterasesª²¢ò (hCEª²¢ò) and characterize its properties. METHODS: BALB/c mice were immunized with human liver microsome protein which contained hCEª²¢ò. The mAb was prepared by hybridoma technique and purified by proteinª²G affinity chromatography. The titer and specificity of mAb was detected by ELISA and Western blot respectively. Tissue localization of antigen was detected by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) matched with Mascot human protein database. PMF was obtained by immunoprecipitation and matrixª²assisted laser desorption/ionization timeª²ofª²flight mass spectrometry (MALDIª²TOFª²MS). RESULTS: One clone of hybridoma secreting specific mAb against hCEª²¢ò was obtained. The Ig subclass of the mAb was IgG1(¦Ê). The titer of the mAb was 1¡Á10-7. Western blot analysis showed one clear belt in the Mr of 62000. Immunohistochemistry demonstrated that the mAb had special combination with the liver cytoplasm protein, but not with the vascular smooth muscle cell protein. Immunoprecipitation showed one clear band in the Mr of 62 000, which was in conformity with the Mr of hCEª²¢òand the antigen was confirmed to be hCEª²¢òafter being analyzed with mass spectrometry. CONCLUSION: The mAb against hCEª²¢òwith high titer and specificity has been obtained, which lays the foundation for investigation of hCEª²¢ò function and diagnosis and therapy of liver cancer. ......