[Generation and characterization of monoclonal antibody against HSD11B1].

ABSTRACT

AIM: To generate and identify monoclonal antibodies (mAb) against homo sapiens hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1). METHODS: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to generate mAb by hybridoma technique. The mAb were characterized by ELISA, Western blot and immunohistochemistry. The antibody specificity was identified by immunoprecipitation (IP), and confirmed by Uni-ZAP expression library screening. RESULTS: One hybridoma CBF245 secreting specific mAb against HSD11B1 was established. The Ig subclass of this mAb was IgG1, and it could be used in ELISA, Western blot, immunohistochemistry. Our data showed that the antigen recognized by CBF245 mAb was localized in the hepatocyte cytoplasm, with molecular weight of M(r) 35 000 in the mitochondria of human liver tissue. The CBF245 mAb was further confirmed by immunoscreening of Uni-ZAP XR liver cDNA expression library. CONCLUSION: A hybridoma cell line stably secreting specific mAb against HSD11B1 is established and characterized. This mAb reacted with HSD11B1 in ELISA, Western blot, immunohistochemistry assay, IP, and would be very useful for the HSD11B1 studies.