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目的:制备抗人11β-羟类固醇脱氢酶1(homo sapi-ens hydroxysteroid 11-beta dehydrogenase 1,HSD11B1)的单克隆抗体(mAb)并初步鉴定其特性。方法:将正常成人肝组织匀浆离心并分离线粒体,用线粒体总蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb,并通过间接ELISA法、Westernblot、免疫组化的方法对mAb进行特性鉴定,通过免疫沉淀、Uni-ZAP XR肝脏cDNA表达文库筛选鉴定抗原。结果:通过间接ELISA筛选获得1株可稳定分泌抗人HSD11B1 mAb的杂交瘤细胞系。其分泌的mAb的Ig亚类(型)为IgG1(κ),Western blot结果显示该mAb可以特异地识别相对分子质量(Mr)为35 000的蛋白;应用mAb CBF245对人肝脏cDNA表达文库(Uni-ZAP XR)进行筛选,阳性噬菌斑测序结果显示5个阳性克隆插入序列均为HSD11B1。结论:mAb BAD062特异地识别抗原HSD11B1,该mAb可用于ELISA检测、Westernblot、免疫组化和免疫沉淀实验,为HSD11B1的研究提供了有力的工具。
Objective: To prepare a monoclonal antibody (mAb) against human 11β-hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1) and preliminary identification of its characteristics. Methods: The normal adult liver homogenate was centrifuged and the mitochondria were isolated. The BALB / c mice were immunized with total mitochondrial protein. The mAb was prepared by hybridoma technique. The mAb was characterized by indirect ELISA, Western blot and immunohistochemistry The antigen was identified by immunoprecipitation, Uni-ZAP XR liver cDNA expression library. Results: One hybridoma cell line stably secreting anti-human HSD11B1 mAb was obtained by indirect ELISA. The mAb secreted mAb has an Ig subclass of IgG1 (κ). The results of Western blot showed that the mAb could specifically recognize a protein with a relative molecular weight of 35 000. The mAb CBF245 was used to detect the expression of human liver cDNA library (Uni -ZAP XR). Positive plaque sequencing showed that all five positive clones had HSD11B1 inserted. Conclusion: mAb BAD062 specifically recognizes the antigen HSD11B1. The mAb can be used for ELISA, Western blot, immunohistochemistry and immunoprecipitation assays, providing a powerful tool for the study of HSD11B1.