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目的:制备小鼠抗人烯脂酰辅酶A水解酶(enoyl CoA hydratase,ECHS1)的单克隆抗体(mAb)并进行初步鉴定。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、Western blot、免疫沉淀、免疫组化和间接免疫荧光等方法对mAb进行特异性鉴定,通过Uni-ZAP XR正常成人肝脏cDNA表达文库筛选鉴定抗原。结果:获得1株可稳定分泌抗人ECHS1 mAb的杂交瘤细胞株BGB095。该mAb的Ig亚类(型)为IgG1(κ),可用于ELISA检测、Western blot、免疫沉淀、免疫组化等实验。结论:成功制备了抗人ECHS1的mAb,为代谢疾病和肿瘤的研究提供了有力的工具。
OBJECTIVE: To prepare a monoclonal antibody (mAb) for mouse anti-human enoyl-CoA hydratase (ECHS1) and preliminary identification. METHODS: Normal adult liver homogenates were centrifuged and isolated from mitochondria. BALB / c mice were immunized with total mitochondrial protein. The mAbs were prepared by hybridoma technique and identified by indirect ELISA, Western blot, immunoprecipitation, immunohistochemistry and indirect immunofluorescence MAb were identified by Uni-ZAP XR method. The antigens were identified by Uni-ZAP XR normal adult liver cDNA expression library. Results: One hybridoma cell line BGB095 that can stably secrete anti-human ECHS1 mAb was obtained. The Ig subclass (type) of this mAb is IgG1 (κ), which can be used for ELISA test, Western blot, immunoprecipitation and immunohistochemistry. Conclusion: The mAbs against human ECHS1 were successfully prepared and provided a powerful tool for the study of metabolic diseases and tumors.