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目的制备抗人丙酮酸脱氢酶复合体E2亚基(pyruvate dehydrogenase complex E2,PDC-E2)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法、Western blot及免疫组化的方法对mAb进行特性鉴定,通过免疫沉淀联合质谱、Uni-ZAP XR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PDC-E2mAb的杂交瘤细胞系。mAb的Ig亚类(型)为IgG1(κ),该mAb可用于ELISA检测、Western blot、免疫组化和免疫沉淀实验。结论成功制备了抗人PDC-E2的mAb,为PDC-E2的研究提供了有力的工具。
Objective To prepare monoclonal antibodies against human pyruvate dehydrogenase complex E2 (PDC-E2) and to identify them. Methods Normal adult liver homogenates were centrifuged and isolated from mitochondria. Balb / c mice were immunized with total mitochondrial protein, and mAb was prepared by hybridoma technique. The mAb was identified by indirect ELISA, Western blot and immunohistochemistry , Identified by immunoprecipitation combined with mass spectrometry, Uni-ZAP XR liver cDNA expression library screening identified antigen. Results A hybridoma cell line stably secreting anti-human PDC-E2 mAb was obtained. The Ig subclass (type) of mAb is IgG1 (κ), which can be used for ELISA test, Western blot, immunohistochemistry and immunoprecipitation experiments. Conclusion The mAb against human PDC-E2 was successfully prepared, which provided a powerful tool for the research of PDC-E2.