抗丙酮酸脱氢酶复合体E2亚基单克隆抗体的制备与鉴定

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-E2 mAb 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.

Preparation and characterization of monoclonal antibody against PDC-E2

Objective To prepare monoclonal antibodies(mAbs)against pyruvate dehydrogenase complex E2(PDC-E2).Methods Normal human liver tissues were homogenized and the mitochondria were isolated by differential centrifugation.The total mitochondrial proteins were used to immunize BALB/c mice for preparation of mAbs by hybridoma technique.The mAbs were analyzed by ELISA,Western blot,and immunohistochemistry.The antibody specificities were identified by immunoprecipitation(IP)and confirmed by Uni-ZAP expression library screening.Results One hybridoma AEB018 secreting specific mAb against PDC-E2 was established.The Ig subclass of this mAb was IgG1.The mAB could be used in ELISA,Western blot,immunohistochemistry,and IP.The protein recognized by AEB018 was about Mr 70000 and localized in bile duct and hepatocytes of human liver tissue.Conclusion A hybridoma cell line stably secreting specific mAb against PDC-E2 is established.The specific mAb against PDC-E2 would be very useful for the studies of PDC-E2 functions and distribution.