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目的:制备抗人氨甲酰磷酸合成酶(CPSI)的单克隆抗体(mAb)并进行初步鉴定与应用。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、免疫组化及免疫荧光染色的方法对mAb进行特性鉴定,通过免疫沉淀联合质谱、Uni-ZAP XR表达文库筛选鉴定抗原,借助免疫捕获的方法用抗体来分离复合物。结果:获得3株可稳定分泌抗人CPSImAb的杂交瘤细胞系。mAb的Ig亚类(型)为IgG1(κ),该mAb可用于ELISA检测、Westernblot、免疫组化、免疫荧光染色、免疫沉淀实验和复合物的分离。结论:成功制备了抗人CPSI的mAb,为CPSI的研究提供了有力的工具。
OBJECTIVE: To prepare a monoclonal antibody (mAb) against human carbamoyl phosphate synthase (CPSI) and preliminary identification and application. METHODS: Normal adult liver homogenates were centrifuged and isolated from mitochondria. BALB / c mice were immunized with total mitochondrial protein. MAbs were prepared by hybridoma technique. The expression of mAbs was detected by indirect ELISA, Western blot, immunohistochemistry and immunofluorescence staining The mAbs were characterized, immunoprecipitated with mass spectrometry, Uni-ZAP XR expression libraries were screened to identify antigens, and antibodies were used to isolate the complexes by immunocapture. Results: Three hybridoma cell lines stably secreting anti-human CPSI mAb were obtained. The Ig subclass (type) of mAb is IgG1 (κ), which can be used for ELISA detection, Western blotting, immunohistochemistry, immunofluorescence staining, immunoprecipitation experiments and complex separation. Conclusion: The anti-human CPSI mAb was successfully prepared and provided a powerful tool for the study of CPSI.