氨甲酰磷酸合成酶单克隆抗体的制备、鉴定及应用  

Preparation,characterization and application of monoclonal antibody against CPS1

作　　者：鞠艳芳[1] 刘蓉[2] 杨金菊[3] 柳晓兰[2] 高建恩[3] 孙启鸿[2] 

机构地区：[1]解放军总医院肿瘤内科,北京100853 [2]军事医学科学院放射与辐射医学研究所十二室,北京100850 [3]北京蛋白质组研究中心,北京102206

出　　处：《细胞与分子免疫学杂志》2008年第5期479-481,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家科技攻关计划资助项目(2004BA711A20;2002BA711A11);国家重点基础研究发展计划(973)资助项目(2001CB510208)

摘　　要：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。AIM： To prepare and characterize the monoclonal antibody （mAb） against human carbamyl phosphate synthetase I （CPSI） and makea study of its application, METHODS： Normal human liver tissues were homogenized, and their mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAb using the routine hybridoma technique, The mAb was detected by ELISA, Western blot immunohistochemistry and immunofluorecent staining, The specificity of mAb was identified by mass spectrometry （MS） and immunoprecipitation （IP） and then confirmed by Uni-ZAP expression library screening. The antibody was used to isolate potential enzymatic complexes by immunocapturing. RESULTS： Three hybridoma cell lines BEH045, ACB271 and BFG021 secreting specific mAb against CPS1 were obtained. The Ig subclass of the mAb was IgG1, which was used in ELISA, Western blot immunohistochemistry, immunoprecipitation, immunofluorecent staining and the isolation of potential enzymatic complexes. CONCLUSION： A hybridoma cell line which can secre specific mAb against CPSI stably has been established. The specific mAb against CPSI is of valne to the research into the functions and distribution of CPSI.

关 键 词：氨甲酰磷酸合成酶(CPSI) 单克隆抗体 特性 

分 类 号：R392.11[医药卫生—免疫学]