烯酰辅酶A水合酶单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibody against enoyl-CoA hydratase 1

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

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：《南方医科大学学报》2009年第4期648-651,共4页Journal of Southern Medical University

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

摘　　要：目的制备抗人烯酰辅酶A水合酶(ECH1)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 blotting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-ZAPXR表达文库筛选鉴定抗原。结果获得1株可稳定分泌抗人ECH1mAb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 blotting、免疫组化和免疫沉淀实验。结论成功制备了抗人ECH1的mAb,为ECH1的研究提供了有力的工具。Objective To prepare monoclonal antibodies （mAbs） against enoyl-CoA hydratase 1 （ECH1）. Methods Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitoehondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry （MS） following immunoprecipitation （IP） and confirmed by Uni-ZAP expression library screening. Results One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitafion. Conclusion A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies ofECH1.

关 键 词：烯酰辅酶A水合酶 单克隆抗体 ELISA 

分 类 号：R392.11[医药卫生—免疫学]