抗人羧酸酯酶_医学论文.docVIP

抗人羧酸酯酶_医学论文 抗人羧酸酯酶_医学论文 作者：武正华, 唐晓波, 王志刚, 纪宏宇, 于磊, 刘慧文, 朱大岭 【关键词】 人羧酸酯酶 　　Preparation and characterization of monoclonal antibody against human carboxylesterasesⅡ 　　[Abstract]AIM: Topreparemonoclonalantibody(mAb) against human carboxylesterasesⅡ (hCEⅡ) and characterize its properties. METHODS: BALB/c mice were immunized with human liver microsome protein which contained hCEⅡ. The mAb was prepared by hybridoma technique and purified by proteinG affinity chromatography. The titer and specificity of mAb was detected by ELISA and Western blot respectively. Tissue localization of antigen was detected by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) matched with Mascot human protein database. PMF was obtained by immunoprecipitation and matrixassisted laser desorption/ionization timeofflight mass spectrometry (MALDITOFMS). RESULTS: One clone of hybridoma secreting specific mAb against hCEⅡ was obtained. The Ig subclass of the mAb was IgG1(κ). The titer of the mAb was 1×10-7. Western blot analysis showed one clear belt in the Mr of 62000. Immunohistochemistry demonstrated that the mAb had special combination with the liver cytoplasm protein, but not with the vascular smooth muscle cell protein. Immunoprecipitation showed one clear band in the Mr of 62 000, which was in conformity with the Mr of hCEⅡand the antigen was confirmed to be hCEⅡafter being analyzed with mass spectrometry. CONCLUSION: The mAb against hCEⅡwith high titer and specificity has been obtained, which lays the foundation for investigation of hCEⅡ function and diagnosis and therapy of liver cancer. 　　[Keywords]human carboxylesterasesⅡ monoclonal antibody matrixassisted laser desorption/ionization timeofflight mass spectrometry 　　[摘 要] 目的: 制备鼠抗人羧酸酯酶Ⅱ（human carboxylesterasesⅡ, hCEⅡ）单克隆抗体（mAb）并对其特性进行鉴定。方法: 以含hCEⅡ的人肝脏微粒体蛋白混合抗原免疫BALB/c小鼠, 采用杂交瘤技术制备鼠抗hCEⅡ的mAb, 并用ProteinG亲和层析法纯化mAb。用间接ELISA法测定mAb的效价, 以Western blot鉴定mAb的特异性、 免疫组化染色法对mAb相应的抗原进行组织定位。用免疫沉淀和基质辅助激光解吸电离飞行时间质谱（matr