抗人gp130单克隆抗体的制备及其生物学特性分析  

Preparation of the monoclonal antibody against human gp130 and analysis for its biological traits

作　　者：苗平[1] 钱柳[1] 赵蓉[1] 余奇文[1] 张继英[1] 陈雪华[2] 陆梅生 张冬青[1] 

机构地区：[1]上海交通大学医学院上海市免疫学研究所,上海200025 [2]上海交通大学医学院附属瑞金医院,上海200025 [3]上海麦柏星生物科技有限公司,上海201203

出　　处：《中国免疫学杂志》2011年第12期1119-1122,1128,共5页Chinese Journal of Immunology

基　　金：上海市免疫学研究所项目(08-A04);上海市科委重点项目(10JC1408500);上海市科委重点学科(外科学)开放课题(S30204-K01)等基金资助

摘　　要：目的:制备小鼠抗人IL-6Rβ(gp130)单克隆抗体,并分析其生物学特性及初步的免疫学功能。方法:用gp130抗原免疫BALB/c小鼠,经三次免疫获得高效价的ELISA结果后,取小鼠的脾细胞和SP2/0骨髓瘤细胞进行融合,经多次亚克隆筛选得到稳定分泌抗gp130单克隆抗体的杂交瘤细胞株。大量扩增杂交瘤细胞,收集细胞上清,随即过亲合层析柱进行纯化,对经纯化的抗人gp130单克隆抗体进行亚型鉴定,用Western blot及间接ELISA、biacore方法分析抗人gp130单克隆抗体的纯度、特异性及亲和力,用FACS术检测抗人gp130单克隆抗体与膜表面富含IL-6受体的U266细胞系的结合率,同时建立IL-6诱导的U266细胞增殖系统,用以检测和分析抗人gp130单克隆抗体的抑制和阻断效应。结果:本研究获得了多株能够稳定分泌抗人gp130单克隆抗体的细胞株,取其中一株(14C4)进行分析,表明其亚型属于IgG2b,轻链为κ链,经Western blot及间接ELISA证明此株抗体有较高的纯度及特异性,biacore结果显示14C4与抗原结合的亲和力为2.62E-10,FACS结果证实能与U266细胞表面的gp130特异性结合,并且呈剂量依赖性,细胞增殖实验说明14C4在一定程度上可抑制IL-6诱导的U266细胞的增殖。结论:初步成功制备了抗gp130单克隆抗体,并且具备较好的生物学特性,为研究人IL-6gp130在抗感染、炎症以及肿瘤和自身免疫病学中的诊断和治疗中的意义提供了有效的工具和手段。Objective：To prepare anti-human gp130 monoclonal antibodies,and preliminary analysis of their biological characteristics and functions.Methods：Immunized the BALB/c mice with gp130,then fusion the spleen cells of the BALB/c mice and the SP2/0 cells,cultured in HAT screened medium,after several subclone,harvested several hybridoma cell lines secreting anti-gp130 monoclonal antibody.Collected the supernatant for affinity purification,using Western blot,indirect ELISA and biacore to analyze the purity,specificity and affinity of antibodies,observed the combination of antibody with U266 cells by FACS,and asessed the proliferation inhibitory effects on U266 cell under conditions of IL-6 existence.Results：Several anti human monoclonal antibodies only against gp130 were harvested,select one of these（14C4） for the following study,the subtypes belonged to IgG2b,κ light chain,Western blot and indirect ELISA demonstrated that it had a higher purity and specificity,biacore showed that the binding affinity was 2.62E-10,FACS results showed that it could specific bind the gp130 to the surface of U266 cells and in a dose-dependent manner,cell proliferation test showed：mAb inhibited IL-6 induced U266 cell proliferation.Conclusion：We preliminary prepared the anti-gp130 monoclonal antibody,and it had a better biologicall characteristics,it supplied effective tools for the study of human IL-6gp130 anti-infective,inflammatory and autoimmune diseases.

关 键 词：IL-6 GP130 单克隆抗体 特异性 亲和力 

分 类 号：R392[医药卫生—免疫学]