拮抗型抗hTNF-α单克隆抗体的筛选和活性鉴定  

Screening and identification of antagonistic monoclonal antibodies against hTNF-α

作　　者：黄蕊[1] 马莉[1] 谷岳[1] 谢印良[1] 佘鸣[1] 邵晓枫[1] 熊冬生[1] 刘汉芝[1] 许元富[1] 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《山东医药》2010年第23期13-16,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(30400405);天津市自然科学基金资助项目(05YFJZJC01200)

摘　　要：目的制备和筛选拮抗型人肿瘤坏死因子-α(hTNF-α)单克隆抗体(McAb)并检测其活性,为临床靶向治疗提供理论依据。方法用经典免疫方法获得抗hTNF-αMcAb,经接种于小鼠腹腔后获得腹水,采用硫酸铵沉淀和蛋白G亲和层析法纯化得到纯度>95%的抗hTNF-αMcAb,采用ELISA方法测定效价、抗体亚型和亲和力,MTT法测定抗体阻断hTNF-α对L929细胞的细胞毒作用和筛选拮抗型抗hTNF-αMcAb,流式细胞仪法测定抗体阻断hTNF-α诱导L929细胞凋亡的作用。结果获得1株能稳定分泌抗hTNF-αMcAb的杂交瘤细胞株XB10,分泌的抗体亚型为IgG2b;该抗体能高亲和性与hTNF-α结合,特异性阻断hTNF-α对L929细胞的细胞毒作用和抑制细胞凋亡,并呈一定剂量依赖性。结论成功制备能稳定分泌拮抗型抗hTNF-α的McAb的杂交瘤细胞株,其分泌的抗体对hTNF-α有特异性拮抗作用,为今后研制临床治疗型抗hTNF-α的基因工程抗体奠定了实验基础。Objective To prepare and screen human TNF-α（hTNF-α） antagonistic monoclonal antibodies （McAbs） and determine its activity, so as to provide theory evidence for clinical targeted therapy. Methods McAbs against hTNF- α were prepared by traditional hybridoma technique, and hybridoma cell line inoculated into the mouse abdominal cavity to prepare ascites,the hTNF-α McAbs with the purity 〉95% were obtained by saturated ammonium sulfate precipitation and protein G affinity column purification, ELISA were used to identify their binding specioficity, immunoglobin subtype, MTT assay was used to detect the inhibition of McAbs on the cell mediated cytotoxicity of hTNF-α to L929 cells , and McAbs inhibitory activity of hTNF-α mediated apoptosis on L929 cells was evaluated by FACS assay. Results Hybridoma cell line secreting hTNF-α（XBIO） was prepared ,the subtype was IgG2b;XB10 antibody showed higher inhibitory activity in hTNF-α mediated cytotoxicity and apoptosis on L929 cells with a dose-dependent fashion. Conclusion Hybridoma cell line secreting hTNF-α antagonistic monoclonal antibody can be prepared and the antibodies which screened can against the hTNF-α specially, this study set up the experimental foundation for the construction of clinical anti-hTNF-α antibody.

关 键 词：人肿瘤坏死因子-Α 单克隆抗体 细胞凋亡 细胞毒性 拮抗剂 

分 类 号：R392.11[医药卫生—免疫学]