鼠抗肿瘤坏死因子-α单克隆抗体的制备及鉴定  

Preparation and Identification of Murine Monoclonal Antibody against TNF-α

作　　者：李萍[1] 蒋琳[1] 马亚茹[1] 鱼轲[1] 郭敏[1] 陆俭[1] 

机构地区：[1]兰州生物制品研究所第四研究室,兰州730046

出　　处：《中国生物制品学杂志》2008年第12期1090-1093,共4页Chinese Journal of Biologicals

摘　　要：目的制备高中和活性的鼠抗肿瘤坏死因子-α(TNF-α)单克隆抗体,并进行鉴定。方法用重组TNF-α免疫BALB/c小鼠,采用杂交瘤技术制备抗TNF-α单抗,纯化后,用间接ELISA法和微量细胞病变MTT法检测抗体效价,ELISA相加试验测定抗原表位,硫氰酸铵洗脱法测定单抗的相对亲和力指数,并进行类及亚类、细胞染色体核型、特异性及稳定性检测。结果筛选出7株分泌抗TNF单克隆抗体的细胞株,其中6株具有较高的中和活性和良好的稳定性,其细胞上清及腹水的中和效价分别为1∶40～1∶160和(1～3)×10-4,7株单抗均为IgG类IgG1亚类,轻链类型均为κ,细胞核型分析符合杂交瘤细胞特征,特异性良好,5H10株与其余6株有不同的抗原表位,相对亲和力指数为0.4～1.5mol/L。结论已成功制备具有高中和活性的鼠抗TNF-α单克隆抗体,为抗人TNF-α嵌合抗体的构建奠定了基础。Objective To prepare and identify the murine monoclonal antibody （McAb） against tumor necrosis factor-α（TNF-α） with high neutralizing activity. Methods BALB/c mice were immunized with recombinant TNF-α, based on which the McAb was prepared by hybridoma technique, then purified and determined for titer by indirect ELISA and MTT method, for antigenic epitopes by additive ELISA, for relative affinity index by ammonium thiocyanate elution method, and analyzed for class, subclass, specificity and stability. The cell strains secreting McAbs were analyzed for chromosome karyotype. Results Seven cell strains secreting McAb against TNF-α were screened. The McAbs secreted by 6 of the 7 strains showed high neutralizing activity and stability. All the McAbs secreted by 7 strains were IgG1 with K chain, and their neutralizing titers were 1 ： 40 - 1 ： 160 and （1 - 3） × 10^-4 in supernatant and ascites respectively. The chromosome karyotypes of the 7 cell strains were consistent with those of hybridoma cells. The antigenic epitope of McAb secreted by 5H10 strain was different from those by the other 6 strains. The relative affinity indexes of McAbs secreted by the 7 cell strains were between 0. 4 and 1.5 mol/L. Conclusion The murine McAb against TNF-α with high neutralizing activity was successfully prepared, which laid a foundation of construction of chimeric antibody against TNF-α.

关 键 词：肿瘤坏死因子-Α 单克隆抗体 中和活性 

分 类 号：R392-33[医药卫生—免疫学]