抗重组人白细胞介素12单克隆抗体的制备及其生物学特性  

Production and biological characteristics of monoclonal antibodies against recombinant human interleukin-12

作　　者：李俐[1] 马颖哲[1] 张桂荣[2] 

机构地区：[1]吉林大学基础医学院生物化学与分子生物学实验中心,吉林长春130021 [2]吉林大学基础医学院生物化学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2007年第4期621-625,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30570688)

摘　　要：目的:以纯化的重组人白细胞介素12(rhIL-12)蛋白为抗原,制备具有生物活性的rhIL-12单克隆抗体(McAb),并对其特性进行研究鉴定,为肿瘤及免疫相关性疾病的诊断与治疗提供具有实用价值的研究工具。方法:以纯化的rhIL-12p70免疫BALB/C小鼠,采用杂交瘤技术,用间接ELISA法筛选抗体阳性的杂交瘤细胞及多次克隆化,培养制备杂交瘤细胞系,用clutathione sepharose 4B进行纯化。采用间接ELISA及Western blotting等方法对McAb的Ig亚类(型)腹水效价及特异性进行鉴定。结果:建立了3株持续分泌rhIL-12p70McAb的杂交瘤细胞株,3株细胞染色体数目均为100条左右,证实为杂交瘤细胞,所分泌的抗体为2株IgG1亚类及1株IgG2a亚类,轻链属k型,小鼠腹水效价测定分别为1∶5.9×106、1∶2.9×107、1∶3.6×107,亲和力依次为EM5>EM6>EV9。结论:成功地制备了3株能稳定分泌抗rhIL-12p70的杂交瘤细胞株,产生的McAb特异性好,亲和力高。Objective To produce hybridoma cell lines of monoclonal antibodies （McAb） against recombinant human interleukin-12 （rhIL-12） by purified rhIL-12 and identify the specificity of anti-rhIL-12 McAb, and provide a valuable tool for the diagnose and treatment of tumor and immune associated disease . Methods BALB/C mice were immunized with purified rhIL-12p70 and hybridomas were generated with traditional technique. McAb were screened by ELISA with limited dilution and subcloning approach and purified with clutathione sepharose 4B affinity chromatography column. The subtype and titers in the ascites and specificity of McAb were identified by kit and Western blotting respectively. Results Three cell lines of hybridoma constantly secreted McAbs against rhIL-12p70. The chromosomal number of three strains were about 100 and cell lines of hybridoma had been confirmed. Two of them belonged to IgG1 and one of them belonged to IgG2a, Kappa isotypes. Ascites titers were 1 ： 5.9×10^6 , 1 ： 2.9×10^7 , and 1 ： 3.6×10^7. The affinity were EM5 〉 EM6 〉 EV9 in proper order. Conclusion Three hybridoma cell strains are constructed successfully which stably secrete high-titer and highspecific McAb against rhIL-12.

关 键 词：白细胞介素12 单克隆抗体 杂交瘤 

分 类 号：R-332[医药卫生]