用于构建抗人TNF嵌合抗体的鼠单克隆抗体中和活性的鉴定  

Characterization of neutralizing activity of murine monoclonal antibodies for construction of anti-human TNF chime- ric antibody

作　　者：朱参胜[1] 欧阳为明[1] 刘雪松[1] 曹云新[1] 赵宁[2] 金伯泉[1] 

机构地区：[1]第四军医大学基础部免疫学教研室,陕西西安710032 [2]第四军医大学科研部生物技术中心,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2003年第6期576-579,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No.39870672);军队医药卫生科研基金重点项目(No.012080)

摘　　要：目的:筛选构建抗人TNF嵌合抗体所需高质量的鼠源性mAb。方法:从一组分泌抗人TNF mAb杂交瘤细胞中,挑取3株D2、E6和F6,按常规方法制备腹水。用饱和硫酸铵盐析后,分别用蛋白A亲和层析和QFF阴离子交换层析法,纯化D2株分泌的mAb(IgG2b)和E6和F6株分泌的mAb(均为IgGl)。纯化前后,3株mAb的效价,采用间接ELISA法测定。纯化的3株mAb的中和活性,通过它们对TNF诱导的L929细胞死亡和上调ECV304细胞上ICAM-1表达的抑制作用来进行鉴定。结果:间接ELISA检测表明,3株mAb D2、E6和F6纯化前的效价分别为1×10-6、1×10-7和1×10-7,纯化后效价分别为0.01、0.002和0.002 mg/L。mAb中和活性的检测表明,浓度为0.16、0.40和0.50 mg/L的D2、E6和F6,可保护2×104U/L TNF诱导的L929细胞半数不发生死亡。1.0 mg/L的mAb D2、E6和F6,对2×105U/L TNF使ECV304细胞上ICAM-1表达上调的抑制率,分别为70.1％、60.1％和60.1％;3株mAb的浓度降到0.1 mg/L时,抑制率仍可达到60.1％、53.7％和59.0％。结论:所选3株mAb的效价及中和活性均较高,可作为抗人TNF嵌合抗体的候选mAb。AIM: To isolate high-quality murine monoclonal antibodies (mAb) for construction of anti-human TNF chimeric antibod-y. METHODS: 3 hybridoma cells D2, E6 and F6 were selected from one group of hybridoma cells secreting anti-human TNF mAbs and then ascitic fluids were prepared according to routine protocol. After salted out with saturated ammonium sulphate, the mAb (lgG2b) secreted by D2 cells was purified through protein A affinity chromatography column. The mAbs (IgG1) secreted by E6 and F6 cells were purified through QFF anion exchange chromatography column. The titers of 3 mAbs were detected by indirect ELISA before and after purification. Inhibition of TNF-in-duced cell death of L929 cells and ICAM-1 upregulation on ECV304 cells were assayed as indexes of neatralizing activity of the mAbs. RESULTS: ELISA results showed that the tilers of 3 mAbs D2, E6, and F6 before purification were 1 × 10 -6, 1 ×10-7, and 1 ×10-7, respeclively, and 0.01, 0.002 and 0.002 mg/L after purificalion, respeclively. 0.16 mg/L D2, 0.40 mg/L E6, or 0. 50 mg/L F6 could prolecl 50% L929 cells from cell dealh induced by 2 × 104 U/L TNF, and the inhibilion rales of ICAM-1 upregulalion by 2 × 105 U/L TNF of 1.0 mg/L D2, E6 or F6 were 70.1%, 60.1%, and 60.1%, re- spectively. The inhibition rates still reached 60.1%, 53.7% and 59.0%, respectively, when the concentrations of 3 mAbs D2, E6 and F6 decreased to 0.1 mg/L. CONCLUSION: 3 mAbs with high liters and neutralizing activities were isocated, which paves the way for construction of anti-human TNF chimeric antibody.

关 键 词：TNF MAB 中和活性 嵌合抗体 

分 类 号：R392.11[医药卫生—免疫学]