静注人免疫球蛋白抗体依赖细胞介导的细胞毒性作用测定方法的建立

Development of the method for determinating ADCC biological activity of IVIG

目的利用转录报告基因细胞,建立静注人免疫球蛋白(pH4)(human Immunoglobulin(pH4)for Intravenous Injection,IVIG)抗体依赖细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity,ADCC)生物学活性测定方法。方法以Jurkat-NFAT-Luc-CD16细胞作为效应细胞,PLC/PRF/5细胞作为靶细胞,将效应细胞、靶细胞和IVIG孵育后,通过检测IVIG Fc段结合效应细胞后活化T细胞核因子释放的荧光素酶,建立IVIG ADCC生物学活性检测方法,同时对试验条件进行优化,以及对该方法进行方法学验证。结果IVIG在该方法中存在量效关系,符合四参数方程。经过试验条件优化,最终确定将PLC/PRF/5细胞作为靶细胞,抗体起始稀释浓度为20 mg/mL,梯度稀释倍数为1∶2,效靶比为1∶3,效应细胞、靶细胞和IVIG共同孵育时间为24 h。三次独立检测的日内和日间起始工作浓度荧光素酶相对光单位(relative light unit,RLU)及半最大效应浓度(concentration for 50%of maximal effect,EC50)的相对标准偏差(relative standard deviation,RSD)均<11%,2个不同稀释组回收率样本相对效价分别(23.50±1.69)%,(49.30±2.97)%,对应的回收率分别为(93.50±6.30)%,(96.24±5.43)%,RSD均<11%。结论本研究利用转录报告基因细胞成功建立IVIG ADCC生物学活性检测方法,该方法具有专属性强、重复性好、准确性高等优点,可作为IVIG ADCC生物学活性检测方法。

Objective To establish a method for determinating the antigen-dependent cell-mediated cytotoxicity(ADCC)of human immunoglobulin(pH4)for intravenous injection(IVIG)on luciferase reporter gene-modified cell assay.Methods As effector cells,Jurkat-NFAT-Luc-CD16 cells were used in the assay,and PLC/PRF/5 cells were used as target cells.After incubation of effector cells and target cells with IVIG,the method for determinating ADCC biological activity of IVIG was established by detecting luciferase released by activated T nuclear factor after binding of IVIG Fc fragment to effector cells.Meanwhile,the experimental assay conditions were optimized,and the methodology was verified subsequently.Results IVIG had a dose-response relationship in this method,which was consistent with four parameter logistic model.And the PLC/PRF/5 cells were finally determined as the target cells.The initial dilution concentration of antibody was 20 mg/mL,and the ratio dilution was 1∶2,and the effector to target ratio was 1∶3,and co-incubation time of two cells and IVIG was 24 hours.Within-run and between-run analysis including three independent tests,initial working concentration relative light unit(RLU)and the relative standard deviation(RSD)of the concentration for 50%of maximal effect(EC50)were less than 11%.The relative titers of the recovery samples of the two different dilution groups were(23.50±1.69)%and(49.30±2.97)%,respectively,and the corresponding recovery rates were(93.50±6.30)%and(96.24±5.43)%,respectively,with RSD less than 11%.Conclusion The method for determinating ADCC biological activity of IVIG based on luciferase reporter gene-modified cell assay was successfully established.It could be applied in determinating the ADCC biological activity of IVIG,and has the advantages of satisfactory linearity,accuracy,precision and specificity.