抗肿瘤坏死因子-α单克隆抗体体外生物学活性检测方法的优化及验证  

Optimization and verification of in vitro biological activity assay for monoclonal antibody against tumor necrosis factor-α

作　　者：潘勇兵[1] 张雅婷[1] 张银川[1] 宋桂芝[1] 桂芳[1] 闭兰[1] 

机构地区：[1]武汉生物制品研究所有限责任公司抗体研究室,湖北武汉430207

出　　处：《中国生物制品学杂志》2015年第11期1202-1205,1210,共5页Chinese Journal of Biologicals

基　　金："十二五""重大新药创制"科技重大专项原创性全人源单克隆抗体药物研发技术平台的建立及应用(2011ZX09506-005)

摘　　要：目的优化抗肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)单克隆抗体体外生物学活性的检测方法,并进行验证。方法对TNF-α单克隆抗体体外生物学活性检测方法中的结晶紫染色液浓度、细胞浓度、放线菌素D(Act D)浓度及TNF-α杀伤浓度进行优化,并对该方法的精密度及专属性进行验证。绘制质量控制图,对抗TNF-α单克隆抗体样品进行20次重复检测。结果结晶紫染色液、细胞浓度、Act D和TNF-α的最适浓度分别为0.2%、1.5×105个/ml,1 ng/ml和0.8μg/ml。同一人员于不同日6次重复测定参考品的半数有效浓度(ED50)均值为(44.18±6.858)ng/ml,相对标准偏差(relative standard deviation,RSD)为15.522%;不同日由3位不同人员重复测定参考品的ED50均值为(41.19±3.05)ng/ml,RSD值为7.40%;在TNF-α浓度一定的情况下,随着抗TNF-α单克隆抗体和阳性对照Humira浓度的降低,细胞的存活率随之降低,其他抗体无此现象。抗TNF-α单克隆抗体样品的20次测定结果均成立。结论成功优化了抗TNF-α单克隆抗体体外生物学活性检测方法的条件,该方法具有良好的精密度及专属性。Objective To optimize and verify the in vitro biological activity assay for monoclonal antibody（Mc Ab）against tumor necrosis factor（TNF）-α.Methods The concentrations of crystal violet as a staining solution,cells,actinomycin D（Act D）and TNF-α in in vitro biological assay for Mc Ab againt TNF-α were optimized,and the assay was verified for precision and specificity,based on which a quality control chart was drawn,and the samples of Mc Ab against TNF-α were tested for 20 times.Results The optimal concentrations of crystal violet,cells,Act D and TNF-α were0.2%,1.5 × 105 cells / ml,1 ng / ml and 0.8 μg / ml respectively.The mean ED50 of reference determined for 6 times by one staff on various working days was（44.18 ± 6.858） ng / ml,with a relative standard deviation（RSD） of 15.522%.However,the mean ED50 determined by three staff on various working days was（41.19 ± 3.05） ng / ml,with a RSD of7.40%.At a specified TNF-α concentration,the survival rate of cells decreased with the decreasing concentrations of anti-TNF-αMc Ab and Humira as positive control,which was not observed in the cells added with other antibodies.All the20 test results of samples of Mc Ab againt TNF-α were valid.Conclusion The condition for in vitro biological activity assay for Mc Ab against TNF-α was successfully optimized,and the assay showed high precison and specificity.

关 键 词：肿瘤坏死因子-Α 单克隆抗体 生物学活性 

分 类 号：R392-33[医药卫生—免疫学]