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

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.