抗狂犬病毒单克隆抗体中残留宿主细胞蛋白ELISA定量检测方法的验证

Validation of quantitative ELISA method in determination of residual host cell proteins in anti-rabies virus monoclonal antibody

目的验证抗狂犬病毒单克隆抗体(单抗)中残留宿主细胞蛋白(host cell protein,HCP)ELISA定量检测方法。方法使用商用中国仓鼠卵巢细胞HCP残留ELISA检测试剂盒,测定2种针对不同表位的抗狂犬病毒单抗原液中的残留HCP,并验证该方法的专属性、准确度、精密度、线性、耐用性、检测限及定量限。结果2种抗狂犬病毒单抗发酵液稀释10000倍后检出的HCP含量分别为31.42和19.36 ng/ml。制剂溶液、HEK293F和E.coli培养上清液中未检出HCP。3种浓度的HCP加标供试品的加标回收率均在80%~120%之间。3种浓度的HCP加标供试品重复性检测结果及中间精密度检测结果相对标准偏差均<20%。分别对3次残余HCP检测绘制四参数标准曲线,决定系数均>0.990。在一定范围内改变孵育时间和显色时间,HCP测定结果在合理的偏差范围内。检测限和定量限分别为2和3 ng/ml。结论抗狂犬病毒单抗中HCP残留量的ELISA定量检测方法专属性、准确度、精密度、线性及耐用性良好。

Objective To verify a quantitative ELISA in determination of the residual host protein(HCP)in anti-rabies virus monoclonal antibody(mAb).Methods The commercial Chinese hamster ovary cell HCP ELISA kit was used to determine the residual HCP in the stock solution of 2 anti-rabies virus mAbs targeting different epitopes,and the specificity,accuracy,precision,linearity,robustness,limit of detection(LOD)and limit of quantitation(LOQ)of the method were verified.Results The HCP content detected after diluting the fermentation broth of 2 anti-rabies virus mAbs by 10000 times was 31.42 and 19.36 ng/ml,respectively.HCP was not detected in the preparation solution,and the culture supernatant of HEK293F and E.coli.The recoveries of the spiked samples with 3 different HCP concentrations were between 80%and 120%.The relative standard deviation of the repeatability test results and the intermediate precision test results of the spiked samples with 3 different HCP concentrations were all<20%.The four-parameter standard curves were drawn for 3 HCP residual tests respectively,and coefficient of determination were all 0.990.The HCP test results were within a reasonable deviation range when the incubation time and the developing time were changed in a certain range.The LOD and LOQ were 2 and 3 ng/ml,respectively.Conclusion A quantitative ELISA detection method for residual HCP in anti-rabies virus mAb is verified,which shows good specificity,accuracy,precision,linearity,and robustness.