森林脑炎病毒单克隆抗体的研制及抗原定量双抗体夹心ELISA检测方法的建立和应用  

Preparation of monoclonal antibody against tick-borne encephalitis virus and development and application of a double antibody sandwich ELISA method for quantitative antigen determination

作　　者：吴月 韩顺子 唐剑光 张秀霞 吴晓娟 孙宏亮 曹玉锋 常军亮 邹勇 WU Yue;HAN Shun-zi;TANG Jian-guang;ZHANG Xiu-xia;WU Xiao-juan;SUN Hong-liang;CAO Yu-feng;CHANG Jun-liang;ZOU Yong(Changchun Institute of Biological Products Co.,Ltd.,Changchun130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2021年第8期963-968,973,共7页Chinese Journal of Biologicals

基　　金：吉林省省级医药健康产业发展专项基金项目(20170311010YY)。

摘　　要：目的制备森林脑炎病毒(tick-borne encephalitis virus,TBEV)单克隆抗体,并建立TBEV抗原定量双抗体夹心ELISA检测方法,初步应用于疫苗生产过程中TBEV抗原含量监测。方法以TBEV"森张"株灭活纯化全病毒作为免疫原免疫BALB/c小鼠,制备小鼠单抗杂交瘤及腹水抗体,纯化后鉴定单抗的特异性及相对亲和力。经单抗叠加ELISA配对,建立双抗体夹心ELISA病毒抗原检测方法。以TBEV抗原标准品作为定量标准,建立剂量-反应曲线,验证该方法的敏感度、线性、特异性、准确性、试验内与试验间精密性,对5批TBEV灭活疫苗原液进行检测,初步验证方法的适用性。结果制备了4株抗TBEV杂交瘤细胞株:5F5、2G12、5E1及2H9;间接ELISA法测定腹水抗体效价为1︰106~1︰10~7,Western blot鉴定4株单抗均与TBEV包膜E蛋白发生特异性反应;4株单抗相对亲和力:2G12> 5F5> 2H9> 5E1;抗体配对筛选后,将5F5作为包被抗体,工作浓度为10μg/mL;2G12作为标记抗体,稀释倍数为1︰3 200倍。该方法对TBEV抗原最低检出限为0.009 8μg/mL;线性范围为0.078 1~2.5μg/mL,R~2> 0.98;检测TBEV疫苗生产相关原辅料成分无交叉反应;准确性验证病毒抗原回收率在96.2%~109.2%之间;试验内和试验间精密性变异系数分别在7.37%~8.40%、8.66%~9.38%之间;用该方法检测5批TBEV灭活疫苗原液呈剂量依赖关系。结论成功制备了TBEV小鼠单克隆抗体,并建立了TBEV抗原定量双抗体夹心ELISA检测方法,该方法检测TBEV抗原准确可靠,为TBEV灭活疫苗研发及生产过程中病毒抗原检测提供了一种简便有效的监测方法。Objective To prepare the monoclonal antibody(mAb)against tick-borne encephalitis virus(TBEV),develop a double-antibody sandwich ELISA method for quantitative determination of TBEV antigen and preliminarily apply to the monitoring of TBEV antigen content during vaccine production.Methods BALB/c were immunized with inactivated and purified whole virus of TBEV“Senzhang”strain as immunogen,and the prepared mouse mAb was purified and identified for specificity and relative affinity.A double antibody sandwich ELISA method for virus antigen detection was developed by antibody pairing through additive ELISA.Using the TBEV antigen standard as a quantitative standard,a dose-response curve was plotted and used for verification of sensitivity,linearity,specificity,accuracy,intra-and inter-test precisions of the developed method.Five batches of bulks of TBEV vaccine were determined to preliminarily verify the applicability of the method.Results Four hybridoma cell lines against TBEV,5F5,2G12,5E1 and 2H9,were prepared,by which the secreted mAbs were at titers of 1∶106~1∶107 in ascites.Western blot showed specific reactions of all the four mAbs with TBEV envelope E protein.In the turn of relative affinity,the four mAbs were 2G12,5F5,2H9 and 5E1.After antibody pair screening,5F5 was used as the coating antibody at a working concentration of 10μg/mL,while 2G12 as the labeled antibody at a dilution of 1∶3200.The minimum detection limit of the developed method for TBEV antigen wa s0.0098μg/mL,while the linear range was 0.0781~2.5μg/mL,with a R2 value of more than 0.98.There was no cross reaction with the raw and auxiliary materials related to TBEV vaccine production.The virus antigen recovery rate in verification for accuracy was 96.2%~109.2%,while the coefficients of variation(CVs)of precision in intra-and interassays were 7.37%~8.40%and 8.66%~9.38%respectively.The determination results of five batches of bulks of TBEV vaccine were dose-dependent.Conclusion Mouse mAb against TBEV was successfully prepared,and a double

关 键 词：森林脑炎病毒 单克隆抗体 双抗体夹心ELISA 抗原定量检测 

分 类 号：R373.31[医药卫生—病原生物学]