摘要
目的建立水痘-带状疱疹病毒(varicella-zoster virus,VZV)糖蛋白E(gE)的双抗体夹心ELISA检测方法,并进行验证。方法以VZV全病毒抗原为免疫原,通过小鼠杂交瘤融合技术筛选可稳定分泌抗VZV-g E抗体的杂交瘤细胞株,间接ELISA法检测小鼠腹水抗体效价,经Hi Trap^(TM)Mabselect^(TM)Su Re和Hi Trap^(TM)Desalting纯化后,12%SDS-PAGE分析单克隆抗体纯度,Western blot法检测特异性,小鼠单克隆抗体分型试剂盒鉴定亚型。经表位叠加试验筛选捕获抗体及酶标抗体,棋盘滴定法确定捕获抗体(0.25、0.5、1、2、5和10μg/m L)和酶标抗体(1∶500、1∶1000、1∶2000、1∶5000和1∶10000稀释)工作浓度后,建立VZV-g E含量的双抗体夹心ELISA检测方法。验证方法的线性范围、准确性、精密性和特异性。采用建立的方法检测3批7 L生物反应器培养1~14 d的CHO-VZV-g E细胞培养上清中g E含量。结果共获得4株稳定分泌抗VZV-gE特异性抗体的阳性杂交瘤细胞株,命名为mAb-B2、mAb-11、K9C7和K9F4,小鼠腹水抗体效价为10^(6)~10^(7),纯化后纯度约为97%,均可与VZV全病毒蛋白发生特异性结合,轻链均为κ链,重链分别为IgG_(2b)、IgG_(1)、IgG_(2b)及Ig G_(2a)。确定mAb-B2作为捕获抗体,HPR标记的mAb-11作为酶标抗体,两者最佳工作浓度分别为1.5μg/mL和1∶5000稀释。g E抗原内部参考品浓度在1.95~1000 ng/mL范围内,与A_(450)呈良好的线性关系,四参数方程为:Y=(0.15-3.99)/[1+(X/67.4)^(1.49)]+3.99,R^(2)为0.999;准确性验证回收率为94.9%~114.0%;精密性验证变异系数(CV)均<15%;除CHO-VZV-gE细胞培养上清、水痘减毒活疫苗和带状疱疹减毒活疫苗外,其他检测样品的A_(450)均<0.15,无交叉反应。3批CHO-VZV-gE细胞培养上清中gE含量随培养时间的延长逐渐升高,且在14 d内与培养时间呈正相关(R^(2)=0.9956,P=0.0001)。结论建立的双抗体夹心ELISA法具有良好的准确性、精密性和特异性,可用于VZV疫苗中g E抗原含量的快速检测。
Objective To develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap^(TM)Mabselect^(TM)Su Re and Hi Trap^(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1000,1∶2000,1∶5000 and 1∶10000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1~14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of 10^(6)~10^(7) with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5000respectively.The internal reference concentration of gE antigen was in the range of 1.95~1000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)^(1.49)]+3.99,and R^(2)value was 0.999.The recoveries of accuracy verification were 94.9%~114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R^(2)=0.9956,P=0.0001).ConclusionThe developed double antibody sandwich ELISA had good accuracy,precision and specificity,which might be used for rapid detection of gE antigen content in VZV vaccine.
作者
张树杰
王梦涵
朱晓文
周宏达
刘洪涛
孙跃秋
夏立新
张夫坤
ZHANG Shujie;WANG Menghan;ZHU Xiaowen;ZHOU Hongda;LIU Hongtao;SUN Yueqiu;XIA Lixin;ZHANG Fukun(Changchun Keygen Biological Products Co.,Ltd.,Changchun 130000,Jilin Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第3期341-346,共6页
Chinese Journal of Biologicals
基金
吉林省科技厅发展计划项目(20200404191YY)。
关键词
水痘-带状疱疹病毒
糖蛋白E
单克隆抗体
酶联免疫吸附试验
Varicella-zoster virus(VZV)
Glycoprotein E
Monoclonal antibodies(mAbs)
Enzyme-linked immunosorbent assay(ELISA)