水痘-带状疱疹病毒糖蛋白E含量双抗体夹心ELISA检测方法的建立及验证

Establishment and verification of double antibody sandwich ELISA for detection of content of varicella-zoster virus glycoprotein E

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.