汉城病毒L99株双抗体夹心ELISA抗原检测方法的建立及应用  

Establishment and application of sandwich ELISA for detection of specific antigen of Seoul virus L99 strain

作　　者：孙晨 唐剑光 孙宏亮 李景良 常军亮 顾建阳 Sun Chen;Tang Jianguang;Sun Hongliang;Li Jingliang;Chang Junliang;Gu Jianyang(Biotechnology Research Laboratory,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,China;Vaccine Room One,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,China;Vaccine Room Three,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,China;Production Management Department,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,China)

机构地区：[1]长春生物制品研究所有限责任公司生物技术研究室,长春130012 [2]长春生物制品研究所有限责任公司疫苗一室,长春130012 [3]长春生物制品研究所有限责任公司疫苗三室,长春130012 [4]长春生物制品研究有限责任公司生产管理部,长春130012

出　　处：《中华微生物学和免疫学杂志》2022年第3期234-240,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的:建立汉城病毒(Seoul virus, SEOV)L99株特异性双抗体夹心ELISA抗原检测方法,验证其检测性能,为双价肾综合征出血热(hemorrhagic fever with renal syndrome, HFRS)疫苗研制、生产和检定过程中Ⅱ型病毒抗原含量检测提供有效手段。方法:小鼠体内诱生法制备L99病毒单克隆抗体(monoclonal antibody, McAb)腹水,Protein-A亲和层析纯化腹水抗体,SDS-PAGE鉴定纯化抗体纯度,间接ELISA法测定McAb效价,Western blot鉴定其特异性;叠加ELISA法对4株McAb进行抗体配对。用辣根过氧化物酶(horseradish peroxidase, HRP)标记单抗后,通过正交试验优化包被和标记抗体使用浓度,建立双抗体夹心ELISA病毒抗原检测方法。以Ⅱ型HFRS疫苗标准品作为定量标准,验证方法的检测限、线性范围、专属性、准确性、精密度;通过检测3批Ⅱ型病毒疫苗灭活原液验证方法适用性。结果:4株McAb腹水效价均>1∶10 6,抗体纯度均>98%;经Western blot鉴定,1D5、3A4、5B7特异识别Ⅱ型L99株出血热病毒核衣壳蛋白,2E3与Ⅰ型PS-6株出血热病毒存在交叉反应;经抗体配对筛选后,选择3A4为包被抗体,1D5为标记抗体;棋盘滴定法确定包被抗体工作浓度为20 μg/ml,HRP标记抗体工作浓度为1∶4 000。该方法对SEOV L99抗原最低检测限为0.078 1 μg/ml;线性范围为0.078 1~2.500 0 μg/ml, R2>0.99;专属性验证其对Ⅱ型HFRS疫苗具有检测特异性;抗原回收率在95.8%~108.7%之间,精密度验证变异系数<10%。用该方法检测3批Ⅱ型HFRS灭活疫苗原液结果均呈剂量依赖性。 结论:成功建立了SEOV L99株型特异性双抗体夹心ELISA抗原检测方法,为地鼠肾双价肾综合征出血热疫苗生产及检定过程中,SEOV L99株病毒抗原含量测定提供了检测方法。Objective To establish a double antibody sandwich ELISA for detecting the specific antigen of Seoul virus(SEOV)L99 strain and to provide a means for antigen detection in the development,production and verification of vaccine against hemorrhagic fever with renal syndrome(HFRS).Methods Monoclonal antibodies(McAbs)aganist L99 virus were induced in mice using four hybridoma cell lines and purified by Protein-A affinity chromatography.The purity,titer and specificity of McAbs were determined by SDS-PAGE,indirect ELISA and Western blot,respectively.Four McAbs were paired with each other and the additivity indices of paired McAbs were analyzed.After labeling McAbs with horseradish peroxidase(HRP),the concentrations of the coated and labeled antibodies were optimized by orthogonal test,and then a double antibody sandwich ELISA for virus antigen detection was established.TypeⅡHFRS inactivated vaccine standard was used as a quantitative standard to verify the sensitivity,linearity,specificity,accuracy and precision of the developed method.The applicability of the method was verified by testing three batches of vaccine stock solutions.Results Four McAbs were at titers of greater than 1∶106 and their purity was all greater than 98%.The McAbs secreted by 1D5,3A4 and 5B7 cells could specifically recognize the nucleocapsid protein of SEOV L99.There was cross-reaction between McAb secreted by 1D5 cells and Hantaan virus PS-6.The McAbs secreted by 3A4 and 1D5 were used as coating and labeling antibodies based on the results of antibody pairs.The working concentrations of the coating antibody and the horseradish peroxidase(HRP)-labeled antibody were 20μg/ml and 1∶4000,respectively.The minimum detection limit of the established method for the detection of SEOV L99 antigen was 0.0781μg/ml,and the linear range was 0.0781-2.5000μg/ml with a R2 value of more than 0.99.There was no cross reaction with other HFRS vaccine.The virus antigen recovery rate was between 95.8%and 108.7%,and the coefficients of variation of precision wa

关 键 词：汉城病毒 单克隆抗体 双抗体夹心ELISA 抗原检测 

分 类 号：R446.6[医药卫生—诊断学]