摘要
目的 建立一种针对水痘-带状疱疹病毒(varicella-zoster virus,VZV)-ORF7蛋白的双抗体夹心ELISA检测方法,并进行验证,以用于水痘减毒活疫苗Oka-7S株研究工艺中各阶段样品ORF7抗原的检测。方法 用重组pET-28a-ORF7质粒诱导表达VZV-ORF7原核蛋白,经Ni^(2+)亲和层析柱进行纯化;以ORF7原核蛋白为免疫原,采用小鼠杂交瘤融合技术,筛选单抗杂交瘤细胞株,制备单抗,间接ELISA法检测抗体效价,Western blot法鉴定抗体特异性;叠加ELISA试验分析单抗抗原表位,经抗体配对筛选建立VZV-ORF7抗原的双抗体夹心ELISA检测方法,对方法的灵敏度、特异性和重复性进行验证;用建立的方法检测3批VZV Oka-7S株收获物。结果 共获得4株稳定分泌抗VZVORF7的杂交瘤细胞株,分别命名为M2B5、M2G5、8C2和1C4,细胞培养上清效价均为10~4~10~5,腹水效价均为10~6~10~7;纯化的单抗在还原和非还原状态纯度均约为97%。4株单抗均可与VZV全病毒蛋白和纯化的ORF7原核蛋白发生特异性结合;确定M2G5为捕获抗体,M2B5-HRP为酶标抗体,二者最佳工作浓度分别为4μg/mL和1∶8 000;确定临界值为A_(450)=0.111,A_(450)≥0.111判定为阳性,<0.111判定为阴性。本方法标准曲线范围为3.9~250 ng/mL,拟合方式为四参数,标准曲线方程y=(0.049 5-2.24)/(1+x/97.8)^(1.27)+2.24,相关系数(R^(2))为0.999,最低检测限为2.2 ng/mL;与四价流感病毒裂解疫苗、腮腺炎病毒、麻疹病毒均无交叉反应;批间及批内CV均<10%。3批VZV Oka-7S株检测结果均为阴性,符合率为100%。结论 建立的VZV-ORF7双抗体夹心ELISA法灵敏度高,特异性好,重复性高,适用于VZV-ORF7蛋白以及VZV Oka和Oka-7S株的快速检测。
Objective To develop a double-antibody sandwich ELISA for the detection of varicella-zoster virus(VZV)-ORF7 protein,and verify the method for the detection of ORF7 antigen across manufacturing stages of live attenuated varicella vaccine Oka-7S strain.Methods Recombinant plasmid pET-28a-ORF7 was used to induce prokaryotic expression of VZV-ORF7 protein,which was then purified by Ni2 affinity chromatography column.Using ORF7 prokaryotic protein as the immu-nogen,the hybridoma cell lines were screened by mouse hybridoma fusion technique,and the monoclonal antibodies(mAbs)were prepared.The antibody titer was detected by indirect ELISA,and the antibody specificity was identified by Western blot.The antigenic epitopes of mAbs were analyzed by ELISA,and the double-antibody sandwich ELISA for VZV-ORF7 antigen was developed by antibody pairing screening.The sensitivity,specificity and repeatability of the method were veri-fied,and three batches of VZV Oka-7S harvested samples were detected by using the developed method.Results Four hybridoma cell lines steadily secreting anti-VZV-ORF7 specific antibodies,named M2B5,M2G5,8C2 and 1C4,were produced.The antibody titers of culture supernatant and mouse ascites were 10*-10 and 10°-10',respectively.The purity of purified mAbs was both about 97%in reduced and non-reduced states.All the four mAbs exhibited specific binding to VZV whole virus protein and purified ORF7 prokaryotic protein.M2C5 was selected as the capture antibody and M2B5-HRP as the enzyme-labeled antibody,with the optimum working concentrations of 4μg/mL and 1:8000,respectively.The cutoff value was 0.111,and when A_(450)≥0.111,it was determined as positive and<0.111 as negative.The linear range of this method was 3.9-250 ng/mL with a four-parameter fit,and the standard curve equation was y=(0.0495-2.24)/(1+x/97.8)^(1.27)+2.24,R^(2)=0.999,with the limit of detection of 2.2 ng/mL.There was no cross-reaction with tetravalent influenza virus split vaccine,mumps virus and measles virus.In addition,the intra-assay and inter-assay CVs of repeatability were all less than 10%.Three batches of VZV Oka-7S strains were all detected to be negative,and the coincidence rate was 100%.Conclusion The developed double-antibody sandwich ELISA against VZV-ORF7 has high sensitivity,good specificity and high repeatability,and can be used for the rapid detection of VZV-ORF7 protein and VZV Oka and Oka-7S strains.
作者
王梦涵
侯安奇
谭仕明
董翔
李爽
张夫坤
WANG Menghan;HOU Anqi;TAN Shiming;DONG Xiang;LI Shuang;ZHANG Fukun(Changchun Keygen Biological Products Co.,Ltd.,Changchun 130000,Jilin Province,China)
出处
《中国生物制品学杂志》
2025年第6期731-737,745,共8页
Chinese Journal of Biologicals
基金
吉林省与中国科学院科技合作高技术产业化专项资金项目(2020SYHZ0010)。
关键词
水痘-带状疱疹病毒
ORF7蛋白
单克隆抗体
酶联免疫吸附试验
Varicella-zoster virus(VZV)
ORF7 protein
Monoclonal antibodies(mAbs)
Enzyme-linked immunosorbent assay(ELISA)
