柯萨奇病毒A组10型抗原双抗体夹心ELISA定量方法的建立及应用  

Establishment and application of a quantitative double-antibody sandwich ELISA for coxsackievirus A10 antigen

作　　者：鲁卫卫 郭会杰 刘善茹 郝春生 杨永娟 张中洋 李秀玲 Lu Weiwei;Guo Huijie;Liu Shanru;Hao Chunsheng;Yang Yongjuan;Zhang Zhongyang;Li Xiuling(Laboratory 2,National Vaccine and Serum Institute,Beijing 101111,China;Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]国药中生生物技术研究院有限公司第二研究室,北京101111 [2]上海生物制品研究所有限责任公司,上海200051

出　　处：《国际生物制品学杂志》2022年第6期310-315,共6页International Journal of Biologicals

基　　金：国家科技重大专项"重大新药创制"(2016ZX09101120)。

摘　　要：目的建立柯萨奇病毒A组10型(coxsackievirus A10,CV-A10)抗原双抗体夹心ELISA定量检测方法,用于CV-A10生产过程样品和四价手足口病疫苗的质量控制。方法以纯化的CV-A10抗原分别免疫绵羊和小鼠获得羊免疫血清和杂交瘤单克隆抗体(单抗)细胞株,以杂交瘤细胞株免疫小鼠制备腹水,羊血清和小鼠腹水分别经亲和层析后获得纯化羊抗CV-A10多克隆抗体(多抗)和小鼠抗CV-A10单抗。采用微量细胞病变法测定纯化多抗和单抗中和抗体效价。分别以纯化羊多抗作为包被抗体、小鼠单抗作为检测抗体,进行配对筛选研究,建立CV-A10抗原定量双抗体夹心ELISA;对方法的线性、专属性、准确度、精密度、范围进行验证,并对CV-A10抗原生产过程中的样品和成品进行检测。结果纯化抗CV-A10单抗和多抗均具有中和抗体活性。以多抗作为包被抗体、7株单抗作为检测抗体进行配对,其中5株单抗配对成功,选择CY166-14R作为检测抗体用于CV-A10抗原定量检测ELISA的建立;对检测方法进行优化,确定以羊多抗作为包被抗体的使用稀释比例为1∶5000~1∶10000、小鼠单抗检测抗体稀释比例1∶2000~1∶4000。建立的CV-A10抗原定量检测方法范围为0.42~10.00 U/ml,线性决定系数≥0.99;该方法仅能特异性检测CV-A10抗原,与其他抗原或物质(肠道病毒71型、CV-A6、CV-A16、M199、DMEM、Vero细胞蛋白、牛血清)无交叉反应;对高、中、低浓度样品进行测定,测定值/理论值在95%~110%之间,相对标准偏差在15%以内。结论建立了CV-A10抗原定量双抗体夹心ELISA,该方法在一定范围内的线性、专属性、准确度、精密度均较好,可用于CV-A10抗原生产过程样品和成品的质量控制,为CV-A10抗原的体外效力评价提供方法学基础。Objective To establish a quantitative double-antibody sandwich ELISA for coxsackievirus A10(CV-A10)antigen,which can be applied to the quality control of CV-A10 antigen production process samples and 4-valent hand-foot-mouth disease(HFMD)vaccine.Methods The hybridoma cell lines against CV-A10 antigen were prepared from BALB/c mice and polyclonal antibodies were prepared from sheep immunized with purified CV-A10 antigen,respectively.Monoclonal antibodies were prepared from ascites of BALB/c mice immunized with hybridoma cells.Sheep anti-CV-A10 polyclonal and mouse anti-CV-A10 monoclonal antibodies were purified by affinity chromatography.Neutralizing anibody activity was determined by micro-cytopathic assay.Double-antibody sandwich ELISA was developed using polyclonal antibody as coating antibody and monoclonal antibody as detection antibody.The method was verified for linearity,specificity,accuracy,precision,range and applied to the production process samples and final product of CV-A10 antigen.Results Both of the purified polyclonal and monoclonal antibodies have neutralizing activity.Match test was performed using polyclonal antibody as coating antibody and monoclonal antibody as detection antibody.5 of the 7 anti-CV-A10 monoclonal antibodies matched to coating polyclonal antibody.CY166-14R was used for estabiishment of quantitaive ELISA for CV-A10 antigen detection.The optimal dilution ratio of coating antibody and detection antibody were 1:5000-1:10000 and 1:2000-1:4000,respectively.The range of the developed ELISA was 0.42-10.00 U/ml,with the coefficient of determination≥0.99.The established method showed high specificity only detecting CV-A10 antigen not other antigens(enterovirus A71,CV-A6,CV-A16)and substances(M199,DMEM,Vero cell protein,newborn calf serum)in the production process.The ratio of measured values for high,medium and low concentration samples to theoretical values were 95%-110%with the relative standard deviation≤15%.Conclusions The double-antibody sandwich ELISA for quantitative determin

关 键 词：柯萨奇病毒 抗体 单克隆 酶联免疫吸附测定 手足口病 抗原 双抗体夹心法 定量分析 

分 类 号：R392.11[医药卫生—免疫学]