柯萨奇病毒A组6型抗原含量双抗体夹心ELISA检测方法的建立、验证及初步应用  

Development,verification and preliminary application of a quantitative double antibody sandwich ELISA for Coxsackievirus A6 antigen

作　　者：鲁卫卫 刘善茹 郭会杰 郝春生 王潇潇 吴蕴怡 张中洋 李秀玲 LU Weiwei;LIU Shanru;GUO Huijie;HAO Chunsheng;WANG Xiaoxiao;WU Yunyi;ZHANG Zhongyang;LI Xiuling(Laboratory 2,National Vaccine&Serum Institute,Beijing 101lll,China;不详)

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201401

出　　处：《中国生物制品学杂志》2024年第11期1341-1348,共8页Chinese 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(Coxsackievirus A,CV-A)6型抗原含量双抗体夹心ELISA检测方法,并进行验证及初步应用,以用于CV-A6生产过程和四价手足口病(hand-foot-mouth disease,HFMD)疫苗的质量控制。方法分别以纯化的CV-A6羊多抗作为包被抗体、小鼠抗CV-A6单抗作为检测抗体,进行配对筛选研究,建立CV-A6抗原定量双抗体夹心ELISA检测方法,并对试验条件进行优化。对方法的线性、范围、专属性、准确性、精密性、耐用性进行验证。采用建立的方法检测CV-A6抗原生产过程样品和成品抗原含量。结果羊多抗和单抗16D1-1E5具有中和抗体活性,其他3株单抗均无中和抗体活性;所有抗体的ELISA效价均不低于1.00×10^(5);除单抗2B6-4F9为IgG3亚型外,其他3株单抗均为IgG2a亚型。以羊多抗作为包被抗体、单抗16D1-1E5作为检测抗体进行配对,可用于方法的建立。确定包被抗体的稀释比例为1∶2000~1∶6000,检测抗体的稀释比例为1∶4000~1∶6000。建立的CV-A6抗原定量检测方法线性范围为0.664~21.263 U/mL,线性R2≥0.99;该方法仅能特异性检测CV-A6抗原,与CVA10、CV-A16和肠道病毒71型(enterovirus 71,EV-71)抗原无交叉反应,生产过程中可能存在的M199、DMEM、Vero细胞蛋白、牛血清蛋白对方法测定无干扰;该方法测定高(21.00 U/mL)、中(10.50 U/mL)、低(1.70 U/mL)浓度样品的测定值/理论值在95%~105%之间,相对标准偏差在15%以内;在0.664~21.263 U/mL范围内,线性R2≥0.99,测定值/理论值在95%~105%之间,精密性验证相对标准偏差在15%以内;该方法在不同孵育温度(35、37、39℃)下测定样品的准确性和精密性无差别。采用建立的方法检测CV-A6抗原生产过程样品及成品抗原含量,可有效反映工艺过程样品及成品抗原浓度变化趋势。结论建立了CV-A6抗原含量双抗体夹心ELISA检测方法,该方法线性、范围、专属性、准确性、精密性、耐用性均较好,可用于CV-A6抗原�Objective To develop a double antibody sandwich ELISA for the determination of the Coxsackievirus A6(CVA6)antigen content,and to verify and preliminarily apply it for the CV-A6 production process and the quality control of tetravalent hand-foot-mouth disease(HFMD)vaccine.Methods Paired antibodies were screened and double antibody sandwich ELISA was developed by using purified CV-A6 sheep polyclonal antibody as coating antibody and mouse anti-CVA6 monoclonal antibody as detection antibody.The experimental conditions were optimized,and the linearity,range,specificity,accuracy,precision and robustness of the method were verified.The developed method was used to detect the antigen content of CV-A6 antigen production process samples and final products.Results Both of the sheep polyclonal antibody and monoclonal antibody 16D1-1E5 had neutralizing antibody activity,while the other three monoclonal antibodies showed no neutralizing antibody activity.The ELISA titers of all antibodies were not lower than 1.00×10^(5).The monoclonal antibody 2B6-4F9 was IgG3 subtype,while the other three monoclonal antibodies were IgG2a subtype.Using sheep polyclonal antibody as coating antibody and monoclonal antibody 16D1-1E5 as detection antibody for pairing,the method was developed.The dilution ratios of coating antibody and detection antibody were 1∶2000-1∶6000 and 1∶4000-1∶6000 respectively.The linear range of the developed method was 0.664-21.263 U/mL,with the R2≥0.99.The method could only detect CVA6 antigen specifically and had no cross-reaction with CV-A10,CV-A16 and enterovirus 71(EV-71)antigens.M199,DMEM,Vero cell protein and bovine serum protein probably existing in the production process had no interference with this method.The measured/theoretical values of samples with high(21.00 U/mL),medium(10.50 U/mL)and low(1.70 U/mL)concentrations were between 95%and 105%,with the RSDs within 15%.In the range of 0.664-21.263 U/mL,the R2was not less than 0.99,the measured/theoretical values were between 95%and 105%,and the RSD

关 键 词：柯萨奇病毒A组6型 抗原含量 酶联免疫吸附测定 四价手足口病疫苗 

分 类 号：R373.2[医药卫生—病原生物学]