口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立  

Development of a sandwich ELISA for detecting 3AB non-structural protein of foot-and-mouth disease virus

作　　者：付元芳[1] 何伟[1,2] 孙普 杨林[1] 包慧芳 曹轶梅[1] 白兴文[1] 李平花[1] 李冬[1] 陈应理[1] 刘磊[2] 卢曾军[1] 刘在新[1] Yuanfang Fu;Wei He;Pu Sun;Lin Yang;Huifang Bao;Yimei Cao;Xingwen Bai;Pinghua Li;Dong Li;Yingli Chen;Lei Liu;Zengjun Lu;Zaixin Liu(State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences(CAAS),Lanzhou 730046,Gansu,China;College of Veterinary Sciences,Gansu Agricultural University,Lanzhou 730070,Gansu,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《生物工程学报》2020年第11期2357-2366,共10页Chinese Journal of Biotechnology

基　　金：国家重点研发计划项目(No.2016YFD0501500);宁夏回族自治区重点研发计划(No.2019BBF02005)资助。

摘　　要：抗原纯净度是口蹄疫(Foot-and-mouth disease,FMD)灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白(Non-structural protein,NSP)抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP 3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒(Foot-and-mouth disease virus,FMDV)NSP 3A单克隆抗体和辣根过氧化物酶(Horseradish peroxidase,HRP)标记的3B单克隆抗体,建立定量检测NSP 3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值(OD)的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7 ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0 ng/mL之间;而纯化后的病毒抗原中3AB蛋白残留量低于最低检测限;33份来自不同厂家的成品疫苗抗原中9份疫苗抗原3AB蛋白含量在9.0–74.0 ng/mL之间,其余24份疫苗抗原中3AB蛋白残留量低于最低检测限。检测3AB蛋白含量的双抗体夹心ELISA方法能够特异、敏感地检测疫苗抗原中的3AB蛋白含量,为疫苗质量控制与纯净度检验提供了一种可供选择的检测方法。Antigenic purity is important for quality control of the foot-and-mouth(FMD)whole virus inactivated vaccine.The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein(NSP)3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine.In this study,we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen,to provide a reference to evaluate the antigenic purity of FMD vaccine.Monoclonal antibody(Mab)of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD.Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve.The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0.Results:The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL.The correlation coefficient R2 is greater than 0.99,and the lowest detectable limit is 4.7 ng/mL.The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains,whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit.The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL,whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens.Conclusion:the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD,which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.

关 键 词：口蹄疫 非结构蛋白 定量ELISA 疫苗抗原纯净度 

分 类 号：S852.65[农业科学—基础兽医学]