牛血清中口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测方法的建立

为研究快速定量检测牛血清中口蹄疫病毒非结构蛋白抗体的方法,试验通过优化抗原表达条件等步骤,在大肠杆菌原核表达系统中表达可溶性的3A-3B融合蛋白,并基于纯化的可溶性融合蛋白建立口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测试剂盒。结果表明:建立的方法能够检测牛血清中的口蹄疫病毒非结构蛋白抗体,敏感性高,特异性强,对其他相关的牛类病原无交叉反应,其组内与组间变异系数分别低于10%和15%,具有良好的重复性。对300份临床牛血清样品进行检测,同Procheck公司的口蹄疫非结构蛋白抗体试剂盒进行比较,阳性样品符合率96%,阴性样品符合率93.3%,总的符合率95.7%。重复性试验组内与组间变异系数均小于10%。文章首次建立了口蹄疫病毒非结构蛋白抗体时间分辨荧光免疫分析检测方法,同传统的ELISA方法相比,该检测方法特异性相当、敏感性更高,操作更简单、快速,具有较高的应用推广价值。

Development of Time-resolved Fluoroimmunoassay Method for Detection of Non-structural Protein Antibodies in Bovine Serum Against Foot-and-mouth Disease Virus

To establish the time-resolved fluoroimmunoassay method detection method for foot-and-mouth disease virus antibody in bovine serum,this experiment expressed the soluble 3A-3B fusion protein in Escherichia coli,through exploration of protein expression conditions.Further based on purified fusion protein,a time-resolved fluoroimmunoassay detection method for foot-and-mouth disease virus antibody was established.The results showed as follovos:this method could detect the antibodies of foot-and-mouth disease virus in bovine serum quickly and quantitatively.It had high sensitivity and specificity,and had no cross reaction to other related bovine pathogens.The coefficient of variation intra batch and inter batch were all below 10%.A total of 300 clinical serum samples were tested,and the ELISA kit(Procheck)was used for comparison.The coincidence rate of the positive samples was 96%,the coincidence rate of the negative samples was 93.3%,and the total coincidence rate was 95.7%.In this paper,a time-resolved fluorescence immunoassay for detection of non-structural protein antibodies of foot-and-mouth disease virus(FMDV) was established for the first time.Compared with the traditional ELISA method,this method has similar specificity,higher sensitivity,simpler operation and higher application value.