PRRSV血清抗体间接ELISA检测方法的建立

用亲和层析纯化的猪繁殖与呼吸综合征病毒(PRRSV)重组核衣壳蛋白GST-N作为诊断抗原,建立检测PRRS血清抗体的间接ELISA方法。抗原最佳包被量为每孔400 ng,待检血清最佳稀释度为1∶100,待检血清样品的D_(490 nm)≥0．43D_(po■) 0．57D_(neg)判为阳性,反之判为阴性。对采自14个猪场的364份猪血清样品进行检测,PRRS阳性率为51．4%,略高于HerdChek ELISA的阳性检出率49．7%。与HerdChek ELISA相比,间接ELISA的敏感性为94．5%,特异性为91．3%,总符合率为92．9%,Youden指数为0．86。同时还证明该方法具有良好的批间和批内重复性,并且不与猪瘟、猪伪狂犬病等阳性血清发生反应。该方法的成功建立将为我国PRRS的防制工作作出贡献。

Development of a recombinant nucleocapsid protein-based indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus in swine sera

Using recombinant nucleocapsid protein GST-N of porcine reproductive and respiratory syndrome virus (PRRSV)as antigen,an indirect ELISA for the detection of antibodies against PRRSV was developed.The optimal coat- ing concentration of antigen was determined by checkerboard titration and amounted to 400 ng of GST-N fusion protein per well,the serum sample for testing was diluted to 1:100 for detection and the cutoff was determined to be 0.43 D_(pos) 0.57 D_(neg).A total of 364 serum samples originating from 14 swine herds were detected by this recombinant nucleocapsid protein- based indirect ELISA,and the positive rate was 51.4%,slightly higher than that(49.7%)detected by HerdChek ELISA. Comparing this indirect ELISA with HerdChek ELISA kit,its sensitivity and specificity were 94.5%,91.3 % respectively, the correlation between these two ELISA methods was 92.9%,and the Youden index was 0.86.All data in this study demonstrated that the recombinant nucleocapsid protein-based indirect ELISA would be a very useful tool for routine diag- nostics,epidemiological surveys and outbreak investigations.