PRRSV血清抗体间接ELISA检测方法的建立

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

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。

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 coating 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 Dpos＋0. 57 Dneg. A total of 364 serum samples originating from 14 swine herds were detected by this recombinant nucleocapsid proteinbased 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 diagnostics, epidemiological surveys and outbreak investigations.