重组N蛋白间接ELISA检测猪繁殖与呼吸综合征病毒抗体

An indirect ELISA based on recombinant nucleoprotein for detection of antibodies to porcine reproductive and respiratory syndrome virus

用纯化的猪繁殖与呼吸综合征病毒重组 N蛋白作为包被抗原 ,建立了检测猪繁殖与呼吸综合征病毒抗体的间接EL ISA方法 ,并确定了 EL ISA最佳工作条件 :抗原包被浓度为 0 .2 7μg/ m l,37℃ 1h加 4℃过夜 ,血清 (1∶ 4 0 )和酶标兔抗猪Ig G(1∶ 4 0 0 )分别在 37℃温育 30 m in,底物溶液 37℃显色 15 min。经重复性试验、交叉试验、阻断试验等试验结果表明该方法重复性好、特异性强、灵敏度高 ;与美国 IDEXX试剂盒相比较 ,特异性和敏感性分别为 97.6 %和 92 .1% ,无显著性差异。用已建立的方法检测临床血清样本 187份 ,总阳性率为 30 .5 %。

PRRSV The recombinant nucleoprotein was expressed by E.coli and purified through high-salt washing method.Using purified nucleoprotein, an indirect ELISA for detection of anti-PRRSV antibodies was developed and its optimal reaction conditions were determined: coating antigen for 37℃1 hour and 4℃ overnight at a concentration of 0.27 μg/ml, serum sample(1∶40) and HRP labeled anti-porcine IgG being incubated at 37℃for 30 min, the substrate for ELISA being incubated at 37℃ for 15 min. The ELISA assay was confirmed to have a good reiterativity, specificity and sensitivity by reiterativity test. cross test and blocking test. And Compared with the IDEXX Test kit, the specificity and sensitivity of the ELISA is 97.6% and 92.1% respectively, which showed no significant difference between the two assays. In addition, 187 serum samples collected from swine farms were detected by the developed assay ,it was showed that the positive rate was 30.5%.for antibody against PRRSV.