重组M蛋白间接ELISA检测猪繁殖与呼吸综合征病毒抗体

用纯化的猪繁殖与呼吸综合征病毒重组M蛋白作包被抗原,建立了检测猪繁殖与呼吸综合征病毒抗体的间接ELISA方法,并确立了ELISA最佳工作条件:抗原包被浓度为3.5μg/mL,37℃1h加4℃过夜,血清(1:40)和酶标SPA(1:80)分别在37℃温育1h,加底物溶液常温显色5min。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、敏感度高;与美国IDEXX公司试剂盒相比较,特异性和敏感性分别为96.3%和93.5%,无显著性差异。用建立的方法检测临床血清样品168份,总阳性率为39.9%。

An indirect ELISA based on recombinant matrix membrane protein for detection of antibody to porcine reproductive and respiratory syndrome virus

PRRSV recombinant matrix membrane protein was expressed by E.coli and purified by simple method.Using the purified matrix membrane protein,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 3.5μg/ml,serum sample(1:40) and HRP labeled SPA(1:80) being incubated at 37℃ 1 hour,the substrate OPD for ELISA reacted at common temperature for 5 minutes.The ELISA assay was confirmed to have a good reproducibility,specificity and sensitivity by reproducibility test cross test and blocking test.And compared with the IDEXX test kit,the specificity and sensitivity of the ELISA is 96.3% and 93.5% respectively,suggesting no significant difference between the two assays.In addition,168 serum samples collected from swine farms were detected by the developed assay,it was shown that the positive rate was 39.9% for antibody against PRRSV.