重组NP蛋白抗原检测SIV抗体间接ELISA方法的建立  

An indirect ELISA method based on recombinant nucleoprotein(NP) for detection of antibodies to swine influenza virus(SIV)

作　　者：王方昆[1,2] 袁秀芳[1] 刘思当[2] 张存[1] 徐丽华[1] 王一成[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [2]山东农业大学动科院,山东泰安272018

出　　处：《浙江农业学报》2008年第6期408-413,共6页Acta Agriculturae Zhejiangensis

基　　金：浙江省科技计划重点项目(2004C22044)

摘　　要：用纯化的H9N2亚型猪流感可溶重组NP蛋白作为包被抗原,建立了检测猪流感抗体的间接ELISA方法。ELISA的最佳工作条件是:抗原包被浓度为12.5μg/mL,37℃1 h,4℃过夜;血清稀释度为1∶40;酶标SPA稀释度为1∶10 000,37℃温育40 min,底物显色37℃15 min。经重复性试验、交叉试验、竞争抑制试验等试验,结果表明该方法特异性强、灵敏度高、重复性好。利用TG-ROC软件确定了自制ELISA(NP-ELISA)酶标板的临界值为0.20,特异性和敏感性分别为95.83%和91.43%。与美国IDEXX试剂盒相比较,符合率为94.00%,无显著性差异。用已建立的ELISA方法检测临床血清样本356份,总阳性率为35.40%。The soluble SIV recombinant nucleoprotein（NP） was expressed by Escherichia. coli and purified through native conditions purification method. Using purified soluble nucleoprotein, an indirect ELISA for detection of anti-SIV antibodies was developed and its optimal reaction conditions were determined： coating antigen concentration was 12.5 μg/ml, at 37℃ for 1 h and 4℃ overnight, serum samples were diluted to 1：40, Pemxidase-Labeled SPA（ 1 ： 10000） was incubated at 37℃ for 40 min, the substrate for showing color was inc, nbated at 37℃ for 15 rain. The ELISA assay was confirmed to have a good, specificity and sensitivity by repeatability, Cross test and competitive-inhibition test. The critical OD values for the ELISA was 0.20, which was determined by using the Two Graph-ROC functions of the CMDT software package, and it was found that the specificity and sensitivity of the ELISA were 95.83 % and 91.43%, respectively. And compared with the IDEXX Test Kit, the accordant is about 94.00% ,which showed no significant difference between the two assays. In addition, 356 serum samples collected from different swine farms were detected by the developed assay, it showed that the positive rate of antibody against SIV was 35.40%.

关 键 词：猪流感 重组NP蛋白 ELISA 

分 类 号：S828[农业科学—畜牧学]