非洲猪瘟病毒抗体检测间接ELISA方法的建立

Development of an indirect enzyme-linked immunosorbent assay for detection antibody against African swine fever virus

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。

In this study,a VP73 recombinant protein-coated indirect ELISA（i-ELISA）for the detection of antibodies to ASFV in porcine sera was developed.The sensitivity assay showed that the maximum dilution of ASFV positive serum for detection was 2 560-fold,which is consistent with results of the commercial ELISA kit.The i-ELISA showed excellent specificity to anti-ASFV antibodies in porcine sera,but negative for the positive samples of APP,PRV,CSFV,FMDV and PRRSV.The coefficient of variation of each sample tested was less than 10% in this assay.The kit can be stable under at 37℃ for 5 days,and the results of each sample showed no big difference.A total of 150 serum samples were detected by the method and the commercial ELISA kit,respectively.Compared with the commercial ELISA kit,the specificity and sensitivity of the iELISA were 99.1% and 94.3%,respectively.The coincidence rate between the two methods was 98%.Above all,the method,with excellent specificity,sensitivity,repeatability and stability can be used for clinical testing.