猪细小病毒抗体IPMA检测方法的建立及应用

Establishment and application of immunoperoxidase monolayer assay for detection of the antibody against porcine parvovirus

建立了一种检测猪细小病毒(PPV)血清抗体的免疫过氧化物酶单层细胞试验(IPMA),对该方法的反应条件、特异性、敏感性和重复性进行了系统试验,并与HI和ELISA的检测结果进行了比较。结果显示,PPV种毒按照1∶100的剂量接种后第48小时固定效果最佳,辣根过氧化物酶-葡萄球菌A蛋白标记物的最适稀释度为1∶1 000,待检血清最适稀释度为1∶100。该IPMA方法与PCV2、PRV、PRRSV和CSFV的参考血清无交叉反应;PPV阳性血清稀释至1∶1 600时仍能检出;批内和批间重复性试验表明,该方法具有良好的重复性。符合性试验结果表明,该IPMA方法与HI和ELISA的符合率分别为96.0%与98.0%。用建立的IPMA方法检测PPV灭活疫苗免疫猪,结果免疫后第2周开始阳转,5周后全部阳转。对现地送检的400份猪血清样本进行检测,结果平均检出阳性率为86.5%。该IPMA方法的建立使PPV的血清学快速检测成为可能,可以用此方法开展PPV的抗体流行病学普查。

An immunoperoxidase monolayer assay（IPMA） was developed to detect antibody against porcine parvovirus（PPV）.The reaction conditions,specificity,sensitivity,and reproducibility of IPMA were optimized,evaluated,and compared with that of HI and ELISA.In the optimized IPMA,the virus was diluted at 1∶100 after 48 hours,HRP-SPA was diluted at 1∶1 000,and swine serum samples were diluted at 1∶100.There was no cross-reaction with reference sera against other porcine viruses.The sensitivity test showed that the positive sera could be detected at 1∶1 600.Reproducibility tests revealed that the established IPMA had good reproducibility.The IPMA method had 96.0% and 98.0% correlation with HI and ELISA method respectively.About 100% positive rate could be achieved by the assay for PPV-immunized porcine sera on the 6th week post-inoculation.A total of 400 serum samples were examined using this method.The positive rate was 86.5%.This method offered a convenient tool for the epidemiological investigation of PPV infections and quick evaluation of antibodies against the virus.