基于非洲猪瘟病毒p72蛋白的阻断ELISA检测方法的建立及初步应用

Establishment and preliminary application of a blocking ELISA based on p72 protein of African swine fever virus

本研究以真核表达的非洲猪瘟病毒(ASFV)p72蛋白为包被抗原,以抗ASFV p72蛋白的特异性单克隆抗体为检测抗体,经条件优化,建立了特异性检测ASFV抗体的阻断ELISA方法。结果显示,最佳抗原包被量为100 ng/孔,待检血清样本最佳稀释度为1∶2,酶标抗体最佳稀释度为1∶10000。该阻断ELISA方法的结果判定标准:当血清样品的PI≥41.84%时,判定为阳性;当血清样品的PI<41.84%时,判定为阴性。特异性试验结果显示,该方法仅能与非洲猪瘟病毒阳性血清反应,与PRV、PCV2、PRRSV、CSFV和PPV等猪病疫苗毒阳性血清无交叉反应;敏感性试验结果显示,该方法最低能检出1∶128稀释的阳性血清;批内和批间重复性试验的变异系数均小于10%;与商品化ELISA试剂盒的对比检测结果表明,两种方法的符合率为100%。上述结果表明,本研究建立的基于ASFV p72蛋白的阻断ELISA方法可以应用于ASFV抗体的检测,为ASFV流行病学调查及疫情监测提供了有效的检测手段。

In order to establish an enzyme-linked immunosorbent assay(ELISA)method for detection of antibodies against African swine fever virus(ASFV),a specifically blocking ELISA method was developed after optimization of the reaction conditions with purified p72 eukaryotic protein as coating antigen and a monoclonal antibody against the p72 protein of ASFV as detecting antibody.The results showed that the optimal antigen coating concentration was 100 ng/well,the optimal serum dilution was1∶2,and the optimal dilution of the enzyme-labelled antibody was 1∶10000.The blocking ELISA judge criteria:the cut off values of average percentage inhibition(PI)≥41.84%was taken as positive result,the PI value<41.84%was negative result.The analysis specificity test showed that this method can only detect ASFV and no cross-reaction with those antibody positive serums against PRV,PCV2,PRRSV,CSFV,PPV.The analysis sensitivity test showed that a 1∶128 dilution of positive serum can be detected.The co-efficient of variation(CV)of the intra-and inter-assay precision test were all less than 10%.Our method had high agreement(100%)comparing with commercial ELISA kit for the detection of ASFV-specific antibodies.In conclusion,the blocking ELISA based on ASFV p72 gene established in this study can be applied to the detection of antibodies against ASFV,which provides an effective detection method for the epidemiological investigation and epidemic monitoring of ASFV.