非洲猪瘟病毒p30蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

Preparation of Monoclonal Antibody against African Swine Fever Virus p30 Protein and Establishment of a Blocking ELISA for Detection of Antibody against ASFV p30 Protein

为建立检测非洲猪瘟病毒(ASFV)抗体的阻断ELISA方法,本研究利用原核表达的ASFV p30重组蛋白免疫BALB/c小鼠制备单克隆抗体。以重组p30蛋白作为包被抗原,以辣根过氧化物酶(HRP)标记的p30单克隆抗体作为检测抗体,经条件优化,建立了一种检测ASFV抗体的阻断ELISA方法。ROC曲线分析显示,该方法最佳阻断率临界值为16.63%。该方法与CSFV、FMDV-O/A、PRRSV、PEDV、SVA的阳性血清均无交叉反应;最低能检出1∶128稀释的阳性血清;批内和批间变异系数(CV)均<10%。用本方法与商品化试剂盒平行检测208份血清样品,Kappa值为0.96,表明具有高度一致性。上述结果表明,本研究建立的阻断ELISA方法具有较高的特异性和敏感性,可用于血清ASFV抗体的检测,为ASFV流行病学调查及猪群疫情监控提供技术支持。

In order to establish a blocking ELISA method for detection of African swine fever virus(ASFV) antibody, the prokaryotic expressed ASFV p30 protein was used to immunize BALB/c mice for preparation of monoclonal antibody(MAb). A blocking ELISA method was developed after optimization of the reaction conditions with the recombinant p30 protein as a coating antigen and the horseradish peroxidase(HRP) conjugated MAb as detecting antibody. The receiver operating characteristic(ROC) curve calculated an optimal cut-off value of 16.63%. This method had no cross-reaction with positive serum samples of CSFV, FMDV-O/A, PRRSV, PEDV or SVA. The sensitivity test showed that a 1∶128 dilution of positive serum can be detected by this method. The coefficient of variation(CV) of the intra-and inter-assay was less than 10%. A total of 208 serum samples were detected in parallel by the method and the commercial ELISA kit, the Kappa value was 0.96, which demonstrated its high consistency. In conclusion, the established blocking ELISA method with high sensitivity and specificity can be applied to the detection of ASFV antibodies in serum, which provides technical support for the epidemiological investigation and epidemic monitoring of ASFV.