非洲猪瘟病毒抗体ELISA检测方法的建立及初步评价

Establishment and Preliminary Evaluation of African Swine Fever Virus Antibody ELISA Detection Method

为建立非洲猪瘟病毒(ASFV)抗体检测方法,分别用ASFV重组蛋白p30、p72、pA104R作为包被原建立间接酶联免疫吸附试验(ELISA)方法,并进行初步评价。结果显示,ASFV重组蛋白p30、p72及pA104R分别按照25 ng/孔、50 ng/孔、50 ng/孔包被,血清样品均100倍稀释,辣根过氧化物酶(HRP)标记的羊抗猪IgG均20000倍稀释作为酶标试剂条件最优。p30-ELISA、p72-ELISA、pA104R-ELISA临界值分别为0.502、0.567、0.550。样品检测S/P值≥临界值判为阳性,否则判为阴性。3种方法分别检测梯度稀释的ASFV阳性血清、ASFV(CD2v缺失)阳性血清,p72-ELISA灵敏度最高,为1∶64、1∶128;3种方法检测猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪伪狂犬病病毒、猪流行性腹泻病毒阳性血清各5份,ASFV阴性血清200份,结果均为阴性;3种方法批间、批内重复性变异系数均小于10%。说明建立的方法均可用于ASFV的血清学检测,为非洲猪瘟的防控提供了多样化的检测方法。

To establish the ELISA methods for African swine fever virus(ASFV)antibody detection,the recombinant proteins p30,p72 and pA104R were used as coating antigens,and then the ELISA methods were evaluated preliminarily.The results showed that the optimal coating concentrations of p30,p72 and pA104R were 25,50 and 50 ng/well,respectively.The optimal dilution ratios of the serum sample and the HRP-labeled sheep anti-pig IgG were 1∶100 and 1∶20000,respectively.The critical values of p30-ELISA,p72-ELISA and pA104R-ELISA were 0.502,0.567 and 0.550,respectively.Sample with the S/P value equal to or higher than the corresponding critical value was considered as positive,otherwise it was judged as negative.In the detection of the series diluted positive serum samples of ASFV and ASFV(CD2v deletion)by the three methods,the method of p72-ELISA showed the highest sensitivity(reached up to 1∶64-1∶128).Positive sera of CSFV,PRRSV,PCV2,PRV and PEDV(5 samples for each virus),as well as 200 negative sera of ASFV were detected by the three methods,and the results indicated all of these samples were negative.The inter batch and intra batch repeatabilities of variation of the three methods were all lower than 10%.Overall,the three ELISA methods could be used for the serological detection of ASFV,and also could provide diversified detection methods for the prevention and control of ASF clinically.