非洲猪瘟病毒p72蛋白阻断ELISA检测方法的建立

Development of a Blocking ELISA for Detecting the Antibodies against African Swine Fever Virus p72 Protein

本研究旨在建立一种非洲猪瘟病毒(ASFV)p72蛋白阻断ELISA抗体检测方法。以纯化的重组p72蛋白作为包被抗原,HRP标记的6E5抗体作为阻断抗体,通过对反应条件进行优化,建立了基于ASFV p72蛋白的阻断ELISA方法。用建立的阻断ELISA方法检测119份临床阴性血清,计算血清阻断率确定临界值,确定了该方法的判定标准:当阻断率PI≥50%时判定为ASFV抗体阳性;PI≤40%时判定为ASFV抗体阴性;当阻断率介于两者之间时,判定为可疑。该方法与猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、猪圆环病毒2型、A型塞内卡病毒、猪口蹄疫病毒、猪大肠杆菌、猪多杀性巴氏杆菌、副猪格拉瑟菌和猪胸膜肺炎放线杆菌的抗体阳性血清无交叉反应,批间、批内试验的变异系数均小于15%。用该方法与商品化ASFV抗体检测试剂盒同时检测447份猪临床血清样品,两种方法的相对敏感性和相对特异性分别为95.3%和94.5%,符合率为94.9%。本研究建立的阻断ELISA方法具有较高的敏感性和良好的特异性,可用于ASFV感染诊断和流行病学调查。

This study aimed to establish a blocking ELISA method for detection of African swine fever virus(ASFV)p72 protein antibodies.With the purified recombinant p72 protein as coating antigen,and the HRP labeled 6E5 antibody as the blocking antibody,a blocking ELISA method based on ASFV p72 protein was established after optimization of the reaction conditions.One hundred and nineteen clinical negative serum samples were detected using the present established method to determine the cut off value by the serum percentage of inhibition(PI)calculation.The criteria of the blocking ELISA:serum samples with PI≥50%were judged as positive;the serum samples with PI≤40%were judged as negative;and the serum samples with PI between 40%and 50%were judged as suspicious.This method had no cross reactions with the serum antibodies against PRV,PRRSV,CSFV,PCV2,SVA,FMDV,Escherichia coil,Pasteurella multocida,Glaesserrella parasuis and Actinobacillus pleuropeumoniae.The intra-assay and inter-assay variation coefficients were both less than 15%.Total 447 swine clinical serum samples were then detected simultaneously by the present established method and commercial ASFV antibody detection kit.The result showed that the relative sensitivity and specificity of the two methods were 95.3%and 94.5%,respectively,and the coincidence rate was 94.9%.In summary,the blocking ELISA method established in this study has high sensitivity and specificity,therefore it is suitable for ASFV diagnosis and epidemiological investigation.