非洲猪瘟病毒CD2v阻断ELISA检测方法的初步建立与应用

Preliminary establishment and application of a blocking ELISA based on CD2v protein of African swine fever virus

为了建立特异性检测CD2v抗体的非洲猪瘟病毒(ASFV)阻断ELISA方法,以杆状病毒表达的CD2v蛋白作为包被抗原,以抗ASFV CD2v蛋白的特异性阻断单克隆抗体为检测抗体,经过条件优化以及敏感性和特异性检测,建立了特异性检测CD2v抗体的ASFV阻断ELISA方法。结果显示,建立的阻断ELISA的最佳抗原包被量为20 ng/孔,待检血清样本最佳稀释度为1∶1,酶标抗体最佳稀释度为1∶10000。当血清样品的PI≥41.23%时,判定结果为CD2v抗体阳性,当血清样品的PI<41.23%时,判定结果为CD2v抗体阴性。特异性试验结果显示,该方法仅能与ASFV阳性血清反应,与伪狂犬病病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪细小病毒和猪瘟病毒等猪病病毒疫苗毒阳性血清无交叉反应;敏感性试验结果显示,该方法最低能检测出128倍稀释的阳性血清;批内和批间重复性试验的变异系数均小于10%;对64份猪血清样品盘样品进行检测,结果显示符合率为96.88%。上述结果表明,本研究中建立的基于CD2v蛋白的ASFV阻断ELISA方法,联合p30等ASFV抗原血清学诊断方法可以用于CD2v缺失毒株的鉴别检测,为我国出现的ASFV CD2v缺失毒株的流行病学调查及疫情监测提供有效的检测技术。

In order to establish a blocking enzyme-linked immunosorbent assay(ELISA)method for specific detection of CD2v antibodies against African swine fever virus(ASFV),CD2v eukaryotic protein was used as coating antigen,and a monoclonal antibody against the CD2v protein of ASFV was used as blocking antibody.After optimization of the reaction conditions,sensitivity and specificity test,a specifically blocking ELISA method was developed.The results showed that the optimal antigen coating concentration was 20 ng/well,the optimal serum dilution was 1∶1,and the optimal dilution of the enzyme-labelled antibody was 1∶10000.The prelimary judge criteria of blocking ELISA method was as follows:the cut off values of average percentage inhibition(PI)≥41.23%was taken as CD2v antibody positive result,the PI value<41.23%was CD2v antibody negative result.The specificity test showed that this method had no cross-reactions with positive sera with pseudorabies virus,porcine circovirus2,porcine reproductive and respiratory syndrome virus,porcine parvovirus and classical swine fever virus,respectively.The sensitivity test showed that a 128-fold dilution of positive serum can be detected by this method.The intra-assay and inter-assay variation coefficients were both less than10%.The coincidence rate of this method to 64 pig sera was 96.88%.In conclusion,the blocking ELISA based on ASFV CD2v protein established in this study can be applied to the identification of low-virulent ASFV strain with CD2v deletion combining with the serological diagnosis method of p30 protein and other antigens of ASFV.This method provides an effective detection technology for the epidemiological investigation and epidemic monitoring of low-virulent ASFV strain with CD2v deletion in China.