基于单克隆抗体的非洲马瘟病毒间接ELISA抗体检测方法的建立与应用

Development and Application of a Mab-Based iELISA for the Detection of Antibodies Against African Horse Fever Virus

【目的】为有效应对非洲马瘟(African horse fever,AHS)传入我国的风险,研究建立一种基于单克隆抗体(Monoclonal antibody,Mab)的非洲马瘟病毒(African horse fever virus,AHSV)特异性的间接ELISA(indirect ELISA,iELISA)抗体检测方法,利用该方法对我国马匹进行非洲马瘟抗体监测。为有效诊断非洲马瘟提供血清学检测手段。【方法】首先利用AHSV VP7抗原免疫小鼠制备针对VP7抗原的单克隆抗体;其次通过对包被抗体浓度、蛋白浓度、血清稀释度、酶标二抗稀释度等反应条件的优化建立AHSV iELISA抗体检测方法。利用1000份血清确定该方法的临界值,并评估该方法的敏感性和特异性。由3位试验操作者分别对AHSV敏控阳性血清进行加速试验测定效价,评估该方法的稳定性。通过利用建立的方法对已知的10份AHSV阳性血清和400份阴性血清进行检测并与国外商品化试剂盒检测结果进行比较;最后用该方法对我国2021年18个省份或地区的947份临床样本进行检测来评估我国马匹中AHSV感染风险。【结果】获得了5株针对AHSV VP7抗原的单克隆抗体。通过对5株单克隆抗体筛选确定3G9单克隆抗体捕获抗原性能最佳。利用3G9抗体作为包被抗体,通过对不同反应条件优化建立了AHSV iELISA抗体检测方法。确定该方法的临界值为0.25;经比对试验证实本研究建立的AHSV iELISA方法的敏感性与商品化试剂盒相当。3位操作者分别对敏控血清进行检测,组内变异系数范围分别为3.19%—7.02%、0%—3.11%、0.27%—5.76%,组间变异系数为1.17%—5.03%。37℃加速试验表明AHSV阳性血清7 d内效价稳定,因此证明该方法具有良好的稳定性。该方法与商品化试剂盒进行比较两者总体符合率为100%。通过对我国18个省或地区的947份马血清进行检测,结果表明AHSV抗体阳性率为0%。【结论】成功筛选到了针对AHSV VP7的单克隆抗体,建立了一种基于单克隆抗体的AHSV iELISA抗体检测方法,该方法具有特异性强、敏感性高、稳定性好,可以实现临床样本中AHSV抗体的检测,因此可以作为AHS血清学监测的一种有效工具。

【Objective】In order to effectively address the risk of introduction of African horse fever into China,this study aimed to develop an indirect ELISA(iELISA)antibody test specific for African horse fever virus(AHSV)based on monoclonal antibodies,so as to monitor the antibody status of African horse fever in horses in China.【Method】Firstly,Mabs against VP7 antigen were prepared by immunizing mice with the VP7 antigen.Secondly,the ELISA method was developed by optimizing the amount of coating Mab,antigen and secondary antibody concentrations.Negative sera(n=1000)were used to establish the baseline for a negative population,and then the specificity and sensitivity of it was estimated.The stability of the method was determined by an accelerated test using AHSV-positive reference sera by three independent operators.10 known positive sera and 400 negative sera for African horse fever were tested using the established method and compared with the results of foreign commercial kits.Finally,the method was tested on 947 clinical samples from 18 provinces or regions in China in 2021 to assess the risk of African horse fever in horses in China.【Result】Five Mabs were obtained against the VP7 antigen of AHSV,and the 3G9 Mab had the best antigen capture performance.Using the 3G9 Mab as the capture antibody,an ELISA method for the detection of AHSV was established by optimizing different reaction conditions.A cut-off value of 0.25 was selected in AHSV iELISA.The sensitivity of ELISA method was comparable with that of commercial kits and the sensitivity of both methods was consistent.The intra-group coefficients of variation from 3 operators ranged from 3.19%to 7.02%,0%to 3.11%,and 0.27%to 5.76%,respectively,and the inter-group coefficients of variation for the three operators ranged from 1.17 to 5.03%.The accelerated test at 37°C demonstrated that the components of the testing kit had good stability over 7 days,thus demonstrating the stability of the method.The results showed that the agreement between the AHSV iELISA and commercial AHSV C ELISA kits were 100%.In this study,a serological survey on AHSV was performed by using 947serum samples collected from 18 provinces of China in 2021,and the results showed that the positive rates were 0%.【Conclusion】A Mab-based AHSV iELISA method was established for the detection of AHSV antibodies,which had the good specificity and sensitivity,and could be a promising candidate tools for use serological surveillance for AHS.