猪伪狂犬病毒间接ELISA抗体检测方法的建立

Establishment of an indirect ELISA antibody detection method for porcine pseudorabies virus

为了满足我国现阶段猪伪狂犬病病毒(PRV)高频突变引发新疫情诊断的需求,本实验通过生物信息学分析比较,设计合成了针对PRV g B糖蛋白高度保守的抗原优势表位区肽段,命名为g B872。以此肽段为包被抗原,经条件优化建立了新型的PRV-g B间接ELISA抗体检测方法。该ELISA方法检测结果显示,其仅对PRV血清检测为阳性,而与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒(O型)、猪细小病毒、猪圆环病毒2型等主要猪源病毒阳性血清均无交叉反应,表明该方法具有较强的特异性;最低检测下限血清稀释度为1∶128,高于IDEXX PRV/ADV g B抗体检测试剂盒检测下限稀释度(1∶4),表明该方法敏感性高;批内、批间重复性试验结果显示,变异系数均低于10%,重复性良好。利用该方法与Bio Chek PRV-g B抗体检测试剂盒检测90份临床血清样品,对比结果分析显示,两者阳性符合率为100%,阴性符合率为97.67%,总体符合率为97.78%;同时,采用该方法与IDEXX-g I(gE)和IDEXX-g B试剂盒分别对野毒感染血清和疫苗免疫血清同时检测,该方法可以准确识别野毒阳性血清和疫苗免疫阳性血清,与IDEXX两种试剂盒的结果一致。本研究建立的间接ELISA检测方法对PRV疫苗免疫效果评价、流行病学调查及防控提供了可靠的方法。

In order to meet the needs of diagnosis for new epidemic situation caused by high frequency mutation of pseudorabies virus(PRV)in China,we designed and synthesized a peptide,named as g B872,targeting the conserved antigenic epitopes of the glycoprotein g B of PRV by comparison of bioinformatic analysis.A novel indirect ELSIA method that using this synthesized peptide as coating antigens for detecting specific PRV-g B antibody was established after optimization of work conditions.The results showed that this ELISA method had good specificity that was specific detecting the PRV positive serum but had no cross-reaction with other positive sera of major porcine viral pathogens including swine fever virus,porcine reproductive and respiratory syndrome virus,foot-and-mouth disease virus(O type),porcine parvovirus and porcine circovirus type 2.The detectable dilution of the primary antibody could reach as low as 1∶128,which was more sensitive than that of IDEXX PRV/ADV g B ELISA kit,1:4.The intra-and inter-assays of coefficient of variation(CV)were less than 10%,showing high reproducibility.The comparative analysis with BioChek PRV-g B detection kit revealed that this indirect ELISA method had 100%positive coincidence rate,97.67%negative coincidence rate and the overall coincidence rate was 97.78%.In addition,this indirect ELISA method,IDEXX-g I(gE)and IDEXX-g B kits were used to detect the serum from wild-type PRV infected or vaccinated pigs,respectively.The results demonstrated that this indirect ELISA can accurately identify the antibody originated from both wild-type PRV infection and vaccination serum,which is consistent with the results obtained from IDEXX ELISA kits.In conclusion,the indirect ELISA assay established in this study providing a reliable method for vaccination evaluation,epidemiological survey,and prevention and control of PRV.