猪圆环病毒Ⅱ型血清抗体ELISA检测方法的建立

Establishment of ELISA method to detect porcine circovirus 2 antibody

为了检测猪圆环病毒Ⅱ型(PCV2)抗体,本试验建立了快速、敏感的ELISA诊断方法.通过比较差速离心、蔗糖不连续梯度离心、氯化铯等密度梯度离心和氯仿抽提结合超速离心等4种方法纯化PCV2抗原的包被效果,来确定最佳的PCV2包被抗原纯化方法,在此基础上,通过全病毒抗原最适包被浓度和二抗HRP-SPA稀释倍数的确定、待测血清稀释倍数的确定、封闭液浓度的确定、封闭时间的确定、HRP-SPA反应时间的确定、临界值的确定、特异性测定、重复性测定、符合率比较等,进一步优化检测PCV2抗体的间接ELISA方法.结果表明:对猪的多种病毒阳性血清进行ELISA检测,只有PCV2阳性血清呈阳性;对6份血清进行4次重复试验,方差分析显示P>0.05,差异不显著;与PCV2-ELISA试剂盒(INGEZIM公司生产)的符合率比较,其总符合率达88.89%.可见,优化建立的检测PCV2抗体的间接ELISA方法特异性强、重复性高、稳定性好,可用于PCV2血清学诊断和血清学普查.

The research established a fast and sensitive enzyme-linked immunosorbent assay（ELISA） method to detect porcine circovirus Ⅱ（PCV2） antibody.The experiment sieved four PCV2 antigens from differential centrifugation,gluco-discontinuous gradient centrifugation, cesium chloride isopycnic gradient centrifugation and CHCl3 extract bonding ultracentrifugation.We determined PCV2 antigen from CHCl3 extract bonding ultracentrifugation was the best antigen.In addition,we also confirmed the optimal coating-antigen concentration,HRP-SPA concentration,test serum dilution,blocking liquid concentration,blocking time,HRP-SPA reaction time,critical value,specificness,stability,accordant rate by ELISA method.The results revealed that for the positive serums of pig′s several viruses detected by this ELISA method,only the positive serum of PCV2 was positive;The six serums were detected four duplicates by this ELISA method,F test presented P0.05,the variation was quiet;The coincidence was 88.89% with ＂INGEZIM＂ PCV2-ELISA Kit.The results indicated the optimized ELISA was a specific,sensitive and stabile method,may detect PCV2 antibody.