猪圆环病毒2型Cap蛋白抗体间接ELISA检测方法的建立与应用  

Development and Application of Indirect ELISA for Detecting Antibody to Porcine Circovirus Type 2 Cap Protein

作　　者：魏艳秋[1] 李丽敏[1] 孙泰然 宋勤叶[1] 逯继成 李潭清[1] 张义明[1] 

机构地区：[1]河北农业大学动物医学院,河北保定071000 [2]河北保定市动物疫病预防控制中心,河北保定071001

出　　处：《中国兽医杂志》2015年第9期3-6,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31172329);河北省科技计划项目(13226603D-1<2013-X>);河北省生猪产业技术体系疫病控制专项资金

摘　　要：为了建立检测PCV-2抗体的间接ELISA方法,本研究以猪圆环病毒2型（PCV-2）重组Cap蛋白作为包被抗原,优化了反应条件及抗原、抗体工作浓度。结果显示,抗原最佳包被浓度为1μg/m L,待检血清的最佳稀释度为1∶80;最佳封闭液和抗体稀释液为含1%明胶、0.05%Tween-20的0.01 mol/L p H值7.4 PBS;当被检血清的OD450nm值≥0.3时为阳性,当OD值〈0.3时为阴性。该方法与猪瘟、猪繁殖与呼吸综合征、伪狂犬病、猪流行性腹泻等病毒抗体无交叉反应,与免疫过氧化物酶单层试验的符合率为98.3%,批内与批间重复性试验的变异系数分别低于5%与8%。应用建立的方法检测了126份2013年采集的猪血清,PCV-2抗体阳性率为40%~100%,表明河北省的猪群中PCV-2感染率很高。In order to develop an ELISA to detect antibody against porcine circovirus type 2（PCV-2）,we coated 96 well ELSIA plates using recombinant PCV-2-Cap protein,optimized reaction conditions and concentration of antigen and antibodies. The optimal coating concentration of antigen and the dilution of tested serum were 1 μg/m L and 1∶80,and the blocking buffer or dilution buffer of antibodies was 0.01 mol/L p H7.4 PBS including 1% gelatin and 0.05% Tween-20.The tested serum was positive with PCV-2 antibody as OD450 nm value≥0.3,and negative without PCV-2 antibody as OD450 nm value〈0.3.It did not show crossing reaction with the antibodies to Classic swine fever virus,Porcine reproductive and respiratory syndrome virus,Pseudorabies virus and Porcine epidemic diarrhea virus.The coincidence rate of ELISA with Immunoperoxidase monolayer assay（IPMA）was 98.3% when detecting PCV-2 antibodies. And variable coefficients of repetitive testing in the same or different batches of recombinant antigen were lower than 5% or 8%.Moreover,126 porcine serum samples collected in 2013 were examined with the developed ELISA,and the results showed that positive rates of PCV-2 antibody were 40%~100%,indicating that there is a high infection rate of PCV-2in pigs in Hebei province.

关 键 词：PCV-2 重组CAP蛋白 间接ELISA 抗体 应用 

分 类 号：S852.651[农业科学—基础兽医学]