猪脑心肌炎病毒抗体间接ELISA检测方法的建立及应用  

Development and Application of an Indirect ELISA for Detection of Antibody Against Porcine Encephalomyocarditis Virus

作　　者：陆文俊[1] 施开创[1] 屈素洁[1] 莫胜兰[1] 李向涛[2] 钟诚[2] 

机构地区：[1]广西动物疫病预防控制中心,广西南宁530001 [2]广西大学动物科学技术学院,广西南宁530005

出　　处：《动物医学进展》2011年第12期6-10,共5页Progress In Veterinary Medicine

基　　金：广西科学基金项目(桂科青0728047)

摘　　要：以猪脑心肌炎病毒(Encephalomyocarditis virus,EMCV)VP1重组蛋白为包被抗原,建立了检测EMCV血清抗体的间接酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)。经优化,获得间接ELISA的最佳反应条件为抗原包被浓度0.625μg/mL,待检血清稀释度为1∶40,封闭液为50g/L脱脂乳,酶标兔抗猪抗体稀释度为1∶6 000,底物显色时间为室温避光15min,血清样本阴阳性临界值为OD450nm值≥0.33。与口蹄疫病毒(Foot and mouth disease virus,FMDV)、猪瘟病毒(Classical swine fevervirus,CSFV)、猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪伪狂犬病病毒(Pseudorabies virus,PRV)、猪圆环病毒2型(Porcine circovirus type 2,PCV-2)抗体没有交叉反应。应用所建立的方法对广西2010年—2011年所采集的2 228份血清进行检测,被检血清样品平均阳性率为91.47%,被检猪场阳性率为100%。表明广西地区猪群感染普遍。An indirect ELISA for detection of antibody against porcine encephalomyocarditis virus （EMCV） was developed with the EMCV recombinant protein VP1 as antigen. After optimization, the optimal reac tion conditions of the indirect ELISA were determined. The results showed that the optimal concentration of the coated antigen was 0. 625μg/mL; the dilution of serum samples was 1：40; the optimal blocking solution was 50 g/L skim milk~ the working concentration of HRP-labeled rabbit anti-porcine IgG was 1 ： 6 000; the coloration time was 15 min at room temperature;the threshold value of positive and negative serum samples was 0.33 at OD450nm. The recombinant antigen had no cross reaction with antibodies against FMDV, CSFV, PRRSV, PRV and PCV-2, respectively. The established indirect ELISA was used to detect EMCV antibody in 2 228 serum samples collected from Guangxi in 2010-2011. The results showed that the positive rate of serum samples was 91. 47% ,and 100% pig farms existed positive serum samples, indicating that EMCV infection was very common in pig farms of Guangxi region.

关 键 词：猪 脑心肌炎病毒 VP1重组蛋白 间接ELISA 血清学调查 

分 类 号：S852.65[农业科学—基础兽医学]