欧洲禽源H1N1亚型猪流感病毒抗体间接ELISA检测方法的建立  

DEVELOPMENT OF AN INDIRECT ELISA FOR DETECTION OF ANTIBODIES TO EUROPEAN AVIAN-LIKE H1N1 SUBTYPE SWINE INFLUENZA VIRUS

作　　者：阮宝阳 王林[2] 宫晓倩[1] 刘晓敏[1] 张鹏[1,2] 汪琪[1] 李泽君[1] 童光志[1] 于海[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]山东农业大学动物科技学院,泰安271000

出　　处：《中国动物传染病学报》2016年第2期13-19,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家青年自然科学基金(31201916);上海市自然科学基金青年项目(12ZR1453500);中央级公益性科研院所基本科研业务费项目(2015JB07);中国农业科学院创新工程科研团队"动物流感病毒病原生态学"项目

摘　　要：本研究利用原核表达并纯化的重组蛋白pCold TF-HA1作为包被抗原,建立欧洲禽源H1N1亚型猪流感病毒抗体的间接ELISA检测方法。通过反应条件的优化,确定了间接ELISA最佳工作条件：抗原最佳包被浓度为800 ng/孔,血清稀释度为1：200,封闭时间为1 h,一抗血清最佳作用时间为2 h,酶标二抗最佳稀释度为1：40 000,二抗最佳作用时间为1 h,TMB显色时间为7 min。阴/阳性血清的判断结果分别为OD450≤0.255和OD450≥0.292,介于两者之间为可疑。符合性试验结果显示,建立的间接ELISA方法与血凝抑制（haemagglutination inhibition,HI）检测的阳性率分别为53.27%和49.53%,两者总复合率达96.27%。特异性试验结果显示,该方法与古典H1N1亚型和H3N2亚型猪流感阳性血清无交叉反应。通过重复性试验表明,同一批制备的重组蛋白包被96孔酶标板,检测OD450值的批内变异系数为1.97%-7.85%,批间变异系数为1.94%-6.93%。总之,本研究建立的禽源H1N1亚型猪流感病毒抗体间接ELISA方法,具有敏感性较高、特异性强、重复性好的特点,为猪流感抗体检测提供了一种准确、快捷、简便、有效的技术手段,可用于禽源H1N1亚型猪流感抗体检测和流行病学调查。In the present study, an indirect ELISA method was developed to detect antibodies to European avian-like H1N1 subtype Swine influenza virus （SIV） using prokaryotic expressed and purified recombinant protein pCold TF-HA1 as the coating antigen. The reaction conditions were optimized, e.g. coating antigen at 800 ng, serum dilution at 1：200, 5% skim milk as the blocking solution, 1.0 h for closed time, 2.0 h for serum reaction time, HRP antibody dilution at 1：40000, 1.0 h for labeled antibody reaction time and 7 min for reaction time of TM. Criteria for determining the negative, positive and suspect results of serum samples were OD450nm≤0.255,OD450nm≥0.292 and 0.255-0.292, respectively. The conformance testing showed the high agreement （96.27%） between the indirect ELISA method and hemagglutination inhibition （HI） test as their individual positive rates were 53.27% and 49.53%. The specific test results demonstrated that the established ELISA method had no cross reaction with positive serum samples of H1N1 and H3N2 subtypes SIV. The coefficient of variation was 1.97%-7.85% between ELISA plates coated with the same batch of recombinant protein and 1.94%-6.93% between batches of recombinant proteins. In conclusion, the indirect ELISA method developed in the present study had high sensitivity, specificity and repeatability and thus could be used as an accurate, fast, convenient and effective technique for detection of antibodies to H1N1 subtype SIV and epidemiological surveillance.

关 键 词：欧洲禽源H1N1猪流感病毒 重组蛋白 间接ELISA 特异性 

分 类 号：S852.659.5[农业科学—基础兽医学]