中国动物传染病学报2016，Vol.24Issue(2)：13-19,7.
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
摘要
Abstract
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Key words
European avian-like H1N1 subtype/Swine influenza virus (SIV)/recombinant protein/indirect ELISA分类
农业科技引用本文复制引用
阮宝阳,王林,宫晓倩,刘晓敏,张鹏,汪琪,李泽君,童光志,于海..欧洲禽源H1N1亚型猪流感病毒抗体间接ELISA检测方法的建立[J].中国动物传染病学报,2016,24(2):13-19,7.基金项目
国家青年自然科学基金(31201916) （31201916）
上海市自然科学基金青年项目(12ZR1453500) （12ZR1453500）
中央级公益性科研院所基本科研业务费项目(2015JB07) （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 （）
