羊附红细胞体双抗体夹心ELISA诊断方法的建立

摘要

为快速准确诊断和检测羊附红细胞体病,及时采取防治措施,建立了检测羊附红细胞体抗原的双抗夹心ELISA诊断方法.选取规模化养殖场羊,镜检附红细胞体红细胞感染率＞90%,无菌采取血液.分离羊附红细胞体抗原,制备纯化兔抗羊附红细胞体抗体,应用辣根过氧化物酶标记抗体,进行双抗体夹心ELISA试验.试验结果表明,双抗体夹心ELISA方法的最佳工作条件为:抗体最佳包被量为82.91μg/mL,酶标抗体最适工作浓度为1:400,抗原最低检出量为7.81μg/mL;而且与支原体、大肠杆菌、葡萄球菌以及牛、猪、兔附红细胞体均不出现交义反应,表明该方法具有良好的特异性,可用于羊附红细胞体病的诊断和群体检测.%To diagnose and detect Eperythrozoonosis on sheep quickly and take preventive measures in time, the double-antibody sandwich ELISA was established. The blood samples of sheep from large-scale farms whose red cell infective rate was over 90 ％ were selected. Eperythrozoon ovis antigen was isolated, rabbit anti-sheep antibody was purified, antibody was labeled with HRP, then double-antibody sandwich ELISA was established. The results showed that the optimal working concentration of IgG was 82.91 μg/mL, while the best enzyme-conjugated antibody concentration was 1: 400, and the detective minimum of antigen was 7.81 μg/mL. The specific test and crossing reaction indicated that the double-antibody sandwich ELISA was a quick, sensitive way adapted to diagnosis and colony check.