禽流感(H9N2亚型)酶标抗体的制备及双抗夹心Dot-ELISA检测方法的建立

Preparation of Enzyme-labeled Antibody and Establishment of Double-antibody Sandwich Dot-EIISA Detecting Avian Influenza Virus(H9N2)

采用AIV灭活油乳苗(H9N2)对AIV非免疫鸡进行接种并获取高免血清,取高免血清采用饱和硫酸铵沉淀法,经过Sephadex G25层析柱提纯后可获得纯度很好的IgG。将IgG应用过碘酸钠法标记辣根过氧化酶(HRP),制备禽流感酶标抗体(IgG-HRP),利用所提取的AI-IgG和所制备的禽流感酶标抗体按照双抗夹心Dot-ELISA试验操作步骤,采用方阵法分别摸索包被抗原及酶标抗体的最佳浓度,以及各步反应时间等最佳反应条件,以建立一种优化的AIV双抗夹心Dot-ELISA检测法。结果表明,所制备的AI高免血清HI效价为10log2;AI酶标抗体克分子比值为2.45;优化的Dot-ELISA各条件为:包被AI-IgG最佳稀释倍数为1∶50;最佳封闭剂为1%牛血清白蛋白;AI酶标抗体最佳稀释倍数为1∶100;待检抗原与2种抗体的感作时间均为0.5h(37℃)。优化后的检测方法可在2.5h内诊断结果,制备好的包被膜在4℃下保存2个月不影响其效果。而且敏感性提高了3倍,与新城疫病毒液、传染性支气管炎及减蛋综合症病毒液无交叉反应。

The chicken were inoculated by Avian Influenza（AI） inactivate vaccine（H9N2） and hyperimmune serum was obtained from these chicken. The hyperimmune serum was treated with saturation （NH4）2SO4 deposition method and Sephadex G25 floor analysis column to obtain purified AI- IgG. Then AI- IgG was labeled with horseradish peroxidase by NaIO4 method to prepare AIV enzyme-labeled antibody （IgG- HRP）. According to the steps of sandwich Dot -ELISA and square matrix method, sandwich Dot - ELISA detecting the antigen of AIV was developed. The results showed that the HI titre of AI hyperimmune serum was 10log2 and HRP/IgG was 2.45. Optimized conditions of Dot - ELISA were as follows, optimal dilution ratio of blocking AI -IgG was 1 ： 50; the best blocking reagent was 1 %BSA; optimal dilution ratio of AI enzyme-labeled antibody wasl ＂ 100; At 37 ℃, interaction time of two kinds of antibody with antigen was about 0. 5 hour. the Dot - ELISA was able to give diagnosis result within 2. 5 hours and the blocked capsules keep effectiveness for two month at 4 ℃. The sensitivity of the Dot - ELISA was 3 times higher than the HA test, there was no specific cross reactivity to newcastle disease virus, infectious bronchitis virus and egg drop syndrome virus.