Zhang Anding, Jin Meilin, Liu Fan fang, Guo Xuebo, Hu Qiaoyun, Han Li, Tan Yadi, Chen Huanchun
Unit of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P.R. China.
Avian Dis. 2006 Sep;50(3):325-30. doi: 10.1637/7473-111605R.1.
Rapid detection of avian influenza virus (AIV) infection is critical for control of avian influenza (AI) and for reducing the risk of pandemic human influenza. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for this purpose. The method employed a monoclonal antibody (MAb) as the capture antibody and rabbit polyclonal IgG labeled with horseradish peroxidase as the detector antibody, and both antibodies were against type-specific influenza A nucleoprotein (NP). The DAS-ELISA could detect minimally 2.5 ng of influenza viral protein in virus preparations treated with Triton X-100, which is equvilent to 2.5 x 10(2) EID50 virus particles. This DAS-ELISA could detect all 15n AIV subtypes (H1-H15) and did not cross react with other avian pathogens tested. The DAS-ELISA were directly compared with virus isolation (VI) in embryonated chicken eggs, the current standard of influenza virus detection, for 805 chicken samples. The DAS-ELISA results correlated with VI results for 98.6% of these samples, indicating a sensitivity of 97.4% and specificity of 100%. The method was further tested with H5N1 and H9N2 AIV experimentally infected chickens, ducks, and pigeons, as well as field samples obtained from central China in 2005. The DAS-ELISA method has demonstrated application potential as an AIV screening tool and as a supplement for virus isolation in Asia.
快速检测禽流感病毒（AIV）感染对于控制禽流感（AI）以及降低人类大流行性流感风险至关重要。为此开发了一种双抗体夹心酶联免疫吸附测定法（DAS - ELISA）。该方法采用单克隆抗体（MAb）作为捕获抗体，用辣根过氧化物酶标记的兔多克隆IgG作为检测抗体，两种抗体均针对甲型流感病毒特异性核蛋白（NP）。DAS - ELISA能够在经Triton X - 100处理的病毒制剂中检测到最低2.5 ng的流感病毒蛋白，这相当于2.5×10²个EID50病毒粒子。这种DAS - ELISA能够检测所有15种甲型流感病毒亚型（H1 - H15），且不与所检测的其他禽病原体发生交叉反应。将DAS - ELISA与鸡胚病毒分离（VI）（当前流感病毒检测的标准方法）直接比较，用于检测805份鸡样本。这些样本中98.6%的DAS - ELISA结果与VI结果相关，表明其敏感性为97.4%，特异性为100%。该方法还在经H5N1和H9N2 AIV实验感染的鸡、鸭和鸽子以及2005年从中国中部获得的现场样本上进行了进一步测试。DAS - ELISA方法已证明作为一种AIV筛查工具以及在亚洲作为病毒分离补充方法的应用潜力。
