甲型流感病毒NS1抗原捕获ELISA的建立

Antigen capture ELISA for specific detection of influenza A virus NS1 protein

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1（NS1）抗原检测的酶联免疫吸附（ELISA）法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97（H5N1）-NS1和A/Vietnam/1194/04（H5N1）-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.

Objective To develop a capture enzyme-linked immunosorbent assay （ELISA） for influenza A virus nonstructural protein NS1 using two anti-NS1 monoclonal antibodies（McAb） that map to distinct sites on the protein and to detect the NS1 in the culture supernatant and cell lysis supernatant of influenza virus. Methods Among the McAb against influenza A virus NS1, antibody pairs were analyzed to choose the optimal coating McAb and detecting McAb by determinating the detection sensitivity and specificity. Results The capture ELISA efficiently detected as little as 240 pg/ml of recombinant NS1 protein and exhibited no cross-reactivity for influenza B virus, parainfluenza virus NS1. Conclusion A sensitive and specific NS1-based capture ELISA for influenza A virus NS1 was successfully established, which could be an important tool for diagnosis of influenza A virus infection.