禽流感病毒双抗体夹心ELISA检测方法的建立

Preliminary Establishment of the Double-antibody Sandwich ELISA for AIV

采用饱和硫酸铵初步纯化的兔抗AIV-H9高免血清中的IgG作为包被抗体,利用抗AIV-NP-7B4单抗作为ELISA的第二抗体,建立了检测禽流感病毒(AIV)抗原的双抗体夹心ELISA方法。经方阵滴定试验测定反应的最佳工作条件:兔抗AIV-H9高免血清IgG包被稀释度为1:8000(浓度为3.531μg/ml),抗AIV-NP-7B4单抗腹水最佳使用稀释度为1:800,酶标二抗的工作浓度为1:4000。与其他禽易感病毒(NDV、IBV、EDS-76V)等均没有交叉反应。结果表明,该方法具有快速、敏感、特异等优点,可应用于AIV的检测和流行病学调查。

A double-antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for detecting avian influenza viruses was developed.In this method,the microplates were coated by IgG of rabbit polyclonal antibodies against AIV-H9 at the concentration of 3.531 μg/ml（1：8 000）.Primary antibody of Mab-7B4 with 1：800 dilutions and antibody-HRP of 1：4 000 dilutions gave the best result.No cross-reaction was observed with NDV,IBV,and EDS-76V.All of these results showed that DAS-ELISA method was quick,sensitive and repeatable,which was applicable to the detection of AIV and epidemiological investigation.