Abstract: ObjectiveTo develop a protein chip-based method for simultaneous detection of avian influenza virus (AIV)from birds and human.MethodsProtein chip was constructed by immobilizing the positive control(PC),negative control(NC)and different antigen subtypes with the optimal concentration onto the aldehyde glass slide according to the predesigned pattern.Then the prepared protein chip was used to detect the serum samples from the different AIV subtype and human.The parameters for the performance of protein chip,such as specificity,sensitivity and repeatability,were tested in details.In addition, interactive validation experiment was performed using the protein chip-based method and avian influenza virus(H subtype) hemagglutination inhibition test kit.ResultsTo construct protein chip,the optimal probe concentrations of H1,H3,H5,N1, N2,NP,NS1 and H7,H9 subtype were 1 mg/mL and 0.5 mg/mL,respectively.The optimal concentrations of horseradish peroxidase enzyme labeled antibody for AIV from birds and human were 1:1000 and 1:1500 dilution.The specificity of prepared protein chip was confirmed by testing the samples of H5N1,H7N7,H9N2 positive serum and normal serum.The results were consistent with the expected and there was no cross-reactivity observed.The prepared protein chip had a detection limitation of 1:40 dilution for H7N7 positive serum and show ed excellent repeatability validated by consecutive test of 10 times for H5,H7,and H9 positive serum with a variation coefficient of <2%.The results of the interactive validation experiments using protein chip-based method were consistent with those of hemagglutination inhibition test.ConclusionThe prepared protein chip has a good specificity,sensitivity,and repeatability and could be used to detect AIV from birds and human simultaneously.