Development of a DAS-ELISA for detection of H9N2 avian influenza virus

•Two monoclonal antibodies named 4D10 and 5G2 against HA of H9N2 AIV were prepared.•A double-antibody sandwich ELISA was developed by 4D50 and 5G2.•The sensitivity and specificity of DAS-ELISA were 98.9% and 98.1% compared to VI. H9N2 avian influenza virus is threatening animals and public health systems. Effective diagnosis is imperative to control the disease. Thus, we developed a panel of monoclonal antibodies (Mabs) against the H9N2 avian influenza virus (AIV) and implemented a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to detect the H9 viral antigen. Hybridomas 4D10 and 5G2 were screened to secrete immunoglobulin G (IgG) and IgA, respectively. Antibody 4D10 was used as the capture antibodies and HRP labeled 5G2 as the detector antibody. The specificity of the optimized DAS-ELISA was evaluated by using AIV subtypes H1, H3, H5, H9 and H10. Specimens containing AIV H9 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limit of the DAS-ELISA is 10−2.3 TCID50 (50% Tissue culture infective doses). Negative-positive threshold was 0.211 (OD630). In comparison with virus isolation the sensitivity and specificity of DAS-ELISA were found to be 98.9% and 98.1% respectively. Taken together, the newly developed Mab-based DAS-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H9 subtype AIV.