Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies

Yixin Xiao, Fan Yang, Fumin Liu, Hangping Yao, Nanping Wu, Haibo Wu

The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.