Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses

ABSTRACT An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.