Development Of Monoclonal Antibody Against H7 Subtype Avian Influenza Viruses And Colloidal Gold Immunochromatographic Strip For The Detection

Avian influenza (AI) is a various of syndromes caused by influenza virus of type A (AIV) after infection in poultry. According to differences in the antigenicity of the two surface glycoproteins, HA and NA, influenza A viruses are categorized into different subtypes. Currently,17 HA subtypes and 10 NA subtypes have been identified. In February 2013, a emerging type A of avian influenza viruses (H7N9) was first identified in China, resulting in the high mortality, which is a great threat to the poultry industry and human health safety. The disease spreads rapidly and needs a lot of time to be diagnosed. Therefore, diagnosing the disease as soon as possible is extremely important for public.1. Development of monoclonal antibody against H7 subtype avian influenza virusesIn this experiment, H7 AIV was propagated in 9-11 day-old SPF chicken embryos, then purified by ultracentrifugation and deactivated with 2% formaldehyde. The virus was used as immunization antigens for antibody production or coating antigens for indirect ELISA. Establish indirect ELISA method to determine the following aspects:H7 AIV antigen and serum were diluted to 1:1600 and 1:400, respectively. The optimal condition of coating was 1.0h in 37℃ and then overnight in 4℃. The best blocking time was 40min. The reaction time for serum was 30min. HRP-IgG was diluted to 1:10000 and the reaction time was 2h. The reaetion time for TMB was 15min.Six hybridoma cell stains 5D2、3G4、11C7、8D10、10E6、6E2 were developed by confusing SP2/0 cells and spleen cells of immunized mouse with H7 AIV, which were determined by indirect ELISA and HI. Western-blot test proved that 5D2、3G4 were against structural protein HA, which HI titers of cell supernatants and ascites were 26、25 and 211、 28. Others were against neither HA nor NA. The ELISA titers of cell supernatants and ascites of six hybridoma cell stains were 6400、1600、128000、6400% 1600% 12800 and 204800、51200、819200、3276800、51200、409600, respectively. The results of indirect ELISA proved that the six hybridoma cell stains had excellent specificity. These strains had no neutralization titers against H7 AIV.2. Development of colloidal gold immunochromatographic test strip to detect H7 subtype avian influenza virusThe gold particle in colloidal solution was coupled with purified monoclonal antibody 5D2, and then sprayed on the glass fibers. The purified polyclonal antibody against H7 AIV from rabbit was coated on the nitrocellulose membrane as the capture-antibody and HRP-IgG as control antibody. Then the colloidal gold immunochromatographic (IC) strip for the detection of H7 AIV was assembled in regular order. The detection results indicated that the IC strip was specific to H7 AIV and the results were observed within 10min. The sensitivity was HA 24 for detecting samples. The IC strip can be used for rapid clinical diagnosis of H7 AIV with high specificity, sensitivity and reproducibility.