Production and evaluation criteria of specific monoclonal antibodies to the hemagglutinin of the H7N2 subtype of avian influenza virus

To enhance the rapidity in diagnosing the spread of avian influenza virus (ATV) in chicken layer flocks, studies were initiated to develop more sensitive and specific immunological and molecular methods for the detection of AIV. In this study, the purification of the hemagglutinin protein (H) from field isolates of H7N2, the production of monoclonal antibodies (MAbs), and their evaluation as diagnostic reagents are reported. Hybridomas were generated by fusion of SP2/0-Ag14 myelomas and spleen cells from immunized mice. Hybridomas secreting antibodies specific for the H protein were assayed by an ELISA and cloned using limiting dilution. The MAbs produced were characterized by hemagglutination inhibition (HI), immunohistochemistry (IHC), indirect fluorescent antibody assay (IFA), Western blots, and IFA flow cytometry using various AIV subtypes (i.e., H4N2, H5N3, H7N2). Of the various MAbs assayed, 6 had consistent and reproducible results in each of the assays used. The results obtained in this investigation enhanced the usage of the MAbs to viral H protein in the surveillance of AIV in chickens.