Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens.

Reference antisera were produced against 15 influenza hemagglutinin (HA) subtypes using DNA vaccination to produce a high-quality polyclonal serum to the HA protein without antibodies to other influenza viral proteins. The HA gene from each of 15 different HA subtypes of influenza virus was cloned into a eukaryotic expression vector and injected intramuscularly, together with a cationic lipid, into 3- to 4-week-old specific-pathogen-free chickens. Birds were boostered twice at 4-week intervals after the initial injection, and in general, antibody titers increased after each boost. The antisera were successfully applied in the hemagglutination inhibition test, which is the standard method for the classification of the HA subtypes of influenza virus. We also demonstrated the HA specificity of the antisera by Western blot and immunodot blot analysis. DNA vaccination also provides a safer alternative for the production of HA-specific antibodies, since it is produced without the use of live virus.