Simultaneous detection of avian influenza virus NP and H5 antibodies in chicken sera using a fluorescence microsphere immunoassay

Avian influenza (AI) surveillance in commercial poultry is accomplished by detecting the presence of antibodies to two group-specific antigens, NP and M1, using the agar gel immunodiffusion test. In order to determine the viral subtype responsible for the infection, positive samples must be further subtyped using the hemagglutination inhibition and neuraminidase inhibition tests. These tests are labor intensive and may take up to 4 days, thus slowing down responses to outbreaks. To expedite the subtyping of chicken sera we have developed a multiplex fluorescence microsphere immunoassay (FMIA), which allows for the simultaneous detection and subtyping of chicken sera to H5 influenza viruses. The FMIA was developed using NP (full length) and H5 (HA1 region) proteins expressed in baby hamster kidney cells using a Venezuela equine encephalitis virus replicon system. Both proteins were tagged with 6xHis at the carboxy-end and purified using cobalt-coated agarose beads. Purified H5 protein showed minimal cross-reactivity with anti-H2 serum, while no cross-reactivity was observed with sera to other AI virus (AIV) subtypes and other important poultry viral pathogens. In addition, and as expected, all the AIV sera tested reacted strongly with purified NP protein. Our results indicate that FMIA can be used for rapid subtyping of chicken sera.