Development of blocking ELISA for detection of antibodies against H9N2 avian influenza viruses.

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies against H9N2 avian influenza viruses (AIVs) based on a monoclonal antibody specific to the hemagglutinin (HA) protein of H9N2 AIV. The specificity of the bELISA was tested using antisera against H3, H4, H5, H7, and H10 AIVs and other avian viruses. The average percent inhibition (PI) value of 116 non-immune serum samples from ducks and specific pathogen-free (SPF) chickens was 2.80%. The selected cut-off PI values for negative and positive sera were 17.6% and 25.0%, respectively. A high correlation of >96% was identified between the bELISA and hemagglutinin inhibition (HI) assay, according to the detection results of sera from infected chickens (n=30) and from chickens vaccinated with an inactivated H9N2 vaccine (n=40). Sera collected from vaccinated chickens and ducks (n=660) at different weeks post vaccination that were positive by the HI assay were confirmed with the bELISA. The results revealed a time-dependent increase in antibody levels. Therefore, the bELISA offers the potential advantage of a high throughput, rapid, sensitive, and specific method for detection of specific antibodies against H9N2.