Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera.

Serologic screening of avian sera for group-specific antibodies to type A influenza is currently accomplished by using the avian influenza (AI) agar gel immunodiffusion (AGID) test. A competitive enzyme-linked immunosorbent assay (CELISA) was developed using a baculovirus vector, Autographa californica nuclear polyhedrosis virus, expressing the nucleoprotein (NP) gene of A/Ann Arbor/6/60 influenza virus. The recombinant NP was obtained by inoculation of Spodoptera frugiperda (Sf9) insect cells or Trichoplusia ni insect larvae with the recombinant baculovirus. A hybridoma cell line producing monoclonal antibody against influenza virus A nucleoprotein was used to generate mouse ascitic fluid for the CELISA. The nucleoprotein and the monoclonal antibody were used without further purification in a CELISA for detection of avian-origin serum antibodies to type A influenza. The AI AGID and CELISA tests were compared for sensitivity and specificity using 1651 experimental and reference antisera. Samples discrepant in AGID and CELISA test results were further evaluated by the AI indirect fluorescent antibody (IFA), hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) tests. The results demonstrated a high degree of correlation between the AGID and CELISA test results, with the IFA, HI, and NI tests offering additional support of CELISA test specificity. The CELISA is a rapid, economical, sensitive, and specific serodiagnostic method for screening large numbers of avian sera for antibodies to avian influenza virus.