Subtyping Influenza A Virus with Monoclonal Antibodies and an Indirect Immunofluorescence Assay

The recent association of certain influenza A virus subtypes with clinically relevant phenotypes has led to the increasing importance of subtyping by clinical virology laboratories. To provide clinical laboratories with a definitive immunofluorescence assay for the subtyping of influenza A virus isolates, we generated a panel of monoclonal antibodies (MAbs) against the major circulating influenza A virus subtypes using multiple inactivated H1N1, H3N2, and 2009 H1N1 strains individually as immunogens. Eleven MAbs that target hemagglutinin (HA) of H1N1 and H3N2 subtypes were selected. These MAbs were combined into three subtype-specific reagents, one each for pan-H1 (seasonal and 2009 strains), H3, and 2009 H1, for the subtyping of influenza A virus-positive specimens by indirect immunofluorescence assay (IFA). Each subtype-specific reagent was tested on 21 prototype influenza A virus strains and confirmed to be specific for its intended subtype. In addition, the subtyping reagents did not cross-react with any of 40 other viruses. The clinical performance of the subtyping reagents was evaluated with 75 archived clinical samples collected between 2006 and 2009 using the D(3) Ultra DFA influenza A virus identification reagent (Diagnostic Hybrids, Inc., Athens, OH) and the influenza A virus subtyping reagents by IFA simultaneously. Sixty-four samples grew virus and were subtyped as follows: 30 as H3N2, 9 as seasonal H1N1, and 25 as 2009 H1N1. RT-PCR was used to confirm the influenza A virus subtyping of these samples, and there was 100% agreement with IFA. This subtyping IFA provides clinical laboratories with a cost-effective diagnostic tool for better management of influenza virus infection and surveillance of influenza virus activity.