Development of an Immunochromatographic Assay Specifically Detecting Pandemic H1N1 (2009) Influenza Virus

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. As of 27 September 2009, more than 4,100 deaths from AH1pdm infection have been reported to the World Health Organization (WHO) (http://www.who.int/csr/don/2009_10_02/en/index.html). Current methods used to diagnose AH1pdm virus in clinical specimens are based on viral RNA analysis targeting hemagglutinin (HA) genes, because the HA genes are among the most specific genes in the influenza virus genome. Although these methods are highly sensitive, they usually take more than 2 to 6 h to complete and require well-equipped laboratories with virologists or well-trained medical technicians and specialized tools for virus genome isolation and amplification (6, 8) (http://www.who.int/entity/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf). Rapid diagnostic kits (RDKs) based on immunochromatography utilize antibodies (Abs) against antigens of interest. Although RDKs are usually less sensitive than genetic assays, they do not require the isolation of a viral genome, thus overcoming the intrinsic difficulties of viral gene analyses. RDKs for many infectious diseases (2, 4, 9, 11-14), including influenza viruses A and B (1), are commercially available. However, RDKs capable of distinguishing AH1pdm viruses from seasonal influenza viruses have yet to be implemented in a clinical setting.Nucleoproteins (NPs) of influenza A, B, and C viruses have important differences in their antigenicities that enable them to be distinguished from one another but are highly conserved within each major serotype. Thus, antibodies to NPs have been utilized in commercially available RDKs to distinguish between influenza A and B viruses (15). In a monoclonal antibody (MAb) preparation procedure targeting NPs derived from highly pathogenic H5N1 avian influenza (HPAI), we obtained 2 MAbs that reacted with NPs of AH1pdm as well as that of HPAI but not those of seasonal influenza A virus. We have therefore utilized these MAbs in the development of novel RDKs for AH1pdm, and we have validated these RDKs in laboratory environments.