HA1-specific MAbs were identified by immunofluorescence assay (IFA) using Sf-9 insect cells infected with a recombinant baculovirus harboring the HA1-encoding region of the HA gene from A/Indonesia/CDC669/06 (H5N1) virus as described previously (16). The N1 neuraminidase (NA)-specific MAb (8H12) recognized a linear epitope comprising the sequence AELPF. This epitope was 99% conserved in the NA of 708 Givinostat hydrochloride analyzed H5N1 viruses, while the epitope was absent in NAs of subtypes N2 through N9. The specificity of the AC-ELISA was examined by using 41 H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. The estimated detection limit was 1 to 2 2 HA titers. It is concluded that this H5N1 AC-ELISA can simultaneously detect H5 and N1 subtype antigens, eliminating the need Givinostat hydrochloride for secondary testing for the NA subtype. Implementation of this assay in ELISA-like formats suitable for field use, such as dot ELISA, immunofiltration, or electrochemical biosensor technologies, would provide dual on-site detection of H5 and N1 in clinical or environmental specimens. Influenza A virus is classified into subtypes H1 to H16 and N1 to N9 based on the antigenic specificity of hemagglutinin (HA) and neuraminidase (NA) (8). Only two influenza A subtypes (H1N1 and H3N2) are currently circulating in the human population (23). However, the emergence of the H5N1 highly pathogenic avian influenza (HPAI) virus in poultry, causing devastating outbreaks and sporadic infection in humans, has raised the Givinostat hydrochloride concern that the H5N1 subtype virus may lead to the next pandemic. As of September 2008, there have been a total of 387 cases Givinostat hydrochloride of confirmed H5N1 infection in humans, resulting in 245 fatalities (25). Rapid and sensitive laboratory and field tests for the diagnosis of H5N1 HPAI infection are essential for disease control. Conventional laboratory methods for H5N1 virus detection include virus isolation in embryonated eggs or Madin-Darby canine kidney (MDCK) cells, followed by subsequent HA and NA subtype identification using serological methods. Molecular detection methods such as reverse transcriptase PCR (RT-PCR) have been widely applied for the laboratory diagnosis of influenza infections and HA subtype identification (11, 21, 22). In addition, several studies have reported the use of real-time PCR assays and DNA microarray analysis for detection of influenza virus in the laboratory (7, 11, 13, 17). However, both conventional and laboratory methods are technically demanding and are not suitable for on-site use in field investigations. The development of rapid H5 subtype influenza virus detection tests in dot ELISA (enzyme-linked immunosorbent assay), AC-ELISA (antigen-capture ELISA), and chromatographic strip formats (5, 6, 10) using H5 monoclonal antibodies (MAbs) have been reported. However, these assays do not directly identify the N1 NA in the H5N1 viruses, and many can yield positive results with H5 avian influenza viruses with other NA subtypes (e.g., H5N2) that have never been reported to cause infection in humans due to the lack of the ability to identify the NA subtype. The identification of the NA subtype is largely done by RT-PCR (3, 20). NA inhibition assay is the conventional method for NA subtype identification (18). However, it is rarely used because the procedure is very cumbersome and NA subtype-specific antisera are not commercially available. Here we describe an AC-ELISA for the rapid diagnosis of HPAI H5N1 virus infection, based on H5- and N1-specific MAbs that mediate positive identification of H5 HA and N1 NA in a single assay. The selection of MAbs for the development of this H5N1 AC-ELISA was based on detailed characterizations of their binding properties. The sensitivity and specificity of this assay were evaluated using multiple HPAI H5N1 strains and other subtypes of influenza A viruses as well as influenza B viruses. The H5N1 AC-ELISA described here is a proof of concept for the future development of a field test. Our results indicate that this antibody pair could be particularly useful for on-site use in field investigations of H5N1 infection, when incorporated into a rapid field test format based on dot ELISA, immunofiltration, or electrochemical biosensor technologies. MATERIALS AND METHODS Virus cultivation. Twenty-four human and two Givinostat hydrochloride avian H5N1 influenza strains (clade 2.1) isolated from Indonesia were obtained from the Ministry of Health, Indonesia. Five non-H5 subtype strains (Table ?(Table1)1) were obtained from the Agri-Food and Veterinary Authority of Singapore. Fourteen non-H5N1 virus strains (multiple subtypes; see Tables ?Tables11 and ?and2),2), sixteen H1N1, six H3N2, and four influenza KSHV K8 alpha antibody B virus strains were isolated from human clinical samples by the Department of Pathology, Singapore General Hospital. Viruses were inoculated into the allantoic cavities of 11-day-old embryonated chicken eggs and.