Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody Anariwa Du a,b , Tomo Daidoji a , Takaaki Koma a,b,1 , Madiha S. Ibrahim b , Shota Nakamura c , U. Chandimal de Silva c , Mayo Ueda a,b , Cheng-Song Yang a,b , Teruo Yasunaga c , Kazuyoshi Ikuta b, * , Takaaki Nakaya a a International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan b Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan c Department of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan article info Article history: Received 29 October 2008 Available online 14 November 2008 Keywords: Avian influenza virus H5N1 Monoclonal antibody Neutralizing antibody ELISA abstract Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb spe- cifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody rec- ognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis dem- onstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses. Ó 2008 Elsevier Inc. All rights reserved. The first outbreak in humans caused by the highly pathogenic avian influenza virus H5N1 occurred in Hong Kong in 1997. During this outbreak, H5N1 caused respiratory disease in 18 people, six of whom died [1]. The H5N1 viruses have now appeared in more than 60 countries across three continents with continued capacity to in- fect humans [2]. The viruses appear to be evolving and diversifying, and while most genes have undergone reassortment yielding many different genotypes, the hemagglutinin (HA) protein, remarkably, has not been replaced in the various isolates since 1996 [2]. Since 2003, more than 380 human H5N1 infections have been identified, of which more than 240 have been fatal (as of September 2008) [3], raising serious worldwide concerns about a severe influenza pandemic. The accurate and prompt diagnosis of an H5N1 infection is a critical component of a disease control plan [4]. Different diagnos- tic approaches, including viral neutralization assay, indirect en- zyme-linked immunosorbent assays (ELISAs), hemagglutination inhibition (HI) assay, and antigen-capture (AC)-ELISAs, are avail- able for avian influenza viruses (AIV) [4]; however, nucleic acid amplification tests, such as reverse-transcriptase polymerase chain reaction (RT-PCR) and loop-mediated isothermal amplification, are considered the most effective and sensitive methods for detecting and subtyping AIV [5]. Reportedly, anti-HA monoclonal antibodies (MAbs) against the H5N1 virus isolated in 1997 (A/Hong Kong/156/97) did not recog- nize recent isolates (A/Hong Kong/213/2003), indicating that there are substantial antigenic differences between the H5N1 strain iso- lated in 1997 and the strains circulating since 2003 [6]. Further- more, the HA antigenic structure differs substantially between the A/Vietnam/1203/04 (H5N1) virus and the low-pathogenic A/ Mallard/Pennsylvania/10218/84 (H5N2) virus [7]. The recently de- fined three-dimensional HA structure of the highly pathogenic H5N1 strain A/Vietnam/1203/04 [8] differs from the HA structure of the A/Duck/Singapore/3/97 (H5N3) virus [9]. Together, these findings suggest that MAbs against the currently circulating H5N1 viruses are necessary for diagnosis. In this study, we developed a panel of MAbs against the H5N1 AIV (A/crow/Kyoto/53/2004; Clade 2.5 [10]) and investigated the reactivity of five anti-HA MAbs. We show that four of the five MAbs, neutralized the H5N1 virus as well as the H5N2 and H5N3 viruses. On the other hand, using the well-established 4G6 MAb, we distinguished the current H5N1 virus from other H5 viruses such as A/Duck/Hong Kong/342/78 (H5N2) and A/Duck/Hong 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.022 * Corresponding author. Fax: +81 6 6879 8310. E-mail address: ikuta@biken.osaka-u.ac.jp (K. Ikuta). 1 Present address: Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine, Sapporo, Japan. Biochemical and Biophysical Research Communications 378 (2009) 197–202 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc