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Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody<sup>
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Qigai He,<sup>1</sup> Sumathy Velumani,<sup>1</sup> Qingyun Du,<sup>1</sup> Chee Wee Lim,<sup>2</sup> Fook Kheong Ng,<sup>2</sup> Ruben Donis,<sup>3</sup> and Jimmy Kwang<sup>1</sup><sup>*</sup> Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore,<sup>1</sup> Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore,<sup>2</sup> Centers for Disease Control and Prevention, Atlanta, Georgia<sup>3</sup>
Received 27 November 2006/ Returned for modification 12 January 2007/ Accepted 15 February 2007
<!-- ABS --> The unprecedented spread of highly pathogenic avian influenza<sup> </sup>virus subtype H5N1 in Asia and Europe is threatening animals<sup> </sup>and public health systems. Effective diagnosis and control management<sup> </sup>are needed to control the disease. To this end, we developed<sup> </sup>a panel of monoclonal antibodies (MAbs) against the H5N1 avian<sup> </sup>influenza virus (AIV) and implemented an antigen-capture enzyme-linked<sup> </sup>immunosorbent assay (AC-ELISA) to detect the H5 viral antigen.<sup> </sup>Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97<sup> </sup>(H5N1) expressed in bacteria or immunized with concentrated<sup> </sup>H5N2 virus yielded a panel of hybridomas secreting MAbs specific<sup> </sup>for influenza virus HA. The reactivity of each MAb with several<sup> </sup>subtypes of influenza virus revealed that hybridomas 3D4 and<sup> </sup>8B6 specifically recognized H5 HA. Therefore, purified antibodies<sup> </sup>from hybridomas 3D4 and 8B6, which secrete immunoglobulin G<sup> </sup>(IgG) and IgM, respectively, were used as the capture antibodies<sup> </sup>and pooled hyperimmune guinea pig serum IgG served as the detector<sup> </sup>antibody. The specificity of the optimized AC-ELISA was evaluated<sup> </sup>by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens<sup> </sup>containing AIV subtype H5 subtype yielded a specific and strong<sup> </sup>signal above the background, whereas specimens containing all<sup> </sup>other subtypes yielded background signals. The detection limits<sup> </sup>of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1<sup> </sup>protein and 124, 62, and 31 50% tissue culture infective doses<sup> </sup>of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively.<sup> </sup>Reconstituted clinical samples consisting of H5 AIVs mixed with<sup> </sup>pharyngeal-tracheal mucus from healthy chickens also yielded<sup> </sup>positive signals in the AC-ELISA, and the results were confirmed<sup> </sup>by reverse transcription-PCR. The tracheal swab samples from<sup> </sup>H9N2-infected chickens did not give positive signals. Taken<sup> </sup>together, the newly developed MAb-based AC-ELISA offers an attractive<sup> </sup>alternative to other diagnostic approaches for the specific<sup> </sup>detection of H5 AIV.
</sup>Qigai He,<sup>1</sup> Sumathy Velumani,<sup>1</sup> Qingyun Du,<sup>1</sup> Chee Wee Lim,<sup>2</sup> Fook Kheong Ng,<sup>2</sup> Ruben Donis,<sup>3</sup> and Jimmy Kwang<sup>1</sup><sup>*</sup> Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore,<sup>1</sup> Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore,<sup>2</sup> Centers for Disease Control and Prevention, Atlanta, Georgia<sup>3</sup>
Received 27 November 2006/ Returned for modification 12 January 2007/ Accepted 15 February 2007
<!-- ABS --> The unprecedented spread of highly pathogenic avian influenza<sup> </sup>virus subtype H5N1 in Asia and Europe is threatening animals<sup> </sup>and public health systems. Effective diagnosis and control management<sup> </sup>are needed to control the disease. To this end, we developed<sup> </sup>a panel of monoclonal antibodies (MAbs) against the H5N1 avian<sup> </sup>influenza virus (AIV) and implemented an antigen-capture enzyme-linked<sup> </sup>immunosorbent assay (AC-ELISA) to detect the H5 viral antigen.<sup> </sup>Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97<sup> </sup>(H5N1) expressed in bacteria or immunized with concentrated<sup> </sup>H5N2 virus yielded a panel of hybridomas secreting MAbs specific<sup> </sup>for influenza virus HA. The reactivity of each MAb with several<sup> </sup>subtypes of influenza virus revealed that hybridomas 3D4 and<sup> </sup>8B6 specifically recognized H5 HA. Therefore, purified antibodies<sup> </sup>from hybridomas 3D4 and 8B6, which secrete immunoglobulin G<sup> </sup>(IgG) and IgM, respectively, were used as the capture antibodies<sup> </sup>and pooled hyperimmune guinea pig serum IgG served as the detector<sup> </sup>antibody. The specificity of the optimized AC-ELISA was evaluated<sup> </sup>by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens<sup> </sup>containing AIV subtype H5 subtype yielded a specific and strong<sup> </sup>signal above the background, whereas specimens containing all<sup> </sup>other subtypes yielded background signals. The detection limits<sup> </sup>of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1<sup> </sup>protein and 124, 62, and 31 50% tissue culture infective doses<sup> </sup>of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively.<sup> </sup>Reconstituted clinical samples consisting of H5 AIVs mixed with<sup> </sup>pharyngeal-tracheal mucus from healthy chickens also yielded<sup> </sup>positive signals in the AC-ELISA, and the results were confirmed<sup> </sup>by reverse transcription-PCR. The tracheal swab samples from<sup> </sup>H9N2-infected chickens did not give positive signals. Taken<sup> </sup>together, the newly developed MAb-based AC-ELISA offers an attractive<sup> </sup>alternative to other diagnostic approaches for the specific<sup> </sup>detection of H5 AIV.
