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Clinical and Vaccine Immunology, May 2007, p. 617-623, Vol. 14, No. 5
1071-412X/07/$08.00+0     doi:10.1128/CVI.00444-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody{triangledown}

Qigai He,1 Sumathy Velumani,1 Qingyun Du,1 Chee Wee Lim,2 Fook Kheong Ng,2 Ruben Donis,3 and Jimmy Kwang1*

Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore,1 Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore,2 Centers for Disease Control and Prevention, Atlanta, Georgia3

Received 27 November 2006/ Returned for modification 12 January 2007/ Accepted 15 February 2007

The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.

* Corresponding author. Mailing address: Animal Health Biotechnology, Temasek Life Science Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore. Phone: 65-68727473. Fax: 65-68727007. E-mail: kwang{at}tll.org.sg

{triangledown} Published ahead of print on 7 March 2007.

Clinical and Vaccine Immunology, May 2007, p. 617-623, Vol. 14, No. 5
1071-412X/07/$08.00+0     doi:10.1128/CVI.00444-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ho, H.-T., Qian, H.-L., He, F., Meng, T., Szyporta, M., Prabhu, N., Prabakaran, M., Chan, K.-P., Kwang, J. (2009). Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies. CVI 16: 726-732 [Abstract] [Full Text]  
  • Mahony, J. B. (2008). Detection of Respiratory Viruses by Molecular Methods. Clin. Microbiol. Rev. 21: 716-747 [Abstract] [Full Text]  


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