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Open Access Highly Accessed Open Badges Methodology article

Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus

Fang He1, Retno D Soejoedono2, Sri Murtini2, Michael Goutama3 and Jimmy Kwang134*

Author Affiliations

1 Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 117604

2 Faculty of Veterinary Medicine, Depart of Infectious diseases & Vet. Public Health, Division of Microbiology, Institute Pertanian, Bogor, Indonesia

3 Tridel Biosciences International Pte Ltd, Singapore

4 Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore

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BMC Microbiology 2010, 10:330  doi:10.1186/1471-2180-10-330

Published: 30 December 2010



Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection.


In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including 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. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples.


Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry.