Development of a Nano-ELISA system for the rapid and sensitive detection of H9N2 avian influenza

Document Type: Original Articles


1 Department of Biology, Faculty of Basic science, Payame Noor University, Tehran, Iran

2 Proteomics and Biochemistry Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

3 Department of Microbiology, School of Specialized Veterinary Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.

4 Department of Proteomics and Biochemistry, Razi vaccine and Serum Research Institute, Karaj, Iran

5 Department of medical Nanotechnology, school of advanced Medical Technology, Tehran University of Medical Sciences, Tehran, Iran

6 Department of cellular and molecular Biology, Faculty of biological sciences, Kharazmi University, Tehran, Iran


Influenza is one of the most important viral diseases that is common among the birds and the mammals
and is caused by specific viruses that belong to the Orthomyxoviridae family. Migratory aquatic birds are
the reservoir of the disease and there is a likelihood of the disease in any region. There are different
methods for detecting the avian influenza, but by the point of detection rates, the ELISA may be one of
the most important current methods. In this work we synthesized Gold nanoparticles and conjugated it
with rabbit-anti-chicken IgG-HRP. An ELISA test was done to compare the bioactivity of Au-antichicken
HRP with anti-chicken HRP in order to detect the antibody against the H9N2 subtype of avian
influenza virus. Using 133 field chicken sera, the sensitivity of nano-ELISA as compared to traditional
ELISA was calculated to be 100%, whereas the specificity was 92%. This method was significantly more
sensitive than the traditional ELISA and didn’t require extra costs. It can therefore be concluded that the
AuNP-HRP conjugate can be applicable in immune analysis procedure where a more confident result is


Main Subjects

Article Title [French]

Développement d’un système Nano-ELISA pour la détection rapide et sensible du virus d’influenza aviaire H9N2

Abstract [French]

L’influenza est l’une des maladies virales les plus importantes et les plus répandues chez les oiseaux et les mammifères. Un virus appartenant à la famille des Orthomyxoviridae est à l’origine de cette pathologie. Les oiseaux migratoires aquatiques représentent le réservoir naturel de ce virus et cette maladie peut donc affecter toutes les régions du monde. Il existe différentes méthodes de détection de l’influenza aviaire, mais l’ELISA semble représenter l’une des méthodes les plus pertinentes avec un fort taux de détection. Dans cette étude, nous avons synthétisé des nanoparticules d’or associées à des anticorps de lapin anti-IgG de poulet marqués à l’HRP. Un test ELISA a été effectué afin de comparer la bio-activité des anticorps associés et non-associés aux particules d’or dans la détection du sous-type H9N2 du virus de l’influenza aviaire. La sensibilité de notre nano-ELISA a été comparée à celle de l’ELISA traditionnel sur 133 sérums de poulets fermiers. Les résultats obtenus montrent que la sensibilité du nano-ELISA est supérieure à l’ELISA traditionnel (6 échantillons positifs supplémentaires détectés) avec une spécificité de 92%. Etant donnée la sensibilité plus importante des anticorps de lapin associés aux nanoparticules d’or, l’utilisation de ces réactifs peut augmenter la fiabilité des essais immunologiques diagnostiques.

Keywords [French]

  • Influenza aviaire
  • nanoparticule d’or

Alexander, D.J., 2007. An overview of the epidemiology of avian influenza. Vaccine 25, 5637-5644.

Ambrosi, A., Airo, F., Merkoci, A., 2010. Enhanced gold nanoparticle based ELISA for a breast cancer biomarker. Anal Chem 82, 1151-1156.

Brown, J.D., Stallknecht, D.E., Berghaus, R.D., Luttrell, M.P., Velek, K., Kistler, W., Costa, T., Yabsley, M.J., Swayne, D., 2009. Evaluation of a commercial blocking enzyme-linked immunosorbent assay to detect avian influenza virus antibodies in multiple experimentally infected avian species. Clin Vaccine Immunol 16, 824-829.

Engvall, E., Perlmann, P., 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871-874.

Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79, 2814-2822.

Ge, F.-F., Zhou, J.-P., Liu, J., Wang, J., Zhang, W.-Y., Sheng, L.-P., Xu, F., Ju, H.-B., Sun, Q.-Y., Liu, P.-H., 2009. Genetic Evolution of H9 Subtype Influenza Viruses from Live Poultry Markets in Shanghai, China. Journal of Clinical Microbiology 47, 3294-3300.

Guan, Y., Shortridge, K.F., Krauss, S., Webster, R.G., 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong? Proc Natl Acad Sci U S A 96, 9363-9367.

Hoang, H., Phan, T.H., Conrad, U., Chu, H.H., 2012. Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development. Nanosci Nanotechnol 3, 45017.

Homayounimehr, A.R., Dadras, H., Shoushtari, A., Pourbakhsh, S.A., 2010. Sequence and phylogenetic analysis of the haemagglutinin genes of H9N2 avian influenza viruses isolated from commercial chickens in Iran. Trop Anim Health Prod 42, 1291-1297.

Jia, C.P., Zhong, X.Q., Hua, B., Liu, M.Y., Jing, F.X., Lou, X.H., Yao, S.H., Xiang, J.Q., Jin, Q.H., Zhao, J.L., 2009a. Nano-ELISA for highly sensitive protein detection. Biosens Bioelectron 24, 2836-2841.

Jia, N., de Vlas, S.J., Liu, Y.X., Zhang, J.S., Zhan, L., Dang, R.L., Ma, Y.H., Wang, X.J., Liu, T., Yang, G.P., Wen, Q.L., Richardus, J.H., Lu, S., Cao, W.C., 2009b. Serological reports of human infections of H7 and H9 avian influenza viruses in northern China. J Clin Virol 44, 225-229.

Kumar, S., Aaron, J., Sokolov, K., 2008. Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat Protoc 3, 314-320.

Liu, M., Jia, C., Huang, Y., Lou, X., Yao, S., Jin, Q., Zhao, J., Xiang, J., 2010. Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes. Analyst 135, 327-331.

Loeffelholz, M.J., 2010. Avian influenza A H5N1 virus. Clinics Lab Med 30, 1-20.

Peiris, M., Yuen, K.Y., Leung, C.W., Chan, K.H., Ip, P.L., Lai, R.W., Orr, W.K., Shortridge, K.F., 1999. Human infection with influenza H9N2. Lancet 354, 916-917.

Peng, C.-F., Duan, X.-H., Pan, Q.-L., Liu, L.-Q., Xue, F., 2013. Ultrasensitive Nano-ELISA for Detecting Sulfadimethoxine in Chicken Tissue. Journal of Chemistry 2013, 5.

Shafer, A.L., Katz, J.B., Eernisse, K.A., 1998. Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Dis 42, 28-34.

Starick, E., Werner, O., Schirrmeier, H., Kollner, B., Riebe, R., Mundt, E., 2006. Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species. J Vet Med B Infect Dis Vet Public Health 53, 370-375.

Tansil, N.C., Gao, Z., 2006. Nanoparticles in biomolecular detection. Nano Today 1, 28-37.

Uyeki, T.M., Chong, Y.H., Katz, J.M., Lim, W., Ho, Y.Y., Wang, S.S., Tsang, T.H., Au, W.W., Chan, S.C., Rowe, T., Hu-Primmer, J., Bell, J.C., Thompson, W.W., Bridges, C.B., Cox, N.J., Mak, K.H., Fukuda, K., 2002. Lack of evidence for human-to-human transmission of avian influenza A (H9N2) viruses in Hong Kong, China 1999. Emerg Infect Dis 8, 154-159.

Webster, R.G., Hulse, D.J., 2004. Microbial adaptation and change: avian influenza. Rev Sci Tech 23, 453-465.

Wu, R., Hu, S., Xiao, Y., Li, Z., Shi, D., Bi, D., 2007. Development of indirect enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection and quantification of antibodies against avian influenza virus. Vet Res Commun 31, 631-641.