Skip to main content
Log in

Adaptations of a Competitive Enzyme-Linked Immunosorbent Assays for the Detection of Antibodies to Influenza A Virus in Horse Sera for Use in Wild Aquatic Birds

  • Original Article
  • Published:
Indian Journal of Virology Aims and scope Submit manuscript

Abstract

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2′-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Abraham A, Sivanandan V, Karunakaran D, Halvorson DA, Newman JA. Comparative serological evaluation of avian influenza vaccine in turkeys. Avian Dis. 1988;32:659–62.

    Article  PubMed  CAS  Google Scholar 

  2. Adair BM, Todd D, McKillop ER, McNulty MS. Detection of influenza A type-specific antibodies in chicken and turkey sera by enzyme linked immunosorbent assay. Avian Patho. 1989;18:455–63.

    Article  CAS  Google Scholar 

  3. Jeong OM, Kim MC, Kang HM, Ha GW, Oh JS, Yoo JE, Park CH, Kwon JS, Pack MR, Kim HR, Kim YJ, Kwon JH, Lee YJ. Validation of egg yolk antibody based C-ELISA for avian influenza surveillance in breeder duck. Vet Microbiol. 2010;144:287–92.

    Article  PubMed  CAS  Google Scholar 

  4. Katz JB, Shafer AL, Eernisse KA. Construction and insect larval expression of recombinant vesicular stomatitis nucleocapsid protein and its use in competitive ELISA. J Virol Methods. 1995;54:145–57.

    Article  PubMed  CAS  Google Scholar 

  5. Marche S, van den Berg T. Evaluation of different strategies for the use of ELISA tests as first screening tools for serologic surveillance of low pathogenic avian influenza in the Belgian poultry sector. Avian Dis. 2010;54:627–31.

    Article  PubMed  Google Scholar 

  6. Noordhuizen JPTM, Frankena K, Van der Hoofd CM, Graat EAM. Application of quantitative methods in Veterinary epidemiology. 1st ed. Wageningen: Wageningen Press; 1997.

    Google Scholar 

  7. Perez-Ramirez E, Rodriguez V, Sommer D, Blanco JM, Acevedo P, Heffels-Redmann U, Hofle U. Serologic testing for avian influenza viruses in wild birds: comparison of two commercial competition enzyme-linked immunosorbent assays. Avian Dis. 2010;54:729–33.

    Article  PubMed  Google Scholar 

  8. Selleck P. Influenza A virus: a competitive ELISA for the detection of antibodies to influenza A viruses in equine sera, Equine influenza c-ELISA protocol. Geelong: Australian Animal Health Laboratory, Disease Diagnostic Project CSIRO Animal Health Laboratory; 2007.

    Google Scholar 

  9. Shafer AL, Katz JB, Eernisse KA. Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Dis. 1998;42:28–34.

    Article  PubMed  CAS  Google Scholar 

  10. Snyder DB, Marquardt WW, Yancey FS, Savage PK. An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus. Avian Dis. 1985;29:136–44.

    Article  PubMed  CAS  Google Scholar 

  11. Song DS, Lee YJ, Jeong OM, Kim YJ, Park CH, Yoo JE, Jeon WJ, Kwon JH, Ha GW, Kang BK, Lee CS, Kim HK, Jung BY, Kim JH, Oh JS. Evaluation of a competitive ELISA for antibody detection against avian influenza virus. J Vet Sci. 2009;10:323–9.

    Article  PubMed  Google Scholar 

  12. Starick E, Werner O, Schirrmeier H, Kollner B, Riebe R, Mundt E. 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. 2006;53:370–5.

    Article  PubMed  CAS  Google Scholar 

  13. Zhou EM, Chan M, Heckert RA, Riva J, Cantin MF. Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein. Avian Dis. 1998;42:517–22.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are very grateful to the Australian Biosecurity Cooperative Research Centre (ABCRC) and Australian Department of Agriculture, Forestry and Fisheries (DAFF) for providing project financial support. We thank AusAID for providing an Australian Leadership Award (ALA) and the ABCRC for a top-up PhD scholarship to MAH. We extend our thanks to the scientific and ethics permit authorities for providing the following project approvals, Eco-access permit No. WISp04374507, Queensland Parks and Wildlife Service, northern Region and ethics permit No. A 1175, JCU. Finally, we extend our heartfelt thanks to the members of our AI research team and other contributors to our research for their excellent co-operation during the study period.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M. A. Hoque.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hoque, M.A., Skerratt, L.F., Garland, S. et al. Adaptations of a Competitive Enzyme-Linked Immunosorbent Assays for the Detection of Antibodies to Influenza A Virus in Horse Sera for Use in Wild Aquatic Birds. Indian J. Virol. 23, 261–269 (2012). https://doi.org/10.1007/s13337-012-0074-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13337-012-0074-3

Keywords

Navigation