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Hemagglutination Inhibition Assay

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Animal Influenza Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2123))

Abstract

The hemagglutination inhibition (HI) assay for influenza A virus has been used since the 1940s. The assay may be utilized to detect or quantify antibodies to influenza A viruses and can be used to characterize differences in antigenic reactivity between influenza isolates. In addition, data from HI assays are routinely used for antigenic cartography, influenza virus surveillance, epidemiology, and vaccine-seed strain selection. For antibody quantification, the HI assay is a fast and inexpensive method; other than a source of red blood cells, no expensive or unusual lab equipment is needed, and results can be obtained within a few hours. Historically, the HI assay has also served as a primary method of subtype identification and is still used widely. However, as gene sequencing technology has evolved to be cheaper and faster, it is replacing the HI assay for this purpose.

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References

  1. McClelland L, Hare R (1941) The adsorption of influenza virus by red cells and a new in vitro method of measuring antibodies for influenza virus. Can Public Health J 32(10):530–538

    Google Scholar 

  2. Young GA Jr, Underdahl NR (1950) Neutralization and hemagglutination inhibition of swine influenza virus by serum from suckling swine and by milk from their dams. J Immunol 65(4):369–373

    PubMed  Google Scholar 

  3. Cohen A, Biddle F (1963) Hemagglutination inhibition: interaction of influenza viruses with horse serum inhibitor. Nature 198:508–509

    Article  CAS  Google Scholar 

  4. OIE (2018) Manual of diagnostic tests and vaccines for terrestrial animals 2018. World Organization for Animal Health (OIE). http://www.oie.int/standard-setting/terrestrial-manual/access-online/. Accessed 20 Sept 2018

  5. Anderson TC, Crawford PC, Katz JM, Dubovi EJ, Landolt G, Gibbs EP (2012) Diagnostic performance of the canine influenza A virus subtype H3N8 hemagglutination inhibition assay. J Vet Diagn Investig 24(3):499–508. https://doi.org/10.1177/1040638712440992

    Article  Google Scholar 

  6. Zacour M, Ward BJ, Brewer A, Tang P, Boivin G, Li Y, Warhuus M, McNeil SA, LeBlanc JJ, Hatchette TF, Public Health Agency of C, Canadian Institutes of Health Influenza Research N (2016) Standardization of hemagglutination inhibition assay for influenza serology allows for high reproducibility between laboratories. Clin Vaccine Immunol 23(3):236–242. https://doi.org/10.1128/CVI.00613-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wikramaratna PS, Rambaut A (2015) Relationship between haemagglutination inhibition titre and immunity to influenza in ferrets. Vaccine 33(41):5380–5385. https://doi.org/10.1016/j.vaccine.2015.08.065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Stephenson I, Das RG, Wood JM, Katz JM (2007) Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine 25(20):4056–4063. https://doi.org/10.1016/j.vaccine.2007.02.039

    Article  CAS  PubMed  Google Scholar 

  9. Stephenson I, Heath A, Major D, Newman RW, Hoschler K, Junzi W, Katz JM, Weir JP, Zambon MC, Wood JM (2009) Reproducibility of serologic assays for influenza virus A (H5N1). Emerg Infect Dis 15(8):1252–1259. https://doi.org/10.3201/eid1508.081754

    Article  PubMed  Google Scholar 

  10. Wood JM, Gaines-Das RE, Taylor J, Chakraverty P (1994) Comparison of influenza serological techniques by international collaborative study. Vaccine 12(2):167–174

    Article  CAS  Google Scholar 

  11. Abdelwhab EM, Grund C, Aly MM, Beer M, Harder TC, Hafez HM (2016) Benefits and limits of egg yolk vs. serum samples for avian influenza virus serosurveillance. Avian Dis 60(2):496–499. https://doi.org/10.1637/11207-060115-ResNote

    Article  CAS  PubMed  Google Scholar 

  12. Kumar M, Chu HJ, Rodenberg J, Krauss S, Webster RG (2007) Association of serologic and protective responses of avian influenza vaccines in chickens. Avian Dis 51(1 Suppl):481–483

    Article  Google Scholar 

  13. Swayne DE, Suarez DL, Spackman E, Jadhao S, Dauphin G, Kim-Torchetti M, McGrane J, Weaver J, Daniels P, Wong F, Selleck P, Wiyono A, Indriani R, Yupiana Y, Sawitri Siregar E, Prajitno T, Smith D, Fouchier R (2015) Antibody titer has positive predictive value for vaccine protection against challenge with natural antigenic-drift variants of H5N1 high-pathogenicity avian influenza viruses from Indonesia. J Virol 89(7):3746–3762. https://doi.org/10.1128/JVI.00025-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tian G, Zeng X, Li Y, Shi J, Chen H (2010) Protective efficacy of the H5 inactivated vaccine against different highly pathogenic H5N1 avian influenza viruses isolated in China and Vietnam. Avian Dis 54(1 Suppl):287–289

    Article  Google Scholar 

  15. Commission E (2006) Commission decision 2006/437/EC of 4 August 2006 approving a diagnostic manual for avian influenza as provided for council directive 2005/94/EC. Off J Eur Union 237

    Google Scholar 

  16. UN OIE (2012) Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees). http://www.oie.int/manual-of-diagnostic-tests-and-vaccines-for-terrestrial-animals/. Accessed 10 Jan 2019

  17. Comin A, Toft N, Stegeman A, Klinkenberg D, Marangon S (2013) Serological diagnosis of avian influenza in poultry: is the haemagglutination inhibition test really the ‘gold standard’? Influenza Other Respir Viruses 7(3):257–264. https://doi.org/10.1111/j.1750-2659.2012.00391.x

    Article  CAS  PubMed  Google Scholar 

  18. Truelove S, Zhu H, Lessler J, Riley S, Read JM, Wang S, Kwok KO, Guan Y, Jiang CQ, Cummings DA (2016) A comparison of hemagglutination inhibition and neutralization assays for characterizing immunity to seasonal influenza A. Influenza Other Respir Viruses 10(6):518–524. https://doi.org/10.1111/irv.12408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shiraishi R, Nishiguchi A, Tsukamoto K, Muramatsu M (2012) Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses. J Vet Med Sci 74(9):1217–1220

    Article  Google Scholar 

  20. Prince HE, Leber AL (2003) Comparison of complement fixation and hemagglutination inhibition assays for detecting antibody responses following influenza virus vaccination. Clin Diagn Lab Immunol 10(3):481–482

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Arnold ME, Slomka MJ, Breed AC, Hjulsager CK, Pritz-Verschuren S, Venema-Kemper S, Bouwstra RJ, Trebbien R, Zohari S, Ceeraz V, Larsen LE, Manvell RJ, Koch G, Brown IH (2018) Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks. Epidemiol Infect 146(3):306–313. https://doi.org/10.1017/S0950268817002898

    Article  CAS  PubMed  Google Scholar 

  22. Swayne DE, Suarez DL, Sims LD (2013) Influenza. In: Swayne D (ed) Diseases of poultry, 13th edn. Blackwell, Ames, IA, pp 181–218

    Chapter  Google Scholar 

  23. Wood JM, Major D, Heath A, Newman RW, Hoschler K, Stephenson I, Clark T, Katz JM, Zambon MC (2012) Reproducibility of serology assays for pandemic influenza H1N1: collaborative study to evaluate a candidate WHO International Standard. Vaccine 30(2):210–217. https://doi.org/10.1016/j.vaccine.2011.11.019

    Article  PubMed  Google Scholar 

  24. Wagner R, Gopfert C, Hammann J, Neumann B, Wood J, Newman R, Wallis C, Alex N, Pfleiderer M (2012) Enhancing the reproducibility of serological methods used to evaluate immunogenicity of pandemic H1N1 influenza vaccines-an effective EU regulatory approach. Vaccine 30(27):4113–4122. https://doi.org/10.1016/j.vaccine.2012.02.077

    Article  CAS  PubMed  Google Scholar 

  25. Noah DL, Hill H, Hines D, White EL, Wolff MC (2009) Qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure. Clin Vaccine Immunol 16(4):558–566. https://doi.org/10.1128/CVI.00368-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Morrison BJ, Martin NJ, Rehman T, Ewing D, Dewar RL, Metcalf J, Sun P, Beigel J, Luke TC, Raviprakash K (2018) Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays. BMC Health Serv Res 18(1):651. https://doi.org/10.1186/s12913-018-3465-3

    Article  PubMed  PubMed Central  Google Scholar 

  27. Wilson G, Ye Z, Xie H, Vahl S, Dawson E, Rowlen K (2017) Automated interpretation of influenza hemagglutination inhibition (HAI) assays: Is plate tilting necessary? PLoS One 12(6):e0179939. https://doi.org/10.1371/journal.pone.0179939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ananthanarayan R, Paniker CK (1960) Non-specific inhibitors of influenza viruses in normal sera. Bull World Health Organ 22:409–419

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Jordan WS Jr, Oseasohn RO (1954) The use of RDE to improve the sensitivity of the hemagglutination-inhibition test for the serologic diagnosis of influenza. J Immunol 72(3):229–235

    PubMed  Google Scholar 

  30. Cai Z, Zhang T, Wan XF (2010) A computational framework for influenza antigenic cartography. PLoS Comput Biol 6(10):e1000949. https://doi.org/10.1371/journal.pcbi.1000949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM (2004) Mapping the antigenic and genetic evolution of influenza virus. Science 1097211

    Google Scholar 

  32. Sitaras I, Kalthoff D, Beer M, Peeters B, de Jong MC (2014) Immune escape mutants of highly pathogenic avian influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein. PLoS One 9(2):e84628. https://doi.org/10.1371/journal.pone.0084628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sitaras I, Duijzer M, Peeters B, de Jong MCM (2019) Influence of inter-animal variability of HI titres on antigenic cartography in the study of avian influenza viruses: towards making a better map. Vaccine Submitted Manuscript

    Google Scholar 

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Authors and Affiliations

  1. Exotic and Emerging Avian Viral Diseases Unit, US National Poultry Research Center, US Department of Agriculture, Agricultural Research Service, Athens, GA, USA

    Erica Spackman & Ioannis Sitaras

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  1. Erica Spackman
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  2. Ioannis Sitaras
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Correspondence to Erica Spackman .

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  1. Exotic and Emerging Avian Viral Diseases Unit, US National Poultry Research Center, US Department of Agriculture, Agricultural Research Service, Athens, GA, USA

    Erica Spackman

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© 2020 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.

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Spackman, E., Sitaras, I. (2020). Hemagglutination Inhibition Assay. In: Spackman, E. (eds) Animal Influenza Virus. Methods in Molecular Biology, vol 2123. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0346-8_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0346-8_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0345-1

  • Online ISBN: 978-1-0716-0346-8

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