An Immunoproteomics Approach to Screen the Antigenicity of the Influenza Virus

  • Kevin M. Downard
Part of the Methods in Molecular Biology book series (MIMB, volume 1061)


The structure and antigenicity of protein antigens of the influenza virus are screened in a single step employing an immunoproteomics approach. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) coupled to gel electrophoresis is used both to identify viral antigens and screen their antigenicity. Earlier evidence that antigen–antibody complexes can survive on MALDI targets has allowed both the primary structure and antigenicity of viral strains to be rapidly screened with the specific localization of protein epitopes. The approach is anticipated to have a greater role in the future surveillance of the virus and should also aid in the development of immunogenic peptide constructs as alternatives to whole virus for vaccination.

Key words

Influenza Flu Virus Surveillance Antigenicity Mass spectrometry Proteomics Immunoproteomics 



The author acknowledges the contributions of Bethny Morrissey, Dr. Alexander Schwahn, Dr. Margaret Streamer, and Joshua Ho as indicated by the cited works. The author thanks Robert Shaw and Dr. Ian Barr of the WHO Collaborating Centre for Reference and Research on Influenza and Dr. Elizabeth Pietrzykowski of CSL Limited for the supply of some virus strains and monoclonal antibodies, respectively. Funding for this work was provided by the Australian Research Council (DP0449800 and DP0770619 to the author) and the University of Sydney.


  1. 1.
    Nicholson KG, Wood JM, Zambon M (2003) Influenza. Lancet 362:1733–1745PubMedCrossRefGoogle Scholar
  2. 2.
    Barry JM (2004) The great influenza: the epic story of the deadliest plague in history. Penguin Viking, New YorkGoogle Scholar
  3. 3.
    World Health Organisation Global Influenza Surveillance Network.
  4. 4.
    Stephenson I, Nicholson KG (2001) Influenza: vaccination and treatment. Eur Respir J 17:1282–1293PubMedCrossRefGoogle Scholar
  5. 5.
    Girard MP, Cherian T, Pervikov Y, Kieny MP (2005) A review of vaccine research and development: human acute respiratory infections. Vaccine 23:5708–5724PubMedCrossRefGoogle Scholar
  6. 6.
    Garman E, Laver G (2004) Controlling influenza by inhibiting the virus’s neuraminidase. Curr Drug Targets 5:119–136PubMedCrossRefGoogle Scholar
  7. 7.
    Webster RG, Walker EJ (2003) Influenza: the world is teetering on the edge of a pandemic that could kill a large fraction of the human population. Am Sci 91:122CrossRefGoogle Scholar
  8. 8.
    Horimoto T, Kawaoka Y (2005) Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3:591–600PubMedCrossRefGoogle Scholar
  9. 9.
    Kiselar JG, Downard KM (1999) Direct identification of protein epitopes by mass spectrometry without immobilization of antibody and isolation of antibody–peptide complexes. Anal Chem 71:1792–1799PubMedCrossRefGoogle Scholar
  10. 10.
    Kiselar JG, Downard KM (1999) Antigenic surveillance of the influenza virus by mass spectrometry. Biochemistry 38:14185–14191PubMedCrossRefGoogle Scholar
  11. 11.
    Morrissey B, Downard KM (2006) A proteomics approach to survey the antigenicity of the influenza virus by mass spectrometry. Proteomics 6:2034–2041PubMedCrossRefGoogle Scholar
  12. 12.
    Morrissey B, Streamer M, Downard KM (2007) Antigenic characterisation of H3N2 subtypes of the influenza virus by mass spectrometry. J Virol Methods 145:106–114PubMedCrossRefGoogle Scholar
  13. 13.
    Downard KM, Morrissey B (2007) Fingerprinting a killer – surveillance of the influenza virus by mass spectrometry. Analyst 132:611–614PubMedCrossRefGoogle Scholar
  14. 14.
    Kiselar JG, Downard KM (2000) Preservation and detection of specific antibody-peptide complexes by matrix-assisted laser desorption ionization mass spectrometry. J Am Soc Mass Spectrom 11:746–750PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang WD, Evans DH (1991) Detection and identification of human influenza viruses by the polymerase chain reaction. J Virol Methods 33:165–189PubMedCrossRefGoogle Scholar
  16. 16.
    Wright KE, Wilson GA, Novosad D, Dimock C, Tan D, Weber JM (1995) Typing and subtyping of influenza viruses in clinical samples by PCR. J Clin Microbiol 133:1180–1184Google Scholar
  17. 17.
    Ruben FL, Jackson GG, Gotoff SP (1973) Humoral and cellular response in humans after immunization with influenza vaccine. Infect Immun 7:594–596PubMedGoogle Scholar
  18. 18.
    Wood JM (2002) Selection of influenza vaccine strains and developing pandemic vaccines. Vaccine 20:B40–B44PubMedCrossRefGoogle Scholar
  19. 19.
    Kodihalli S, Justewicz DM, Gubareva LV, Webster RG (1995) Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective. J Virol 69:4888–4897PubMedGoogle Scholar
  20. 20.
    Julkunen I, Pyhala R, Hovi T (1985) Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis. J Virol Methods 10:75–84PubMedCrossRefGoogle Scholar
  21. 21.
    Li J, Chen S, Evans DH (2001) Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR. J Clin Microbiol 39:696–704PubMedCrossRefGoogle Scholar
  22. 22.
    O’Connell J (ed) (2002) RT-PCR protocols, methods in molecular biology, vol 193. Humana, NJGoogle Scholar
  23. 23.
    Poddar SK (2002) Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis. J Virol Methods 99:63–70PubMedCrossRefGoogle Scholar
  24. 24.
    Ho JWK, Morrissey B, Downard KM (2007) A computer algorithm for the identification of protein interactions from the spectra of masses (PRISM). J Am Soc Mass Spectrom 18:563–566PubMedCrossRefGoogle Scholar
  25. 25.
    Pappin DJC, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3:327–332PubMedCrossRefGoogle Scholar
  26. 26.
    Mackun K, Downard KM (2003) Strategy for identifying protein–protein interactions of gel-separated proteins and complexes by mass spectrometry. Anal Biochem 318:60–70PubMedCrossRefGoogle Scholar
  27. 27.
    Sheshberadaran H, Payne LG (1988) Protein antigen–monoclonal antibody contact sites investigated by limited proteolysis of monoclonal antibody–bound antigen: protein “footprinting”. Proc Natl Acad Sci 85:1–5PubMedCrossRefGoogle Scholar
  28. 28.
    Cotter RJ (1997) Time-of-flight mass spectrometry: instrumentation and applications in biological research. American Chemical Society, Washington, DCGoogle Scholar
  29. 29.
    Macken C, Lu H, Goodman J, Boykin L (2001) The value of a database in surveillance and vaccine selection. In: Osterhaus ADME, Cox N, Hampson AW (eds) Options for the control of influenza IV. Elsevier Science, Amsterdam, pp 103–106Google Scholar
  30. 30.
    Downard KM (2004) Chapter 4 – tandem mass spectrometry, in Mass spectrometry – a foundation course. Royal Society of Chemistry, CambridgeGoogle Scholar
  31. 31.
    Morrissey B, Downard KM (2008) Kinetics of antigen-antibody interactions employing a MALDI mass spectrometry immunoassay. Anal Chem 80:7720–7726PubMedCrossRefGoogle Scholar
  32. 32.
    Schwahn AB, Downard KM (2009) Antigenicity of a type A influenza virus through comparison of hemagglutination inhibition and mass spectrometry immunoassays. J Immunoassay Immunochem 30:245–261PubMedCrossRefGoogle Scholar
  33. 33.
    Swaminathan K, Downard KM (2012) Anti-viral inhibitor binding to influenza neuraminidase by MALDI mass spectrometry. Anal Chem 84:3725–3730PubMedCrossRefGoogle Scholar
  34. 34.
    Polley JR (1969) Preparation of stable noninfective influenza virus antigens for typing by hemagglutination–inhibition. Can J Microbiol 15:203–207PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Kevin M. Downard
    • 1
  1. 1.School of Molecular BioscienceUniversity of SydneySydneyAustralia

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