Detection and Titration of Influenza A Virus Neuraminidase Inhibiting (NAI) Antibodies Using an Enzyme-Linked Lectin Assay (ELLA)

  • Bryan S. KaplanEmail author
  • Amy L. VincentEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2123)


The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.

Key words

Swine influenza A virus Neuraminidase Neuraminidase inhibition ELLA 


  1. 1.
    Walia RR, Anderson TK, Vincent AL (2018) Regional patterns of genetic diversity in swine influenza A viruses in the United States from 2010 to 2016. Influenza Other Respir Viruses 13:262. Scholar
  2. 2.
    Anderson TK, Nelson MI, Kitikoon P, Swenson SL, Korslund JA, Vincent AL (2013) Population dynamics of cocirculating swine influenza A viruses in the United States from 2009 to 2012. Influenza Other Respir Viruses 7(Suppl 4):42–51. Scholar
  3. 3.
    Anderson TK, Campbell BA, Nelson MI, Lewis NS, Janas-Martindale A, Killian ML, Vincent AL (2015) Characterization of co-circulating swine influenza A viruses in North America and the identification of a novel H1 genetic clade with antigenic significance. Virus Res 201:24–31. Scholar
  4. 4.
    Rajao DS, Gauger PC, Anderson TK, Lewis NS, Abente EJ, Killian ML, Perez DR, Sutton TC, Zhang J, Vincent AL (2015) Novel Reassortant human-like H3N2 and H3N1 influenza A viruses detected in pigs are virulent and antigenically distinct from swine viruses endemic to the United States. J Virol 89(22):11213–11222. Scholar
  5. 5.
    Anderson TK, Macken CA, Lewis NS, Scheuermann RH, Van Reeth K, Brown IH, Swenson SL, Simon G, Saito T, Berhane Y, Ciacci-Zanella J, Pereda A, Davis CT, Donis RO, Webby RJ, Vincent AL (2016) A phylogeny-based global nomenclature system and automated annotation tool for H1 hemagglutinin genes from swine influenza A viruses. mSphere 1(6):e00275-16. Scholar
  6. 6.
    Lewis NS, Russell CA, Langat P, Anderson TK, Berger K, Bielejec F, Burke DF, Dudas G, Fonville JM, Fouchier RA, Kellam P, Koel BF, Lemey P, Nguyen T, Nuansrichy B, Peiris JM, Saito T, Simon G, Skepner E, Takemae N, Consortium E, Webby RJ, Van Reeth K, Brookes SM, Larsen L, Watson SJ, Brown IH, Vincent AL (2016) The global antigenic diversity of swine influenza A viruses. elife 5:e12217. Scholar
  7. 7.
    Abente EJ, Rajao DS, Santos J, Kaplan BS, Nicholson TL, Brockmeier SL, Gauger PC, Perez DR, Vincent AL (2018) Comparison of adjuvanted-whole inactivated virus and live-attenuated virus vaccines against challenge with contemporary, antigenically distinct swine H3N2 influenza A viruses. J Virol 92:e01323-18. Scholar
  8. 8.
    Gauger PC, Vincent AL, Loving CL, Lager KM, Janke BH, Kehrli ME Jr, Roth JA (2011) Enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (delta-cluster) H1N2 vaccine and challenged with pandemic 2009 H1N1 influenza virus. Vaccine 29(15):2712–2719. Scholar
  9. 9.
    Zanin M, Marathe B, Wong SS, Yoon SW, Collin E, Oshansky C, Jones J, Hause B, Webby R (2015) Pandemic swine H1N1 influenza viruses with almost undetectable neuraminidase activity are not transmitted via aerosols in ferrets and are inhibited by human mucus but not swine mucus. J Virol 89(11):5935–5948. Scholar
  10. 10.
    Monto AS, Petrie JG, Cross RT, Johnson E, Liu M, Zhong W, Levine M, Katz JM, Ohmit SE (2015) Antibody to influenza virus neuraminidase: an independent correlate of protection. J Infect Dis 212(8):1191–1199. Scholar
  11. 11.
    Chen YQ, Wohlbold TJ, Zheng NY, Huang M, Huang Y, Neu KE, Lee J, Wan H, Rojas KT, Kirkpatrick E, Henry C, Palm AE, Stamper CT, Lan LY, Topham DJ, Treanor J, Wrammert J, Ahmed R, Eichelberger MC, Georgiou G, Krammer F, Wilson PC (2018) Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell 173(2):417–429 .e410. Scholar
  12. 12.
    Wohlbold TJ, Nachbagauer R, Xu H, Tan GS, Hirsh A, Brokstad KA, Cox RJ, Palese P, Krammer F (2015) Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice. MBio 6(2):e02556. Scholar
  13. 13.
    Rajendran M, Nachbagauer R, Ermler ME, Bunduc P, Amanat F, Izikson R, Cox M, Palese P, Eichelberger M, Krammer F (2017) Analysis of anti-influenza virus neuraminidase antibodies in children, adults, and the elderly by ELISA and Enzyme inhibition: evidence for original antigenic sin. MBio 8(2):e02281-16. Scholar
  14. 14.
    Fonville JM, Wilks SH, James SL, Fox A, Ventresca M, Aban M, Xue L, Jones TC, Le NM, Pham QT, Tran ND, Wong Y, Mosterin A, Katzelnick LC, Labonte D, Le TT, van der Net G, Skepner E, Russell CA, Kaplan TD, Rimmelzwaan GF, Masurel N, de Jong JC, Palache A, Beyer WE, Le QM, Nguyen TH, Wertheim HF, Hurt AC, Osterhaus AD, Barr IG, Fouchier RA, Horby PW, Smith DJ (2014) Antibody landscapes after influenza virus infection or vaccination. Science 346(6212):996–1000. Scholar
  15. 15.
    Wan H, Gao J, Xu K, Chen H, Couzens LK, Rivers KH, Easterbrook JD, Yang K, Zhong L, Rajabi M, Ye J, Sultana I, Wan XF, Liu X, Perez DR, Taubenberger JK, Eichelberger MC (2013) Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses. J Virol 87(16):9290–9300. Scholar
  16. 16.
    Wohlbold TJ, Podolsky KA, Chromikova V, Kirkpatrick E, Falconieri V, Meade P, Amanat F, Tan J, tenOever BR, Tan GS, Subramaniam S, Palese P, Krammer F (2017) Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2(10):1415–1424. Scholar
  17. 17.
    Sandbulte MR, Westgeest KB, Gao J, Xu X, Klimov AI, Russell CA, Burke DF, Smith DJ, Fouchier RA, Eichelberger MC (2011) Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. Proc Natl Acad Sci U S A 108(51):20748–20753. Scholar
  18. 18.
    Rajao DS, Chen H, Perez DR, Sandbulte MR, Gauger PC, Loving CL, Shanks GD, Vincent A (2016) Vaccine-associated enhanced respiratory disease is influenced by haemagglutinin and neuraminidase in whole inactivated influenza virus vaccines. J Gen Virol 97(7):1489–1499. Scholar
  19. 19.
    Sandbulte MR, Gauger PC, Kitikoon P, Chen H, Perez DR, Roth JA, Vincent AL (2016) Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type. Vaccine 34(33):3773–3779. Scholar
  20. 20.
    Couzens L, Gao J, Westgeest K, Sandbulte M, Lugovtsev V, Fouchier R, Eichelberger M (2014) An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera. J Virol Methods 210:7–14. Scholar
  21. 21.
    Gao J, Couzens L, Eichelberger MC (2016) Measuring influenza neuraminidase inhibition antibody titers by enzyme-linked lectin assay. J Vis Exp (115).
  22. 22.
    Sandbulte MR, Gao J, Straight TM, Eichelberger MC (2009) A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing reverse genetics-derived antigens. Influenza Other Respir Viruses 3(5):233–240CrossRefGoogle Scholar
  23. 23.
    Hoffmann E, Krauss S, Perez D, Webby R, Webster RG (2002) Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 20(25–26):3165–3170CrossRefGoogle Scholar
  24. 24.
    Kosik I, Angeletti D, Gibbs JS, Angel M, Takeda K, Kosikova M, Nair V, Hickman HD, Xie H, Brooke CB, Yewdell JW (2019) Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies. J Exp Med 216(2):304–316. Scholar
  25. 25.
    Chen YQ, Lan LY, Huang M, Henry C, Wilson PC (2018) Hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance. J Virol 93:e01526-18. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Virus and Prion Research Unit, National Animal Disease CenterUnited States Department of Agriculture, Agriculture Research ServiceAmesUSA

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