Advertisement

Analytical and Bioanalytical Chemistry

, Volume 407, Issue 20, pp 6021–6029 | Cite as

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

  • Sonia Amaya-González
  • Noemí de-los-Santos-Álvarez
  • Arturo J. Miranda-Ordieres
  • María Jesús Lobo-CastañónEmail author
Research Paper

Abstract

Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free, providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods.

Graphical Abstract

Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors

Keywords

Aptamer Coeliac disease Electrochemical detection Gliadin analysis Gluten 33-mer peptide 

Notes

Acknowledgments

SAG thanks the Spanish Government for a predoctoral FPI grant. This work was financed by Spanish Government Project CTQ2012-31157 and the European Regional Development Fund.

References

  1. 1.
    Rostom A, Murray JA, Kagnoff MF (2006) American Gastroenterological Association (AGA) Institute technical review on the diagnosis and management of celiac disease. Gastroenterology 131(6):1981–2002CrossRefGoogle Scholar
  2. 2.
    Commission Implementing Regulation (EU) 828/2014 (2014). L228:5–8Google Scholar
  3. 3.
    Schofield JD, Bottomley RC, Timms MF, Booth MR (1983) The effect of heat on wheat gluten and the involvement of sulfhydryl-disulfide interchange reactions. J Cereal Sci 1(4):241–253CrossRefGoogle Scholar
  4. 4.
    Mena M, Lombardia M, Hernando A, Mendez E, Albar J (2012) Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody. Talanta 91:33–40CrossRefGoogle Scholar
  5. 5.
    Garcia E, Llorente M, Hernando A, Kieffer R, Wieser H, Mendez E (2005) Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods. Eur J Gastroenterol Hepatol 17(5):529–539CrossRefGoogle Scholar
  6. 6.
    Tye-Din JA, Stewart JA, Dromey JA, Beissbarth T, van Heel DA, Tatham A, Henderson K, Mannering SI, Gianfrani C, Jewell DP, Hill AVS, McCluskey J, Rossjohn J, Anderson RP (2010) Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease. Sci Transl Med 2(41):41–51CrossRefGoogle Scholar
  7. 7.
    Shan L, Molberg Ø, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C (2002) Structural basis for gluten intolerance in celiac sprue. Science 297(5590):2275–2279CrossRefGoogle Scholar
  8. 8.
    Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AVS (2000) In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope. Nat Med 6(3):337–342CrossRefGoogle Scholar
  9. 9.
    Skerrit JH, Hill AS (1990) Monoclonal-antibody sandwich enzyme immunoassays for determination of gluten in foods. J Agric Food Chem 38(8):1771–1778CrossRefGoogle Scholar
  10. 10.
    Valdes I, Garcia E, Llorente M, Mendez E (2003) Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol. Eur J Gastroenterol Hepatol 15(5):465–474CrossRefGoogle Scholar
  11. 11.
    Moron B, Cebolla A, Manyani H, Alvarez-Maqueda M, Megias M, Thomas MC, Lopez MC, Sousa C (2008) Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. Am J Clin Nutr 87(2):405–414Google Scholar
  12. 12.
    Spaenij-Dekking EHA, Kooy-Winkelaar EMC, Nieuwenhuizen WF, Drijfhout JW, Koning F (2004) A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin. Gut 53(9):1267–1273CrossRefGoogle Scholar
  13. 13.
    Haas-Lauterbach S, Immer U, Richter M, Koehler P (2012) Gluten fragment detection with a competitive ELISA. J AOAC Int 95(2):377–381CrossRefGoogle Scholar
  14. 14.
    Dona VV, Fossati CA, Chirdo FG (2008) Interference of denaturing and reducing agents on the antigen/antibody interaction. Impact on the performance of quantitative immunoassays in gliadin analysis. Eur Food Res Technol 226(3):591–602CrossRefGoogle Scholar
  15. 15.
    Amaya-González S, de-los-Santos-Álvarez N, Miranda-Ordieres AJ, Lobo-Castañón MJ (2014) Aptamer binding to celiac disease-triggering hydrophobic proteins: a sensitive gluten detection approach. Anal Chem 86(5):2733–2739Google Scholar
  16. 16.
    van Eckert R, Berghofer E, Ciclitira P, Chirdo F, Denery-Papini S, Ellis H, Ferranti P, Goodwin P, Immer U, Mamone G, Mendez E, Mothes T, Novalin S, Osman A, Rumbo M, Stern M, Thorell L, Whim A, Wieser H (2006) Towards a new gliadin reference material-isolation and characterisation. J Cereal Sci 43(3):331–341CrossRefGoogle Scholar
  17. 17.
    Gessendorfer B, Koehler P, Wieser H (2009) Preparation and characterization of enzymatically hydrolyzed prolamins from wheat, rye, and barley as references for the immunochemical quantitation of partially hydrolyzed gluten. Anal Bioanal Chem 395(6):1721–1728CrossRefGoogle Scholar
  18. 18.
    Wieser H, Koehler P (2009) Is the calculation of the gluten content by multiplying the prolamin content by a factor of 2 valid? Eur Food Res Technol 229(1):9–13CrossRefGoogle Scholar
  19. 19.
    Redondo MCB, Griffin PB, Ransanz MG, Ellis HJ, Ciclitira PJ, O’Sullivan CK (2005) Monoclonal antibody-based competitive assay for the sensitive detection of coeliac disease toxic prolamins. Anal Chim Acta 551(1-2):105–114CrossRefGoogle Scholar
  20. 20.
    Amaya-González S, de-los-Santos-Álvarez N, Lobo-Castañón MJ, Miranda-Ordieres AJ, Tuñón-Blanco P (2011) Amperometric quantification of gluten in food samples using an ELISA competitive assay and flow injection analysis. Electroanalysis 23(1):108–114Google Scholar
  21. 21.
    Laube T, Kergaravat SV, Fabiano SN, Hernandez SR, Alegret S, Pividori MI (2011) Magneto immunosensor for gliadin detection in gluten-free foodstuff: towards food safety for celiac patients. Biosens Bioelectron 27(1):46–52CrossRefGoogle Scholar
  22. 22.
    Amaya-González S, López-López L, Miranda-Castro R, de-los-Santos-Álvarez N, Miranda-Ordieres AJ, Lobo-Castañón MJ (2015) Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: influence of immobilization and labeling tags. Anal Chim Acta 873: 73–60. doi: 10.1016/j.aca.2015.1002.1053
  23. 23.
    Osman AA, Uhlig HH, Valdes I, Amin M, Mendez E, Mothes T (2001) A monoclonal antibody that recognizes a potential toxic repetitive pentapeptide epitope in gliadins. Eur J Gastroenterol Hepatol 13(10):1189–1193CrossRefGoogle Scholar
  24. 24.
    Fric P, Gabrovska D, Nevoral J (2011) Celiac disease, gluten-free diet, and oats. Nutr Rev 69(2):107–115CrossRefGoogle Scholar
  25. 25.
    Real A, Comino I, de Lorenzo L, Merchan F, Gil-Humanes J, Gimenez MJ, Angel Lopez-Casado M, Isabel Torres M, Cebolla A, Sousa C, Barro F, Piston F (2012) Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease. PLoS ONE 7(12):e48365CrossRefGoogle Scholar
  26. 26.
    Moron B, Bethune MT, Comino I, Manyani H, Ferragud M, Lopez MC, Cebolla A, Khosla C, Sousa C (2008) Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide. PLoS ONE 3(5):e2294CrossRefGoogle Scholar
  27. 27.
    Real A, Comino I, de Lourdes MM, Angel Lopez-Casado M, Lorite P, Isabel Torres M, Cebolla A, Sousa C (2014) Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer. PLoS One 9(6):e100917CrossRefGoogle Scholar
  28. 28.
    Tanner GJ, Colgrave ML, Blundell MJ, Goswami HP, Howit CA (2013) Measuring hordein (gluten) in beer—a comparison of ELISA and mass spectrometry. PLoS One 8:e56452CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Sonia Amaya-González
    • 1
  • Noemí de-los-Santos-Álvarez
    • 1
  • Arturo J. Miranda-Ordieres
    • 1
  • María Jesús Lobo-Castañón
    • 1
    Email author
  1. 1.Departamento de Química Física y AnalíticaUniversidad de OviedoOviedoSpain

Personalised recommendations