Comparative study of commercially available gluten ELISA kits using an incurred reference material

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Z. Bugyi
K. Török
L. Hajas
Z. Adonyi
B. Popping
S. Tömösközi


data variability, effects of processing, method validation, quality assurance


Handling major hypersensitivity reactions (e.g. celiac disease) triggered by proteins of wheat and other cereals is a challenging task for healthcare systems, legislative forces and the related fields of food analysis as well. In spite of the fact that there are available official threshold levels for labelling the absence of gluten, which is considered to be the toxic protein fraction of wheat, barley and rye, validation of the analytical methodology supporting regulatory requirements is currently problematic. The main limiting factors of method validation are the lack of reference methods and reference materials. The objective of this study was to provide a solution to this problem. An incurred reference material in a model food matrix was developed and studied by commercially available ELISA test kits as a part of the activity of the Food Allergen Working Group within the FP6 funded EU project MoniQA. After successful completion of the reference material development process, the incurred material was used as a basis of a comparative study examining the performance and applicability of seven commercially available ELISA kits designed for quantification of gluten/gliadin. In certain cases the obtained data showed discrepancies from the expected gliadin content that may carry both a food safety and an economic relevance. The evaluation of the effects of heat treatment on the analytical results is also presented, highlighting the fact that the food processing steps may have a considerable impact on the analytical data, thus should be carefully handled during method development and validation.

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Abbott, M., Hayward, S., Ross, W., Godefroy, S.B., Ulberth, F., Van Hengel, A.J., Roberts, J., Akiyama, H., Popping, B., Yeung, J.M., Wehling, P., Taylor, S.L., Poms, R.E. and Delahaut P., 2010. Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. Journal of AOAC International 93: 442-450.
Allred, L. and Ritter, B., 2010. Recognition of gliadin and glutenin fractions in four commercial gluten assays. Journal of AOAC International 93: 190-196.
Berger, R. and Schmidt, G., 1996. Evaluation of six anti-gliadin antibody assays. Journal of Immunological Methods 191: 77-86.
Breitender, H. and Radauer, C., 2004. A classification of plant food allergens. Journal of Allergy and Clinical Immunology 113: 821-830.
Briani, C., Samaroo, D. and Alaedini, A., 2008. Celiac disease: from gluten to autoimmunity. Autoimmunity Reviews 7: 644-650.
Bugyi, Zs., Török, K., Hajas, L., Adonyi, Zs., Poms, R.E., Popping, B., Diaz-Amigo, C., Kerbach, S. and Tömösközi, S., 2012. Development of incurred reference material for improving conditions of gluten quantification. Journal of AOAC International 95: 382-387.
Ciclitira, P.J., Johnson, M.W., Dewar, D.H. and Ellis, H.J., 2005. The pathogenesis of coeliac disease. Molecular Aspects of Medidine 26: 421-458.
Codex Alimentarius Commission, 1979. Codex standard for foods for special dietary use for persons intolerant to gluten. Codex Stan 118 - 1979 (revised 2008), 3 pp.
Commission of the European Communities, 2009. Commission Regulation (EC) No. 41/2009 of 20 January 2009 concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten. Official Journal of the European Union 52: 3-5.
Denery-Papini, S., Nicolas, Y. and Popineau, Y., 1999. Efficiency and limitations of immunochemical assays for the testing of gluten-free foods. Journal of Cereal Science 30: 121-131.
Geng, T., Westphal, C.D. and Yeung, J.M., 2008. Detection of gluten by commercial test kits: effects of food matrices and extraction procedures. In: Siantar, D.P., Trucksess, M.W., Scott, P.M. and Herman, E.M. (eds.)Food contaminants: mycotoxins and food allergens, volume 1001. American Chemical Society, Washington, DC, USA, pp. 462-475.
Hischenhuber, C., Crevel, R., Jarry, B., Mäki, M., Moneret-Vautrin, D.A., Romano, A., Troncone, R. and Ward, R., 2006. Safe amounts of gluten for patients with wheat allergy or coeliac disease. Alimentary Pharmacology and Therapeutics 23: 559-575.
Janssen, F., 2006. Detecting wheat gluten in food. In: Koppelman, S.J. and Hefle, S.L. (eds.) Detecting allergens in foods. Woodhead Publishing Ltd., Cambridge, UK, pp. 244-273.
Kieffer, R., Schurer, F., Köhler, P. and Wieser, H., 2007. Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: studies on gluten, gliadin and glutenin. Journal of Cereal Science 45: 285-292.
Lagrain, B., Thewissen, B.G., Brijs, K. and Delcour, J.A., 2008. Mechanism of gliadin-glutenin cross-linking during hydrothermal treatment. Food Chemistry 107: 753-760.
Mills, E.N.C., Sancho, A.I., Rigby, N., Jenkins, J.A. and Mackie A.R., 2009. Impact of food processing on the structural and allergenic properties of food allergens. Molecular Nutrition and Food Research 53: 963-969.
Monaci, L., Brohée, M., Tregoat, V. and Van Hengel A., 2011. Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA. Food Chemistry 127: 669-675.
Petersen, N.H., Hansen, P.R. and Houen, G., 2011. Fast and efficient characterization of an anti-gliadin monoclonal antibody epitope related to celiac disease using resin-bound peptides. Journal ofImmunological Methods 365: 174-182.
Poms, R.E. and Anklam, E., 2004. Effects of chemical, physical and technological processes on the nature of food allergens. Journal of AOAC International 87: 1466-1474.
Poms, R., Emons, E. and Anklam, E., 2006. Reference materials and method validation in allergen detection. In: Koppelman, S.J. and Hefle, S.L. (eds.) Detecting allergens in foods. Woodhead Publishing Ltd., Cambridge, UK, pp. 348-355.
Scaravelli, E., Brohée, M., Marchelli, R. and Van Hengel, A.J., 2008. Development of three real-time PCR assays to detect peanut allergen residue in processed food products. European Food Research and Technology 227: 857-869.
Silano, M. and De Vincenzi, M., 1999. In vitro screening of food peptides toxic for coeliac and other gluten-sensitive patients: a review. Toxicology 132: 99-110.
Skerritt, J.H. andHill,A.S.,1991.Enzyme-immunoassay for determination of gluten in foods; collaborative study.Journal of AOAC International 74:257-264.
Takács, K., Szamos, J., Janaky, T., Polgar, M. and Gelencsér, É., 2010a. Immune-analytical detection of the cross-reactive major cereal allergens. Food and Agricultural Immunology 21: 317-334.
Takács, K., Szamos, J., Kováca, E., Janáky, T., Polgár, M. and Gelencsér, É., 2010b. F?bb gabona allergének immunanalitikai kimutatása. Süt?iparosok, pékek 57: 27-32.
Thomas, K., Herouet-Guicheney, C., Ladics, G., Bannon, G., Cockburn, A., Crevel, R., Fitzpatrick, J., Mills, C., Privalle, L. and Vieths, S., 2007. Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report. Food and Chemical Toxicology 45: 1116-1122.
Thompson, T. and Mendez, E., 2008. Commercial assays to assess gluten content of gluten-free foods: why they are not created equal. Journal of the American Dietetic Association 108: 1682-1687.
Tye-Din, J.A., Stewart, J.A., Dromey, J.A., Beissbarth, T., Van Heel, D.A., Tatham, A., Henderson, K., Mannering, S.I., Gianframi, C., Jewell, D.P., Hill, A.V.S., McCluskey, J., Rossjohn, J. and Andreson R.P., 2010. Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease. Science Translational Medicine 41: 41-51.
Van Eckert, R., Berghofer, E., Ciclitira, P.J., Chirdo, F., Denery-Papini, S., Ellis, H.J., Ferranti, P., Goodwin, P., Immer, U., Mamone, G., Méndez, E., Mothes, T., Novalin, S., Osman, A., Rumbo, M., Stern, M., Thorell, L., Whim, A. and Wieser, H., 2006. Towards a new gliadin reference material-isolation and characterisation. Journal of Cereal Science 43: 331-341.
Van Eckert, R., Bond, J., Rawson, P., Klein, Ch.L., Stern, M. and Jordan, T.W., 2010. Reactivity of gluten detecting monoclonal antibodies to a gliadin reference material. Journal of Cereal Science 51: 198-204.
Wal, J.-M., 2003. Thermal processing and allergenicity of foods. Allergy 58: 727-729.
Werner, S. and Wieser, H., 2003. Comparative investigations of gluten proteins from different wheat species. IV. Reactivity of gliadin fractions and components from different wheat species in a commercial immunoassay. European Food Research and Technology 217: 360-364.
Wieser, H., 2001. Comparative investigations of gluten proteins from different wheat species. III. N-terminal amino acid sequences of a-gliadins potentially toxic for coeliac patients. European Food Research and Technology 213: 183-186.