Development of a quantitative immunoassay for gluten - free food analysis based on a new antigen-binding strategy

DOÑA V; URRUTIA M; GOLDBAUM F; CHIRDO F

Amsterdam

Congreso; 13th International Coeliac Disease Symposium; 2009

International Coeliac Disease Symposium

The current effective therapy in CD is a diet excluding the toxic proteins, commonly named gluten-free diet. To this end, food aimed for coeliac patients must be analysed using high detectability quantitative tests. The extraction from food samples is commonly performed with 60% ethanol though denaturing agents can be added to increase solubility of these proteins. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In this work, we present the generation of a new analytical tool to evaluate the presence of gliadins in food based on single domain antibodies (VHH) obtained in llama. A phage display library was obtained from llama inmunized with gliadin.Specific VHH binding was evaluated in the presence of ethanol, 2-ME and GuHCl .Those VHHs showing more than 50% binding in ethanol 60% and 50% in 2-ME 0,5% and 0,5 mol/L GuHCl were selected for further studies. Sequence analysis revealed the presence of three different clones, all recognizing specifically gliadins but not another related or not related proteins. A quantitative competitive ELISA was developed based on these VHHs, with detection limits ranging from 3,5 to 24 ng/ml.VHHs were recloned and expressed as soluble proteins in E. Coli. After purific ation, affinity was measured against tree sources of gliadin: gliadin Sigma, PWG and ethanolic wheat extract. Dissociation constants were next to 10-7 M  in all cases. A capture ELISA based on soluble VHH26-3B4 mAb was developed with a detection limit of 10 ng gliadins/ml. The calibration curve for gliadin standard solution prepared with commercial gliadin overlapped with that prepared using the European PWG standard. When the performance of this quantitative assay was evaluated in the presence of 15 % ethanol, no change was found in the slope of the regression analysis, demonstrating that the assay is fully operative in the presence of 15% ethanol. This article reports the development of a method to determine toxic gluten peptides in food based in a new analytical strategy using high stable single domain antibodies, which can be used for other compounds when specific recognition units in stringent condition are required.