2.3. Methods

All food samples were processed for gluten content determination by the Ridascreen Gliadin sandwich R5 enzyme-linked immunosorbent assay (ELISA) R-7001 (R-Biopharm, Darmstadt, Germany) at our Celiac Disease Research Laboratory, Polytechnic University of Marche, Ancona. During each run of ELISA, the manufacturer’s guidelines were strictly followed. The Ridascreen R5 ELISA was performed as previously described [6].

The gluten content of analyzed food samples was expressed as ppm. The lower limit of quantification was 5 ppm of gluten. All products with a gluten level higher than 20 ppm were re-extracted and analyzed for a second time.

Finally, we estimated the 24-h amount of gluten consumed by participating children using the following formula to convert ppm of gluten into mg of gluten/day for all the meals with measurable gluten contamination: mg/day gluten = ppm gluten in the food portion × food sample weight (g)/1000.

IgA anti-tissue transglutaminase (anti-tTG) antibody assay was performed in all participating children in our Laboratory by fluorescence enzyme immunoassay ≤30 days prior to the start of the study (normal values <7 U/mL) as part of routine follow-up visits.