Agraquant gluten g12 assay

The gluten content of each sample was analysed using the AgraQuant^® Gluten G12^® Assay (AACC International Method 38–52.01) (Romer Labs UK Ltd., Runcorn, Cheshire, UK). Samples were extracted in duplicate according to the manufacturer’s standard method. Briefly, samples were blended by a blender (Essex Scientific Laboratory Supplies Ltd., Essex, UK) for about five min and 0.25 g weights were mixed with 2.5 mL of extraction solution before incubating at 50 °C for 40 min. This was followed by the addition of 7.5 mL of 80% ethanol and agitation for 60 min at room temperature, and centrifuged at 2000× g for ten min. The clear aqueous layer between the particulate sediment and supernatant was obtained and stored at 4 °C until analysis. The extracted bread samples were diluted to 1:5000 and 1:10,000 with diluent buffer and loaded on to antibody coated microwells in duplicate along with gluten standards (0, 4, 20, 80 and 200 ppm) before analysis.

Gluten quantitation was performed with two commercially available ELISA test kits: RIDASCREEN Gliadin Assay (limit of detection (LOD): 0.5 mg/kg of gliadin, limit of quantitation (LOQ): 2.5 mg/kg) (R7001, R-Biopharm, Darmstadt, Germany) and AgraQuant Gluten G12 Assay (LOD: 2.0 mg/kg of gluten, LOQ: 4.0 mg/kg) (COKAL0200, Romer Labs, Tulln, Austria). These two kits apply different antibodies (R5 mAb and G12 mAb, respectively) and different calibrators (PWG-gliadin and wheat gluten extract, respectively). ELISA procedures were carried out according to the kit instructions. Rye flour extracts were additionally diluted 10.000-fold to obtain a sample concentration in the calibration range. The absorbances were determined using a microplate reader (iMarkTM Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA). The gluten concentrations were calculated from the absorbance values by the Bio-Rad Microplate Manager 6 software (Bio-Rad) using the curve fit and calculations suggested by the test kit manufacturer, respectively. The gluten content is calculated using the conversion factor of 2 (gliadin content × 2) for the RIDASCREEN Gliadin Assay.

Two commercially available ELISA test kits were used for gluten quantitation: RIDASCREEN Gliadin Assay (limit of detection (LOD): 0.5 mg/kg of gliadin, limit of quantitation (LOQ): 2.5 mg/kg and gluten content calculation: gliadin content × 2) (R7001, R-Biopharm, Darmstadt, Germany) and AgraQuant Gluten G12 Assay (LOD: 2.0 mg/kg of gluten, LOQ: 4.0 mg/kg and gluten content calculation directly from the calibration) (COKAL0200, Romer Labs, Tulln, Austria). ELISA procedures were carried out according to the kit instructions. These two kits apply different monoclonal antibodies (R5 and G12, respectively) and different calibrators (PWG-gliadin and wheat gluten extract, respectively). To obtain a concentration in the calibration range, the barley flour extracts were additionally diluted 10.000-fold. The absorbances were determined using a microplate reader (iMarkTM Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA). The gluten concentrations were calculated from the absorbance values by the Bio-Rad Microplate Manager 6 software (Bio-Rad) using the curve fit and calculations suggested by the test kit manufacturer.