R7001

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.

The prolamin solution containing gliadin from wheat (G3375, Sigma) was extracted according to the ELISA’s manufacturer’s advice, using the patented cocktail developed by Dr. Enrique Mendez (R7006, R-Biopharm AG, Darmstadt, Germany), which is the official method according to the AOAC. Then, the extract was diluted to a final concentration of 150 µg/mL in phosphate buffer pH 6.8 containing 250 mL of 0.2 M KH₂PO₄ and 112 mL of 0.2 M NaOH in a final volume of 1000 mL.
The different fractions (coarse, standard = G-PUR^®, and fine) and a coarse powder of PCT were used in various amounts, as noted in Table 3. They were also diluted in phosphate buffer pH 6.8.
Prior to mixing, the various solutions were pre-heated to 37 °C to simulate human body temperature.
After 30 min of incubation rotating (25 rpm/min, rotator SB3, Stuart) at 37 °C, the samples were centrifuged at 4100× g (MEGA STAR 1.6R, VWR) for 2 min and the supernatants were analyzed by ELISA (R7001, R-Biopharm AG, Darmstadt, Germany), as recommended by the manufacturer, to examine their adsorption value. Measurements were performed at a wavelength of λ = 450 nm by using a plate reader (Biotek synergy HT).

The determination of a possible influence of acidity or alkalinity on gliadin adsorption to purified clinoptilolite-tuff was performed by incubation of 125 µg/mL gliadin and 0.1 g/mL PCT for 30 min at 37 °C under rotation (25 rpm/min, rotator SB3, Stuart) in different buffers, including pH 7.8, 6.8, 5.8, 4.5, and a solution of pH 1.5 (Table 2). Then, the samples were centrifuged at 4100× g (MEGA STAR 1.6R, VWR) for 2 min and the supernatants were analyzed by ELISA (R7001, R-Biopharm AG, Darmstadt, Germany), according to the manufacturer’s guidelines, to examine their adsorption value. Measurements were performed at a wavelength of λ = 450 nm by using a plate reader (Biotek synergy HT).