Enhancing gluten detection assay development through optimization of gliadin extraction conditions

Figures

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  Fig. 1. Optimization of direct ELISA. A. Three coating buffers (70% ethanol, carbonate-bicarbonate buffer, and 70% ethanol + carbonate-bicarbonate buffer) and two blocking buffer conditions (1% BSA and SuperBlock) are tested in combinations to find the optimum ELISA condition. The signal intensities were read at 426 nm. 1 (●): 70% ethanol + SuperBlock, 2 (▲): 70% ethanol + BSA, 3(■): carbonate-bicarbonate buffer + SuperBlock, 4 (▬): carbonate-bicarbonate buffer + BSA, 5 (♦): 70% ethanol + carbonate-bicarbonate buffer + SuperBlock, 6 (X): 70% ethanol + carbonate-bicarbonate buffer + BSA. B. Optimization of gliadin coating on 96-well plates: 20 μg/mL gliadin (black), 10 μg/mL gliadin (blue) and 5 μg/mL gliadin (green).

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  Fig. 2. Optimization of Mab coating concentration for sandwich ELISA. A. Four different Mab coating concentrations tested on 96-well plate: 0.18, 0.5, 1, and 5 μg/mL. Absorbance was read at 426 nm. Columns for each Mab concentration represents the dilution rates of gliadin (5 μg/mL (black), 1.25 μg/mL (blue), 0.31 μg/mL (green), and blank (grey), respectively). B. Comparison of in-house sandwich ELISA (grey) and commercial R5 ELISA detection kit (black).

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  Fig. 3. Evaluation of different gliadin extraction conditions using 2-mercaptoethanol and guanidine hydrochloride from wheat flour (A) and crouton (B). Gliadin extraction from wheat flour (A) and crouton (B) [1]: gliadin standard (starting concentration: 5 μg/mL) [2], combination of PBS and 60% ethanol [3], 1 M guanidine hydrochloride [4], 2 M guanidine hydrochloride [5], 50 mM 2-mercaptoethanol [6], combination of 50 mM 2-mercaptoethanol and 1 M guanidine hydrochloride [7], combination of 50 mM 2-mercaptoethanol and 2 M guanidine hydrochloride [8], 250 mM 2-mercaptoethanol [9], combination of 250 mM 2-mercaptoethanol and 1 M guanidine hydrochloride [10], combination of 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride. Columns for each condition represents the dilution rates of gliadin. Dilution rates are 1:4 (black) and 1:16 (blue) for gliadin standard and 1:10 (black) and 1:100 (blue) for other conditions.

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  Fig. 4. Gliadin extraction from gluten free food sample. (1) gliadin standard (starting concentration: 10 μg/mL) [2], only PBS [3], combination of PBS and ethanol [4], combination of 50 mM 2-mercaptoethanol, 2 M guanidine hydrochloride and ethanol [5], 0.1% EDTA in PBS [6], 0.1% EDTA in ethanol [7], 0.1% Tween 20 in PBS [8], 0.1% Tween 20 in ethanol [9], 0.1% Tween 80 in PBS [10], 0.1% Tween 80 in ethanol [11], 0.1% Triton X-100 in PBS, and [12] 0.1% Triton X-100 in ethanol. Dilution rates are 1:1.

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  Fig. 5. Testing different combinations of ChCl-DESs for extraction of gliadin from crouton. [1] blank [2], only PBS [3], combination of PBS + ethanol [4], combination of 50 mM 2-mercaptoethanol, 2 M guanidine hydrochloride and ethanol [5], 2 M ethylene glycol [6], combination of 2 M ethylene glycol and 1 M choline chloride [7], 1 M choline chloride [8], 2 M urea [9], combination of 2 M urea and 1 M choline chloride. Dilution rates are 1:1000 except 1 (blank).