Quantification of Barley Contaminants in Gluten-Free Oats by Four Gluten ELISA Kits

Figures

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  Figure 1. Characterization of the total hordein isolate by reverse-phase liquid chromatography (panel A) and SDS-PAGE (panel B). Lane 1, Novex Sharp Prestained protein standard. Lane 2, albumin + globulin extract of barley flour. Lane 3 and dotted line 3, total hordein extract from barley flour with albumin + globulin removed. Lane 4 and black line 4, total hordein isolate. Lane 5, Mark 12 unstained protein standard. The letters indicated the classification of hordeins, D, D-hordein; C, C-hordein; B/γ, B/γ-hordein.

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  Figure 2. Immunoblot of total hordein isolate by an antibody-conjugate from four ELISA kits. Protein load to the gel lane A 2.5 μg and lane B 0.5 μg.

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  Figure 3. Comparison of each kit’s own calibration curve and total hordein calibration curve in four ELISA kits: (A) R5 gliadin vs total hordein ng/mL, (B) G12 gluten vs total hordein ng/mL, (C) Total Gluten total gluten vs total hordein, (D) Morinaga wheat proteins vs total hordein ng/mL. The total hordein was hordein isolate × protein content ng/mL. The curves were matched on a gravimetric basis. The curve was produced using agonist response-variable slope (four parameters) nonlinear fit, and the EC50 value was calculated from the best-fit curve values by GraphPad Prism 8.0.2 (San Diego, CA).

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  Figure 4. Youden plots of the z-score of two levels (16 mg/kg vs 80 mg/kg) of barley-spiked oats measured with four ELISA kits using the (A) kits’ own calibration and (B) total hordein isolate calibration. The black cross set at z = 0 was the theoretical spiking content, and the black rectangle was set to|z| ≤ 2, which was considered satisfactory in the proficiency assessment (ISO 13528). The blue cross was set at the mean value of all z-scores, and the blue rectangle was set at 2 times standard deviation of all z-scores. Three points from each ELISA kit indicated three individual extraction replicates.