Barley C-Hordein as the Calibrant for Wheat Gluten Quantification

Abstract

The lack of certified reference materials has been one major challenge for gluten quantification in gluten-free products. In this study, the feasibility of using barley C-hordein as the calibrant for wheat gluten in R5 sandwich enzyme-linked immunosorbent assay (ELISA) was investigated. The gluten composition and total gluten R5 reactivity ranged largely depending on the genotypes and the growing environment. The conversion factor of gliadin to gluten averaged 1.31 for common wheat, which is smaller than the theoretical factor of 2. Each gluten group had varying reactivity against the R5 antibody, where ω1.2-, γ- and α-gliadins were the main reactive groups from wheat gluten. A mixture of wheat cultivars or one single cultivar as the reference material can be difficult to keep current. Based on the average R5 reactivity of total gluten from the 27 common wheat cultivars, here we proposed 10% C-hordein mixed with an inert protein as the calibrant for wheat gluten quantification. In spiking tests of gluten-free oat flour and biscuits, calibration using 10% C-hordein achieved the same recovery as the gliadin standard with its cultivar-specific conversion factor. For its good solubility and good affinity to the R5 antibody, the application of C-hordein increases the probability of developing a series of reference materials for various food matrices.

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

“…The isolation procedure was lightly modified from a previous wheat gluten isolation process. 33 The seeds were milled with a Retsch ZM 200 (Haan, Germany) to a particle size 0.5 mm screen followed by a defatting step with defatting solution (methanol/diethyl ether 1:1, v/v) at a ratio of 1:5 (w/v) for 60 min at ambient temperature with constant magnetic stirring. After filtration, the defatted flour was dried overnight and then washed using 67 mM phosphate buffer (pH 7.4) with 0.4 M NaCl for 30 min at a ratio of 1:5 (w/v) three times at ambient temperature to remove the albumins and globulins.…”

“…Brage was isolated and used as a common calibrator in all ELISA kits. The isolation procedure was lightly modified from a previous wheat gluten isolation process . The seeds were milled with a Retsch ZM 200 (Haan, Germany) to a particle size 0.5 mm screen followed by a defatting step with defatting solution (methanol/diethyl ether 1:1, v/v) at a ratio of 1:5 (w/v) for 60 min at ambient temperature with constant magnetic stirring.…”

“…A C-hordein isolate (40% mixed with an inert protein, which does not react with R5) has been proposed for total barley hordein calibration, 30 and this calibrant (10% mixed with an inert protein) has been applied in wheat gluten calibration in R5 ELISA. 33 However, the origin of the gluten contaminant is usually unknown. An ELISA kit, Total Gluten, with multiple antibodies was recently developed to solve this problem that the total gluten contents of wheat, barley, and rye are detected in an oat matrix.…”

“…Quantification of barley contamination by R5 ELISA with gliadin standards led to severe unacceptable overestimation. − One reason was that the high binding affinity of R5 antibody against barley C-hordein was observed due to its high number of QQPFP epitope repeats, and another reason was that the composition of barley hordeins differs from the calibrant, which is wheat gliadin. In addition, a conversion factor of 2 from prolamin content to total gluten content is not valid for barley, or not even always for bread wheat. , The calibration using a barley total hordein standard could correct the overestimation in R5 ELISA , when testing for barley contamination. A C-hordein isolate (40% mixed with an inert protein, which does not react with R5) has been proposed for total barley hordein calibration, and this calibrant (10% mixed with an inert protein) has been applied in wheat gluten calibration in R5 ELISA .…”

“…In addition, a conversion factor of 2 from prolamin content to total gluten content is not valid for barley, or not even always for bread wheat. , The calibration using a barley total hordein standard could correct the overestimation in R5 ELISA , when testing for barley contamination. A C-hordein isolate (40% mixed with an inert protein, which does not react with R5) has been proposed for total barley hordein calibration, and this calibrant (10% mixed with an inert protein) has been applied in wheat gluten calibration in R5 ELISA . However, the origin of the gluten contaminant is usually unknown.…”