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.