title: Proposal for C-Hordein as Reference Material in Gluten Quantification.
abstract: The concentration of residual barley prolamin (hordein) in gluten-free products is overestimated by the R5 ELISA method when calibrated against the wheat gliadin standard. The reason for this may be that the composition of the gliadin standard is different from the composition of hordeins. This study showed that the recognition of whole hordein by R5 antibody mainly came from C-hordein, which is more reactive than the other hordeins. The proportion of C-hordein in total hordein ranged from 16 to 33% of common Finnish barley cultivars used in this study and was always higher than that of ω-gliadin, the homologous protein class in the gliadin standard, which may account for the overestimation. Thus, a hordein standard is needed for barley prolamin quantification instead of the gliadin standard. When gluten-free oat flour was spiked with barley flour, the prolamin concentration was overestimated 1.8-2.5 times with the gliadin standard, whereas estimates in the correct range were obtained when the standard was 40% C-hordein mixed with an inert protein. A preparative-scale method was developed to isolate and purify C-hordein, and C-hordein is proposed as a reference material to calibrate barley prolamin quantification in R5-based assays.
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki , Agnes Sjöberginkatu 2, PL 66, Helsinki FI-00014, Finland.
authors.forename: Xin
authors.identifier: http://orcid.org/0000-0003-0869-840X
authors.initials: X
authors.lastname: Huang
authors.affiliation: Fazer Mills, Oy Karl Fazer Ab , Kasakkamäentie 3, P.O Box 40, Lahti FI-15101, Finland.
authors.forename: Päivi
authors.initials: P
authors.lastname: Kanerva
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki , Agnes Sjöberginkatu 2, PL 66, Helsinki FI-00014, Finland.
authors.forename: Hannu
authors.initials: H
authors.lastname: Salovaara
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki , Agnes Sjöberginkatu 2, PL 66, Helsinki FI-00014, Finland.
authors.forename: Frederick L
authors.initials: FL
authors.lastname: Stoddard
authors.affiliation: Department of Food and Environmental Sciences, University of Helsinki , Agnes Sjöberginkatu 2, PL 66, Helsinki FI-00014, Finland.
authors.forename: Tuula
authors.initials: T
authors.lastname: Sontag-Strohm
mesh_terms: D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005983:Glutens / Q000032:analysis*; D001467:Hordeum / Q000737:chemistry; D012015:Reference Standards; D014908:Triticum / Q000737:chemistry
chemical_list: D005983:Glutens
keywords: C-hordein; R5; gluten quantification; gluten-free; hordein; prolamin