Browsing by Subject "R5"

The literature part of the study reviewed the recommended gluten quantification method, immunological ELISA R5. R5 is a monoclonal antibody that recognizes mainly the epitope that is abundant in especially gluten protein subgroup, ω-gliadin. The current PWG-gliadin reference material used in ELISA leads to inaccuracy of the gluten content, because it cannot represent sample materials that differ in their gliadin composition. The aim of the experimental study was to compare the prolamin compositions of different wheat cultivars and their reactivity against R5 antibody in sandwich ELISA. The aim was to find the most suitable ratio of barley prolamin, C-hordein, to be used as a reference material for wheat gluten quantification. The ω-gliadin proportions of different cultivars were calculated from RP-HPLC-chromatograms. In order to compare the total wheat gluten reactivity of the cultivars in ELISA R5 with gliadin standard and C-hordein in different ratios (10, 20 and 30% in BSA), Km-values that measure the rate of sensitivity in the assay, were calculated. The method to separate gliadin- and glutenin subgroups in RP-HPLC was optimized (solvent to extract gliadin and glutenin, temperature, injection volume, gradient). For cv. Crusoe the ω-, α/β- and γ-gliadins and HMW- and LMW-glutenins were identified. The selected wheat cultivars were categorized into four groups. The proportion of ω-gliadin in total gliadin ranged from 0.8 to 14.1% between the cultivars, whereas for PWG-gliadin this has been reported to be 7.7%. In terms of similar reactivity (Km-value) in ELISA, 20% C-hordein was found to be the most suitable reference material (Km 90) for the selected wheat cultivars (Km average 92), instead of the current gliadin standard (Km 68). The advantage of C-hordein standard is that the concentration and thus reactivity can be adjusted to match the sample materials with different prolamin profiles. Unlike with current gliadin reference material, it can be used without any conversion factors, which improves the method accuracy.