Browsing by Subject "R5 reactivity"

The literature review deals with coeliac disease and wheat, rye and barley prolamins. The characteristics and analysis methods of the prolamins were discussed. Furthermore, the ELISA types, antibodies and reference material in ELISA were introduced in literature review. The aim of this master thesis study was to determine the feasibility of barley C-hordein as reference material in the quantification of wheat gluten in a sandwich ELISA method based on R5 antibody. RP-HPLC was used to determine the compositions of wheat prolamins from 27 wheat cultivars. SDS-PAGE was used for wheat prolamin subgroups identification. The R5 antibody reactivity of prolamins and the same prolamin group from different cultivars were tested. By comparing the R5 reactivity of total gluten of the 27 wheat cultivars, 10% barley C-hordein was used to calibrate the gluten content in spiking test of oat flour and oat biscuits. Omega 1,2-gliadin (Km 13) and γ-gliadin (Km 21) showed rather strong R5 reactivity while ω 5-gliadin (Km 203) and LMW glutenin subunits (Km 523) showed almost no reactivity against R5 antibody. However, the subgroup R5 reactivity differences between cultivars were not significant. The reactivity of total gluten from 27 cultivar varied from Km 14 to 192, with a logarithmic average Km 53. Thus, 10% C-hordein (Km 49) had similar reactivity of the average of all cultivars. In flour spiking test, the recoveries calibrated with PWG gliadin were 57-187%, comparing to 30-115% with 10% C-hordein calibration. In biscuit spiking test, the recoveries calibrated with PWG gliadin and 10% C-hordein were 84-145% and 44-76%.