ABSTRACT
BACKGROUND: Anti-gliadin IgE are expressed in patients with food allergy associated to skin immediate hypersensitivity to hydrolyzed wheat proteins (IHHWP). It is not known if they react with omega5-gliadins, the major allergens in wheat dependant exercise-induced food anaphylaxis (WDEIA), encoded on wheat chromosomes 1B. METHODS: Unmodified gliadins from 14 wheat varieties expressing most of the 1B omega-gliadin alleles, were immunoprobed after SDS-PAGE and blotting, with four sera from patients with IHHWP, and two with WDEIA. Gliadins reacting with IgE were visualized using chemiluminescence and identified according to their mobility and typical SDS-PAGE pattern. The resulting signal was also measured to compare their IgE reactivity. RESULTS: IHHWP and WDEIA sera exhibited distinct patterns of reactivity. IgE of patients with IHHWP reacted mainly with all omega-gliadins alleles and one gamma-gliadin encoded respectively on chromosomes 1D and 1B, but not with any omega5-gliadins alleles as for WDEIA. A few other reactive alleles of omega-gliadins were encoded on chromosomes 1A. Unassigned additional bands of the whole gliadin pattern were also reactive. The four patients with IHHWP exhibited almost the same pattern of reactivity. Main differences concerned band reactivity which modulated the overall reactivity of each wheat variety. CONCLUSIONS: The IgE epitopes involved in IHHWP and WDEIA are different. This suggests that the protein state and the route of exposure to very similar gluten structures, probably orientate the pattern of epitope reactivity and the wheat food allergy manifestations.
Subject(s)
Gliadin/genetics , Gliadin/immunology , Hypersensitivity, Immediate/genetics , Wheat Hypersensitivity/genetics , Alleles , Allergens/adverse effects , Allergens/genetics , Allergens/immunology , Anaphylaxis/genetics , Anaphylaxis/immunology , Dermatitis, Atopic/etiology , Dermatitis, Atopic/genetics , Dermatitis, Atopic/immunology , Electrophoresis, Polyacrylamide Gel/methods , Exercise , Humans , Hypersensitivity, Immediate/blood , Hypersensitivity, Immediate/immunology , Immunodominant Epitopes/genetics , Immunodominant Epitopes/immunology , Immunoglobulin E/blood , Immunoglobulin E/genetics , Immunoglobulin E/immunology , Luminescent Measurements/methods , Triticum/adverse effects , Triticum/genetics , Triticum/immunology , Wheat Hypersensitivity/blood , Wheat Hypersensitivity/immunologyABSTRACT
The detection of glycoproteins based on the periodate oxidation of their carbohydrate moiety, and their conjugation to digoxigenin hydrazide directly on blots, leads to high background staining, especially with polyvinylidene difluoride (PVDF) membranes. The addition of Tween-20 to all incubation solutions, except at the oxidation step, strongly reduced the background staining and allowed us to detect lower amounts of glycans fixed on the blots through the protein moiety. The presence of polysaccharides during the oxidation step was shown to produce side reactions that led to the staining of nonglycosylated proteins; it demonstrated risks that may occur with crude extracts when periodate oxidation is performed in solution. The phenomenon was used to design a total protein-staining assay that can be included along with positive and negative controls in a general strategy based on blotting, which was delineated for the identification of glycoproteins in complex tissue extracts.
