Objectives: Aim of my thesis was the characterization of IgE-reactive cDNA clones which were isolated from a wheat seed cDNA library. After recombinant production of wheat allergens, my aim was to characterize them regarding biochemical, biophysical and immunological properties and for the use in diagnosis of allergy. Material and Methods: A cDNA library (Triticum aestivum) from wheat seeds was constructed and screened with serum IgE antibodies from patients with baker's asthma or wheat-dependent food allergy to identify novel wheat allergens. After recombinant protein expression using a prokaryotic system (E.coli cells) or a eukaryotic system (baculovirus-infected insect cells), allergens were biochemically analyzed using mass spectrometry, circular dichroism spectroscopy, size exclusion chromatography and chemical crosslinking. RAST-based, non-denaturing dot blot experiments were used to determine their IgE-reactivity and basophil degranulation experiments were performed to investigate the allergenic activity. Additionally, allergen-specific rabbit antibodies were raised to study the course of protein expression during wheat seed maturation, cross-reactivity, their stability when subjected to gastric or duodenal digestion, as well as their heat stability in different bread sorts.
Results: In the context of my thesis, five baker's asthma allergens (thioredoxin h, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin) were characterized. The cross-reactive allergen 1-Cys-peroxiredoxin was identified as the most relevant allergen according to prevalence of IgE-recognition and basophil degranulation assays. Additionally, we identified and characterized a new wheat food allergen alpha purothionin (Tri a 37) which was found to serve as a serologic marker for the diagnosis of severe wheat food allergy.
Conclusion: The use of recombinant wheat allergens in microarray-based component-resolved diagnostic tests allows a detailed diagnosis and may distinguish between various clinical manifestations of wheat allergies.