The impact of tyrosine nitration on the allergenicity of airborne allergens.

Controlled nitration of allergens will be performed using an established method, followed by protein analysis and determination of the nitration grade. Using these proteins, the reactivity of serum antibodies in samples from allergy patients towards these proteins will be analysed using ELISA and Western blot analysis. In addition, cell culture will be used to analyse the potential to nitrate proteins in vitro and the nitration grade of allergens directly derived from pollen will be determined. Moreover, protein analysis will be performed using various modes of capillary electrophoresis (CE), e.g. CZE, CIEF, tCITP, affinity-CIEF and CZE-ESI-microTOF. In addition, circular dichroism spectrometry will be used for protein characterization. Our main goal will be to address the effects of allergen nitration on the protein itself and on immune reactions induced by these modified proteins. This study will include three main parts. The first part focuses on the question whether patients allergic to several airborne allergens have enhanced levels of IgE to the disease related nitrated allergens compared to unmodified allergens. Secondly, the heterogeneity of nitrated allergen species, the effects of nitration on the conformation and aggregation of different allergens will be investigated. In the final part, the effect of the nitration grade on the immunological and physico-chemical factors will be addressed and these levels will be compared with those found for natural allergens, whereby the focus will be on birch pollen allergens.