Epitope mapping of allergens and antigens of timothy pollen extract

For the identification of allergens in crude timothy pollen extract, 2D immunoblotting was performed with subsequent use of patients' IgE. Only about 15 of more than 70 detectable components with molecular weights of 55, 38, 35, and 32 kD showed IgE-reactivity. The 55 and 35 kD allergens were identified as glycoproteins by a glycan detection kit. To investigate the protein structure of the allergens, proteins of the pollen extract were cleaved by CNBr and endoproteinases. The cleavage products were identified by patients' IgE and monoclonal antibodies (moab) raised against timothy pollen proteins. While the use of endoproteinases resulted in a complete loss of some allergens, CNBr produced smaller IgE-reactive proteins, e.g. the 38 kD component was split into a 32 kD component and the original 32 kD protein was partially cleaved into IgE-reactive fragments of 30, 21, and 15 kD. In order to investigate the IgE-binding epitopes we intend to determine the amino acid sequence of the cleavage products.