To support its ongoing monitoring program of therapeutic and diagnostic allergen preparations, LIB has initiated a multifaceted study of the relationship between the structure and function of allergens. These include: 1. Physico-chemical identification of allergens. We have initiated studies on the use of HPLC, MALDI-TOF mass-spectroscopy and microarray analyses in the characterization of target allergens. 2. Role of glycosylation in allergen recognition and processing. Recent advances in the bioengineering and cloning of allergens have raised new questions about the immunogenicity of non-glycosylated proteins. Dr. Soldatova has studied the role of glycosylation in the immunogenicity of recombinant bee venom hyaluronidase. Active site and glycosylation sites mutants were produced, and mutant proteins are being expressed in insect cells using recombinant baculovirus. Site-specific mutagenesis of these and other recombinant proteins will expand our current studies in this area. 3. Enzyme activity as a measure of allergenicity. Current standards require the presence of active hyaluronidase and phospholipase in venom extracts. We have initiated studies to address the issue of the relevance of these activities for allergenicity. 4. Molecular cloning of a new bee venom allergen, acid phosphatase. The panel of available recombinant allergens from honey bee venom will be extended to three major allergens including phospholipase A2, hyaluronidase and acid phosphatase. Each of these key components of bee venom as well as a melittin (a minor allergen) can then be evaluated for inclusion in a diagnostic panel to detect honeybee allergy, and for the immunotherapy of allergic individuals. 5. The extended stability study of house dust mite extracts has been completed. The results of the study show that lyophilized extracts are more stable than glycerinated solutions, and that allergen stability in glycerinated specimens is enhanced by the lowering the storage temperature from 4oC to - 20oC.