BEE VENOM PHOSPHOLIPASE-A2 - A MODEL FOR THE STANDARDIZATION OF RECOMBINANT ALLERGENS

Over hundred genes encoding allergens have been isolated so far and re combinant (r)allergens have been produced. To evaluate the usefulness for their in vivo or in vitro application a thorough analysis of these molecules has to be performed by comparing the function of (r)allerge ns in vitro and in vivo to the natural (n) counterpart. We have perfor med such an analysis using the main bee venom allergen phospholipase A 2 (PLA) as a model system. Detailed knowledge about the structure, the enzymatic activity as well as the antigenicity/allergenicity of this molecule is available. We thus produced rPLA with or without enzymatic activity in its correctly folded or denatured form and compared these products to the natural counterpart. In addition, we have produced en zymatically inactive but correctly folded PLA using molecular biologic al techniques. We show that enzymatic active or inactive correctly ref olded rPLA has very similar characteristics compared to purified natur al PLA when assayed in vitro by serological or enzymatic assays and in vivo as determined by skin test activity. In contrast, incorrectly fo lded rPLA had no in vitro or in vivo activity. In particular incorrect ly folded rPLA did not bind human monoclonal or polyclonal antibody of IgG or IgE and consequently did not induce positive type I skin react ivity. The data show the importance of comparatively analysing the nat ural and (r)allergens for the intrinsic nature of allergens such as en zymatic activity or binding to their natural substrate. Both natural o r rPLA bound equally well to synthetic phospholipid bilayers indicatin g their similar binding capacity to the natural substrate the cell sur face of eukaryotic or prokaryotic cells. Hence, a thorough in vitro an alysis comparing the natural to the (r)allergen has to precede the in vivo use. The prospects and limitations of (r)allergens for in vitro o r in vivo diagnostics and potentially for immunotherapy is discussed.