Abstract
BackgroundAllergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.MethodologyTherefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.ConclusionThe experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.
Highlights
IgE-mediated hypersensitivity reactions, the most common cause of allergy, affect more than 25% of the population in industrialized countries
The allergic disease is typically divided into a sensitization phase, where patients do not experience allergic symptoms but become primed to react with certain allergenic molecules, and the immediate phase of the allergic reaction
This resulted in denaturation of Bet v 1.0401 (Figure 1), the protein refolded after lowering the pH to 8.5 using citric acid
Summary
IgE-mediated hypersensitivity reactions, the most common cause of allergy, affect more than 25% of the population in industrialized countries. A detailed immunologic characterization led to a classification of the Bet v 1 family in high, medium, and low IgE binding molecules [8,9]. Based on this data, naturally occurring low IgE binding Bet v 1 isoforms appeared most suitable for safe application in SIT. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity
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