Methods: Der p 1-specific single-chain variable fragments (scFv) were isolated from combinatorial libraries constructed from the IgE repertoire of mite allergic patients and displayed on filamentous phage. They were produced fused to immunoglobulin Fc domains and analyzed for allergen binding by direct and inhibition immunoassays. A scFv was expressed in yeast Pichia pastoris and purified by affinity chromatography.
Results: Two isolated scFv bound strongly to natural Der p 1 by ELISA with different epitope specificity, by comparing with Der p 1-specific mAb of known specificity. A soluble Der p 1-specific scFv did not bind the homolog Der f 1, despite interfering with the binding of mAb-4C1, which recognizes a cross-reacting epitope in both allergens. The overlap of both epitopes was proven by: a) its lack of recognition of the allergen captured by mAb-4C1 and b) the inhibition of the scFv binding to nDer p 1 by mAb-4C1. The scFv was expressed in yeast Pichia pastoris in mg amounts suitable for crystallographic analysis.
Conclusions: Recombinant IgE from combinatorial libraries provide tools for analysis of antigenic determinants in the context of the human IgE antibody repertoire and for the design of immunotherapy.