Analyses of the molecular basis underlying allergenicity and allergen cross-reactivity, as well as improvement of allergy diagnostics and therapeutics, are hampered by the lack of human monoclonal IgE antibodies and knowledge about their epitopes. Here, we addressed the consecutive generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1.Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107 mononuclear cells. An obtained scFv was converted into human immunoglobulin formats including IgE. Using variants of Bet v 1, the epitope of the antibody was mapped and extrapolated to other PR10 proteins.The obtained antibodies exhibited pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein Mal d 1. The epitope as defined by the IgE paratope and a set of chimeric Bet v 1 fusion proteins and fragments could be assigned to a C-terminal helix-structured motif comprised by amino acid residues 132-154, including the critical residue E149. Grafting this motif re-established the reactivity of the per se nonreactive Mal d 1 framework. Cross-reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses.The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the IgE epitope nature of Bet v 1 and homologues allows for detailed insights into molecular aspects of allergenicity and cross-reactivity within the PR10 protein family.     «

Analyses of the molecular basis underlying allergenicity and allergen cross-reactivity, as well as improvement of allergy diagnostics and therapeutics, are hampered by the lack of human monoclonal IgE antibodies and knowledge about their epitopes. Here, we addressed the consecutive generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1.Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107...     »