S U N D A Y 568 Antigenic Determinants On Der p 1 Identified By Mutagenesis Analysis Based On The Structure Of Allergen-Antibody Complexes Dr. Anna Pomes, PhD, FAAAAI, Ms. Jill Glesner, Ms. Lisa D. Vailes, Dr. Wladek Minor, PhD, Dr. Maksymilian Chruszcz, PhD, Dr. Martin D. Chapman, PhD, FAAAAI; Indoor Biotechnologies, Inc., Charlottesville, VA, University of Virginia, Charlottesville, VA, University of South Carolina, Columbia, SC. RATIONALE: Der p 1 is a major dust mite allergen, cross-reactive with the homolog Der f 1. Two structures of Der p 1 in complex with Fab fragments of either monoclonal antibody (mAb) 4C1 or 5H8, that partially inhibit IgE antibody binding, were solved by X-ray crystallography. The goal was to identify the residues involved in allergen-antibody interactions, and IgE antibody binding sites by site-directed mutagenesis. METHODS: Single or multiple mutants of Der p 1 residues involved in mAb 5H8 and/or 4C1 binding were expressed in Pichia pastoris and purified by affinity chromatography. IgE and mAb binding assays were performed by ELISA using sera from mite allergic patients. RESULTS: The capacity of the mutants to bind the mAbs was reduced up to 40 times for 5H8 and almost completely for 4C1, depending on the mutation, and according to the displacement of the mAb direct binding or inhibition curves. This result confirmed the importance of specific residues in the allergen-antibody interaction. Specific mutations in each epitope also reduced IgE antibody binding in different degrees (up to 40%) depending on the patient’s serum and the mutation, and proved an overlap between IgE and mAb binding sites. CONCLUSIONS: Site-directed mutagenesis analysis of residues from epitopes identified by X-ray crystallography revealed mechanisms of antibody recognition. Amino acids important for allergen-antibody interaction were identified for the expression of allergen mutants with reduced IgE antibody reactivity with potential use for immunotherapy.