Epitope mapping of region 11-70 of ovalbumin (Gal d I) using five synthetic peptides.

Abstract

Five successively located peptides, in region 11-70 of the major allergen of ovalbumin (OA) Gal d I (11-19, 20-33, 34-46, 47-55, 56-70), were obtained by manual solid-phase peptide synthesis. These peptides together with the previously reported OA region 1-10 comprise a segment of 70 amino acid residues located at the N-terminal of ovalbumin. The crude peptides were purified by gel filtration and reversed-phase high-performance liquid chromatographies and their sequences were verified. Polyclonal antibodies against the peptides conjugated to carrier protein (BSA) were raised in rabbits. Rocket line immunoelectrophoresis showed that four peptides (20-33, 34-46, 47-55 and 56-70), could deflect OA-line immunoprecipitates. The peptide's affinity to rabbit polyclonal Ig was examined by quantitative precipitation inhibition and the results suggested that an epitope was encompassed in segments 34-55 and 47-55. Allergenicity was tested by inhibition of specific IgE binding of ovalbumin, using several sera and a serum pool from 16 egg-allergic patients. The results showed that the allergenicity was distributed over the whole region. These findings suggested that: (a) the region 11-70 of OA seemed not to encompass continuous epitopes; (b) the antigenicity of this region was convincing for peptides 34-46 and 47-55; (c) the allergenicity, though dependent on the patient serum used, was distributed over the whole of region 11-70; (d) peptide 11-19, although weak antigenically was capable of specific IgE inhibition; (e) human and rabbit polyclonal antibodies did not show analogous affinities to the present peptides.