The conformational IgE epitope profile of soya bean allergen Gly m 4

BackgroundBirch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. ObjectiveTo identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. MethodsSera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. ResultsAll subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kU(A)/L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. Conclusion and Clinical RelevanceSerum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.