Mapping of the IgE and IgG4 Sequential Epitopes of Ovomucoid with a Peptide Microarray Immunoassay

Abstract

Background: Ovomucoid (Gal d 1) has been demonstrated to be the most important allergen in IgE-mediated egg allergy. Peptide microarray analysis is a novel method that can provide useful information on the nature of specific allergens. Methods: A peptide microarray immunoassay was performed using a 15- and 20-amino acid (aa) library of overlapping peptides (3-offset) of the primary sequence of ovomucoid. Sera from 50 patients with IgE-mediated egg allergy and reactivity to ovomucoid, with more than 1 year of follow-up, and sera from 10 controls were tested. Peptides were considered major epitopes when the average weighted Z-score was greater than 3 and recognized by at least 20% of the patient’s sera. Specific IgE epitopes were established on the basis of the IgE/IgG4 Z-score ratio. Results: The IgE and IgG4 recognition pattern was similar in both sets of peptides, but the signal intensity was generally higher in the 20-aa set. Thirty-four percent of the patients did not recognize any IgE sequential peptide and 20% of the patients recognized more than 10 sequential peptides. We identified 3 major IgE B-cell epitopes in domains I and II of ovomucoid. IgE/IgG4 ratio analysis showed that peptides 1–2 (aa 4–20) and peptides 29–31 (aa 91–104) were specific IgE epitopes. Conclusion: By using peptide microarray immunoassay in egg-allergic patients, we established that 34% of the patients do not have any linear epitope recognized by IgE. Further studies are needed to determine the clinical relevance of this finding.

Common food allergens and their IgE-binding epitopes

“…These observations indicated that the presence of sequential epitope-specific serum IgE antibodies represents the basis of a screening method for persistent egg allergy. In a recent study using microarray techniques, two major Bcell epitopes (AA4e20 and AA46e59) that coincided with the epitopes described previously were identified, 37 indicating that AA4e20 and AA46e59 are clinically relevant epitopes. Although AA4e20 and AA46e59 were recognized by 24% and 32% of patients who tested positive for OM-specific IgE, one third of the patients did not recognize any linear epitopes of OM.…”

“…Although AA4e20 and AA46e59 were recognized by 24% and 32% of patients who tested positive for OM-specific IgE, one third of the patients did not recognize any linear epitopes of OM. 37 The 3-D conformation of OM, including carbohydrate modifications, is preferentially recognized by IgE in egg allergy patients. Thus, the detection of conformational epitopes is also of importance for the diagnosis and prognosis of egg OM allergy.…”

A Novel Multipeptide Microarray for the Specific and Sensitive Mapping of Linear IgE-Binding Epitopes of Food Allergens

“…Because of these obvious limitations, peptide microarrays have recently been developed and used for allergen epitope identification, like in other fields of nucleic acid [32], protein/allergen [33] and peptide [34] research. Selected examples of published microarrays with synthetic overlapping peptides for human IgE epitope mapping of food allergens were on milk lactoglobulin and casein subunits [20,35], egg ovomucoid [36], peanut and walnut allergens [37,38], including Ara h 1 [37], shrimp allergens [39] and Atlantic salmon and cod parvalbumins [40,41]. Most of the published and above-cited microarray-based analysis of linear IgE epitopes of food allergens is based on 1 previous microarray development for milk allergens [20].…”

Hen's Egg Allergy

“…OVM has several unique characteristics, such as stability against heat and digestion by proteinases. There have been a few studies on the IgE-binding epitopes in OVM [7][8][9][10][11] , and the reported binding sites resemble each other ( table 2 ) [12] .…”