Methods: IgE and IgG4 immunanalysis of allergen proteins and peptides were performed using antibodies and sera from patients with confirmed peanut and tree nut allergy. The protein extracts of these samples were all normalized according to protein content and subjected to SDS-PAGE, Spot blot, microarray and western blot analysis with sera from multiple patients. Antibody or IgE-reactive bands were identified using mass spectroscopy. Potentially cross-reactive peptides were identified computationally and with peptide-microarray analysis.
Results: Previously unknown IgE and IgG4 epitope maps for multiple nut allergens were developed. IgE cross-reactivity was seen at both at protein and peptide levels of the different nuts. Several proteins that showed cross-reactivity with various antibodies were observed, excised, and shown to be previously identified allergens in the corresponding nut extracts. Previously unidentified, strongly cross-reactive peptides and peptide motifs with various levels of sequence identity were found to be cross-reactive often in proteins that are not in the same protein families (Pfam).
Conclusions: Extensive IgE cross-reactivity was seen between peanuts and tree nuts within the known major allergen protein families. However, previously unidentified cross-reactive proteins seem to also exist. Novel IgE epitopes and peptide motifs were identified that could account for cross reactivity.