Immunoblotting in the diagnosis of cross-reactivity in children allergic to birch

Cudowska B, Kaczmarski M, Restani P

Abstract

Purpose: The scientific experiments with new immunological methods (immunoblotting, RAST inhibition) and isolation of recombinant allergens suggest structural similarities in the allergenic components responsible for cross-reactions. Immunochemical and molecular biology studies indicate that epitopes of major allergen (Bet v 1, Mal  d 1) contain more IgE binding epitopes than minor allergens (Bet v 2, Mal d 2), what explained clinical importance of major birch and apple allergens, but it is individual. The important role in cross-reactivity play also proteins with low molecular weight; a potentially dangerous allergen is lipid transfer protein (LTP) inducing severe systemic reactions in allergic subjects. The recent studies indicate that the IgE cross-reactivity patterns and the clinical relevance is still not clear and that only some of patients with confirmed IgE cross allergy to Bet v 1 and Mal d 1 demonstrated clinical symptoms after ingesting of apple. The aim of study was to establish the pattern of cross-reactivity between major (Bet  v 1) and minor (Bet v 2) birch pollen allergens and apple proteins in children allergic to birch using recombinant allergens and immunoblotting method. 
Material and methods: The prospective study were carried out on the group of 13 children aged 4-16 years, referred to the IIIrd Department of Paediatrics in Biaystok and outpatient clinic with clinical symptoms of food and inhalant allergy. Inclusion criteria to the study were: allergy to birch pollen recombinant allergens and apple, confirmed by presence of specific IgE in the sera of patients. The allergens from peel and pulp of apple and birch were separated and loaded onto the polyacrylamide electrophoretic gel and than transferred to membranes by western blotting. Antigen-IgE complex was detected using goat anti-human IgE antibodies labelled with alkaline phosphatase.
Results: Only few sera presented strong reactions in immunoblotting to birch pollen proteins with a molecular weight of 17-18 kDa, corresponding to the main birch allergen Bet v 1. Most of sera having positive reaction vs Bet v  1 showed cross-reactivity with Mal d 1. All sera recognized specifically the main allergen of apple peal Mal d 3 with molecular weight <10 kDa (Lipid Transfer Protein).
Conclusions: Immunoblotting method allows to verification of cross-reactivity recognized by presence of specific IgE. The nature of proteins responsible for sensitization can influence the spectrum of offending foods and the clinical features of allergic reactions.

Key words: 	cross-reactivity, birch-apple syndrome, immunoblotting, recombinant allergens, lipid transfer protein.