Progress in the Analysis of Food Allergens through Molecular Biology Approaches

Abstract

Food allergies associated with class E immunoglobulins (IgE) are a serious health problem that affects between 1% and 10% of the population of developing countries, with a variability that depends on the geographical area and age range considered. These allergies are caused by a cross-link reaction between a specific food protein (the allergen) and the host IgE. Allergic reactions can range from mild itching to anaphylactic shock and there are no clues to predict the effects of an allergen. Strict avoidance of allergenic food is the only way to avoid possible serious allergic reactions. In the last 30 years a growing number of molecular studies have been conducted to obtain information on the diffusion of food allergens and to establish the structural basis of their allergenicity. At the same time, these studies have also allowed the development of molecular tools (mainly based on synthetic peptides and recombinant allergens) that can be of great help for diagnostic and therapeutic approaches of food allergies. Accordingly, this review focuses on advances in the study of food allergens made possible by molecular technologies and how results and technologies can be integrated for the development of a systematic food molecular allergology. The review may be of interest both to scientists approaching this field of investigation and to physicians who wish to have an update on the progress of research in diagnosis and therapy of food allergies.

Keywords: component resolved diagnosis; epitope mapping; food allergen; food allergy; immunotherapy.

Figure 1

The onset of food allergy reactions. In the gastrointestinal tract, the food antigenic proteins are captured by dendritic cells (DCs) and hydrolysed into small peptides, which are displayed on the cell surface associated with the molecules of the major histocompatibility complex. After migration to the mesenteric lymph nodes, DCs interact with T cells to initiate the adaptive immune response. In the absence of particular stimulatory signals, T cells differentiate into suppressive T cells (Treg, Th3, Tr1) originating a tolerance status (green area). It may happen, however, that in the presence of Th2-promoting cytokines (occurring in the gastrointestinal tract, in the skin and respiratory tract, especially in the presence of epithelial injuries) T cells become Th2-type helper T cells. The Th2-type cells migrate to different tissues and produce specific cytokines (IL-4, IL-5, IL-13) that can induce differentiation of food antigen-specific B cells into plasma cells producing antigen-specific IgE. In the so-called sensitization phase (yellow area), the produced IgE molecules bind to specific receptors on the tissue mast cell. From now on, a subsequent interaction of the antigen with its specific IgE bound to the mast cells will cause mast cell degranulation with release of molecules, such as histamine, prostaglandins, tryptase and cytokines, leading to allergic reaction (red area). More details on the pathogenesis of food allergy can be found in specific review articles [2,3].

Figure 2

Scheme of a possible experimental workflow of molecular approaches for the identification of allergens from food sources, their characterization and development of diagnostic and therapeutic tools. Main procedures are reported in white boxes above the corresponding product, indicated in the green boxes. The results obtained can be then used for the development of molecular tools (yellow boxes) required for CRD and immunotherapy. Dashed arrows indicate the use of data. Red arrows indicate the application of the molecule to CRD or immunotherapy.

Figure 3

Synthesis and screening of a combinatorial peptide library. The synthesis of an n-mer peptide combinatorial library is schematized. Coloured triangles stand for specific amino acids (AA) used for a randomized synthesis of n-mer peptides. White triangles represent any possible amino acid in synthesized peptides. n = peptide length; m = total number of peptides in the library (m = 20ⁿ). Amino acids in red triangles constitute a specific peptide used as a bait for the selection from the library of the peptide with the highest affinity (the length of the bait is not necessarily “n” amino acids). In general, any kind of molecule able to interact with peptides can be used as a bait. The library or the bait are fixed on a solid support to allow selection. In the first case the positions of each peptide on the solid support is known. In DNA based combinatorial libraries, a selected number of codons is randomized in order to obtain after expression the corresponding combinatorial peptide library. In phage display libraries, for example, the gene of interest (containing the chosen degenerated codons) is fused with the gene for a phage surface protein. In this way, after expression of phage particles in Escherichia coli cells, a combinatorial phage library is produced and can be used in the selection step.