First, the terms “allergenic source,” “allergenic extract,” and “allergen” should be appropriately defined.
The term allergen source refers to the material container of allergens; for example, peach, dog, egg, and milk are allergen sources.
Allergenic extracts, commonly used in in vivo and in vitro laboratory diagnostics, come from defined allergenic sources (peach, dog, mites, grass pollen, etc.) and are obtained by extraction and purification processes. Subsequently, the extracts are standardized according to different methods in the various diagnostic industries, with consequent results that are not comparable with each other due to the present lack of a harmonization process.
The quality of these extracts has improved substantially over the years; however, it still presents criticalities and limitations that are difficult to eliminate. The criticalities are intrinsically linked to the extraction processes, which cause the loss of some allergenic proteins, the acquisition of proteins from unknown sources, and the different concentrations and protein composition from one batch to another. Only recently, the concentrations (expressed in μg/mL) of major allergenic proteins have been measured in the extracts, but not of minor proteins, which might even be absent. The absence or low concentration of allergenic proteins in the extract may cause false negatives during diagnosis and con sequent ineffectiveness of hyposensitizing therapies when the proteins contained in the extract are not present at the concentrations necessary to induce desensitization.
The results of in vitro specific IgE according to the extracts used vary according to the type of extract used, and the method used (CAP, Thermo Fisher Scientific; Immulite, Siemens etc.) and are therefore not comparable. To these variables, which can be defined as “dependent on the diagnostic technology,” must be added those related to the clinical symptoms, the age of the patient, the time when the tests are performed (onset of the disease or follow-up), the prevalence of allergy in the population studied, the clinical complexity of the allergic pathology, and its globality.
The limitation, so far insuperable, consists in the impossibility of establishing, in a patient showing polysensitization to specific IgE in vitro, whether the polysensitization is due to cosensitization (sensitization to distinct and unique molecules from different allergen sources) or a mechanism of co-recognition (sensitization to different allergen sources containing homologous molecules).
The term allergen refers to a protein, glycoprotein, or carrier-conjugated haptene, with a molecular weight of 5–150 kDa and an isoelectric point between 2 and 10, capable of binding specific IgE and inducing an allergic reaction.
Thus, each allergen source contains different allergenic proteins, and each allergen may have a different number of antigenic determinants or epitopes.
An epitope is defined as an amino acid sequence recognized by a specific antibody; for example, milk-specific IgE recognizes specific epitopes contained in milk.
Generally, epitopes can be distinguished into linear, when IgE recognizes a contiguous amino acid sequence in the primary structure of the antigen, and conformational, when IgE recognizes a noncontiguous amino acid sequence characteristic of the three-dimensional structure of the protein (Fig. 1).
Fig1. Conformational epitopes consist of noncontiguous amino acids in the protein’s primary structure, but they to be adjacent in the tertiary structure due to the folding of the protein chain. Their formation, therefore, depends on the three-dimensional structure of the allergen. Linear epitopes, on the other hand, are made up of amino acids that are adjacent in the primary structure of the protein. (Copyright EDISES 2021. Reproduced with permission)
The primary sequence of an allergen can be easily found in online search sites such as Allergome (AllergomeAligner, www.allergome.org/ script/tools.php?tool = blaster) or BLAST in UniProt (www.uniprot.org).
The structural folds of a protein are of primary importance in provoking immunological sensitization and the relative antibody response. Many allergenic proteins, if subjected to heat or the action of proteolytic enzymes, as occurs during food preparation or the digestive process, undergo modifications that can determine the loss of conformational epitopes but also the possible unmasking of linear epitopes.
Food allergens can be divided into:
• Class 1 food allergens: these are made up of proteins resistant to digestion and heat and can act as sensitizers in the gastrointestinal tract. To this class belong, for example, the major allergenic proteins of milk, egg, fish, crustaceans, and some vegetables.
• Class 2 food allergens consist of proteins that are not resistant to heat and digestion and are generally incapable of causing systemic symptoms. They are present in plants and foods of animal origin (thermolabile proteins of milk, meat, and eggs) and cause symptoms mainly localized to the oral cavity (oral allergy syndrome) as they lose their antigenic power following degradation in the stomach. The symptoms appear after sensitization to homologous allergens contained in pollens (nonsensitizing elicitors). This phenomenon, defined as cross-reactivity, explains why some patients can present even severe reactions when taking allergenic foods never before ingested.
The allergenicity of a single protein, therefore, depends on:
• its epitopes;
• its spatial conformation upon exposure to antigen- processing cells, such as macrophages, dendritic cells, or B lymphocytes;
• avidity (degree of reaction) between IgE and epitopes, which in turn depends on the number of allergenic epitopes on the molecule (valence), the size, and conformation of the molecule;
• degree of affinity between antibodies and epitopes, which increases in the course of the humoral immune response.
In recent years, 2503 molecular allergens have been characterized at the molecular level, and the latest update is January 6, 2017.
Identifying and characterizing allergenic sources have led to the subsequent industrial production and marketing of natural allergens purified or produced with recombinant DNA technologies.
The recombinant molecules thus obtained have a sensitivity of over 70% in mimicking the allergenic source.
Allergenic molecules are divided into genuine, true markers of a specific source (e.g., Ole e 1 is the marker protein of allergy to olive tree pollen and other Oleaceae), and panallergens, proteins shared by allergenic sources even taxonomically unrelated to each other, responsible for apparent polysensitization to tests performed with extracts. For example, profilin is a panallergen shared by pollens and plant foods. Its recognition by a patient allergic to pollens will cause positivity to all types of pollens and plant foods tested without the patient experiencing symptoms upon exposure to them.
The naming of the components observes an international convention:
• The first three letters (such as Phl, Bet, etc.) correspond to the first three letters of the Linnean name of the allergenic source (in the example, Phleum, Betula);
• The fourth letter (in lower case, as defined by the nomenclature of living organisms) indicates the first letter of the second name of the allergenic source. For this reason, a molecular component of Phleum pratense is defined as Phl p;
• A number is added to the letters to distinguish each com ponent from all the others: Phl p1 indicates the first com ponent identified (and usually cloned) in Phleum pratense;
• Other numbers can be used to define the component further: for example, Amb a 1, from Ambrosia artemisiifolia, has some isoallergens: Amb a 1.01, Amb a 1.02, Amb a 1.03, and Amb a 1.04. For Amb a 1.01, three different variants have been described (Amb a 1.0101, Amb a 1.0102, and Amb a 1.0103), characterized by a very high homology in the primary sequence;
• Finally, the letter “r” or “n” preceding the name of the component indicates its origin (r for recombinant or n for natural). Recombinant components are allergens cloned into eukaryotic or prokaryotic vectors using genetic engineering techniques; when a component is produced in prokaryotes (e.g., Escherichia coli), it does not have glycosylated chains.
Extractive natural molecules are highly purified (in this case, posttranslational modifications, such as glycosylations, are present).
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