Analytical methodology for assessment of food allergens: opportunities and challenges

This review summarizes the available in vitro, in vivo, and informatic methods designed to evaluate different aspects of the capacity of proteins to act as true food allergens. By now, there is no single method to fully assess the potential allergenicity of proteins. The characterization of many food allergens will help to uncover the sequential and structural motifs that determine the behaviour of proteins as food allergens.     

A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens

Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.     

Allergenic activity of mites of the genus Dermatophagoides

The allergenic activities of the laboratory batches of D. farinae allergens have been studied by the methods of indirect mast-cell degranulation, neuroglial cytocrit, electrophoretic mobility changes. D. farinae allergens have been shown to possess specific activity. The method of changes in the electrophoretic mobility of sheep red blood cells has demonstrated that D. pteronyssimus and D. farinae allergens possess common allergenic properties.     

Methods of detection of tick allergens in house dust

Different detection methods of housing dust allergens (belonging to the tick species Dermatophagoides pteronyssinus, D. farinae) were considered. As shown in this study, the immunochemical method in combination with acarological analysis proved to be most promising. On the basis of the data on the cross activity of tick allergens and changes in their content in dust, grounds were given for the expediency of detection by the immunochemical method with the use of polyclonal antibodies to specially obtained immunogen.     

Adverse reactions to foods

Allergic reactions to foods represent a prominent, actual and increasing problem in clinical medicine. Symptoms of food allergy comprise skin reactions (urticaria, angioedema, eczema) respiratory (bronchoconstriction, rhinitis), gastrointestinal (cramping, diarrhea) and cardiovascular symptoms with the maximal manifestation of anaphylactic shock. They can be elicited by minute amounts of allergens. The diagnosis of food allergy is done by history, skin test, in vitro allergy diagnosis and — if necessary — oral provocation tests, if possible placebo-controlled. Avoidance of respective allergens for the allergic patient, however, is often complicated or impossible due to deficits in declaration regulations in many countries. Increasing numbers of cases including fatalities, due to inadvertent intake of food allergens are reported. It is therefore necessary to improve declaration laws and develop methods for allergen detection in foods. Allergens can be detected by serological methods (enzyme immunoassays, in vitro basophil histamine release or in vivo skin test procedures in sensitized individuals). The problem of diagnosis of food allergy is further complicated by cross-reactivity between allergens in foods and aeroallergens (pollen, animal epithelia, latex etc.). Elicitors of pseudo-allergic reactions with similar clinical symptomatology comprise low-molecular-mass chemicals (preservatives, colorings, flavor substances etc.). For some of them (e.g. sulfites) detection assays are available. In some patients classic allergic contact eczema can be elicited systemically after oral intake of low-molecular-mass contact allergens such as nickel sulfate or flavorings such as vanillin in foods. The role of xenobiotic components in foods (e.g. pesticides) is not known at the moment. In order to improve the situation of the food allergic patient, research programs to elucidate the pathophysiology and improve allergen detection strategies have to be implemented together with reinforced declaration regulations on a quantitative basis.     

Comparative characteristics of the diagnostic properties of staphylococcal allergens in an experiment

The diagnostic value of five staphylococcal allergens prepared from a single S. aureus strain by different methods and in different institutions has been tested on the experimental models of delayed, immediate and mixed (immediate and delayed) hypersensitivity in guinea pigs. The advantages of the allergens prepared in Kazan (USSR) for the detection of delayed hypersensitivity and the ultrasonicated allergen, as well as the allergen made in Czechoslovakia, for the detection of immediate hypersensitivity have been noted.     

High performance concentration and gel filtration of rat urinary protein allergens

Allergies to laboratory animals, notably rats, have become an increasingly recognized occupational problem. Identification and isolation of the individual proteins causing allergic reactions, could form the basis for early recognition of sensitivity, diagnosis, control of degree of pollution of the environment and desensibilization treatments. Frequently, allergens originate from dried rat urine. Because earlier published methods were found unsatisfactory we have developed a new strategy for isolation of rat urinary proteins including a high performance technique for their mild concentration on hydroxyapatite. The concentrated allergens have been fractionated according to molecular size by high performace gel filtration and according to carbohydrate content by wheat germ lectin-Sepharose 6 MB affinity chromatography. The obtained fractions have been examined by denaturing and non-denaturing polyacrylamide gradient gel electrophoresis followed by sensitive staining procedures, and tested with respect to allergenicity by skin tests on allergic patients.     

A comparative evaluation of the biological action of allergens and allergoids prepared from plant pollen by the double radial immunodiffusion method

Antisera to allergens and allergoids prepared from timothy, orchard grass, birch and wormwood pollen have been obtained and used in the double radial immunodiffusion test. The preparations of the allergoid row have been found capable of inducing immune response in laboratory animals (rabbits). Both forms of pollen preparations, allergens and allergoids, have been shown to possess common antigenic determinants reacting with antibodies present in antisera to allergens and allergoids. The absence of identity in the ratio of manifestations of gel precipitation reactions for allergoid with respect to the initial forms of allergens of individual pollen preparation has been noted.     

In vivo and in vitro techniques to determine the biological activity of food allergens

Methods for determination of the biological activity of food allergens comprise both determination of the allergenic potency, i.e. the capability to elicit an allergic reaction in an already sensitized individual, and the allergenic potential, i.e. the risk for sensitizing a hitherto non-allergic individual. Several methods are discussed for determination of potency including the double-blinded placebo-controlled food challenge, skin testing, in vitro effector cell assays such as basophil histamine release, and IgE-based techniques such as RAST and RAST inhibition. No reliable methods have yet been developed which can predict the allergenic potential of a food or a food allergen. The progress in the areas of stability studies and animal models for food allergy are discussed.     

Fractionation of Neisseria allergenic preparations by a gel chromatographic method on sepharose 4B

The fraction composition of allergens obtained by different methods from the microbial mass of N. meningitidis, N. gonorrhoeae and N. perflava has been studied. Each method produced its characteristic number of fractions, irrespective of the Neisseria species used. Their molecular weights: more than 2,000,000 daltons, 160,000 daltons, 31,000 daltons and 14,000 daltons. All these fractions are biologically active.     

Study of allergens from vegetables, fruits and berries

Different methods of obtaining allergens from vegetables, fruit and berries have been evaluated. Two technologies for obtaining food allergens have been considered; of these, Coca's method has proved to be more technological, economical and less labor-consuming. The newly developed technology has made it possible to obtain stable, safe and specifically active allergens which may be used for diagnosing food allergy.     

Use of isoelectric focusing for assessing the composition of pollen allergens

The preparations of allergens and allergoids obtained from ragweed, timothy and wormwood pollen, as well as the preparations of allergens from birch and orchard grass pollen differing in the method of their production, have been studied with the use of analytical isoelectric focusing in a thin gel layer. The composition of the preparations of allergoids differs from that of the allergenic preparations from the pollen of the same plant species by the decreased content of protein components detected in this investigation. The main proteins contained in the preparations of allergoids are distributed in the zone of pH 3.5-4.5. Differences in the composition of different batches of the same allergens, manifested by variations in some protein bands or by their absence, have been noted. Protein components with the isoelectric point in the alkaline zone have been detected only in the preparations of ragweed pollen allergens.     

Safety and sensitizing properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine and its effect on hematological indices

The acute and chronic toxicity, influence on hematological characteristics and sensitizing properties of P. aeruginosa polyvalent corpuscular vaccine have been studied in experiments on 3 species of animals. The acute experiment has shown that the LD50 of the preparation contains not less than 7800 million cells, which is almost 160 times higher than the recommended immunizing dose (500 million cells). The safety of the preparation is confirmed by the data obtained in the histological and histochemical investigations of the tissues and organs of animals subjected to multiple immunizations with the vaccine. These investigations have revealed no pathological changes in the animals. During the study of the chronic toxicity of the preparation the hematological characteristics of the animals have been found to remain within normal limits. The vaccine has been shown to possess low sensitizing activity, which is manifested by the absence of severe reactions to allergic skin tests with different bacterial allergens (specific allergens obtained from P. aeruginosa and allergens obtained from other bacterial species), made on completion of the course of immunization with the vaccine.     

Inter-laboratory optimization of protein extraction,separation, and fluorescent detection of endogenous rice allergens

In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52–63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14–16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.     

Allergen exposure and control

Allergens produced by the house dust mites (family Pyroglyphidae) are probably the single most important allergens associated with asthma world wide. If exposure to these allergens in houses could be sufficiently reduced, then asthma symptoms may be markedly reduced and even prevented from being initiated. Only about half of the many attempts to reduce mite allergens in houses have shown any clinical benefit. One reason may be that exposure was not reduced enough-however exposure to mite allergens has never been measured in any trial. This review summarises previous allergen control trials and then provides an outline of allergen exposure, including the nature of exposure, the analytical methods available and the recognised risks of allergen exposure. This provides a perspective to evaluate the individual methods used to kill mites and to reduce exposure to the allergens. The object is to provide a framework to improve and develop allergen avoidance as an effective component of asthma management.The author is supported by a project grant from the National Heath and Medical Research Council of Australia.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Methods of assessment and control of house dust mites population and mite allergen exposure

The work is devoted to analysis of various methods of assessment and control of house dust mites population and mite allergens in city dwellings using own results and the literature data. Data about in-house dust mites biology and ecology, mechanisms of their migration and circulation in modern city conditions are presented. The comparison of several methods of mites number assessment and mite allergens exposure (classical acarological analysis, colorimetric method of guanine detection in house dust, immunochemical methods) has been performed and their advantages and disadvantages analyzed. As choice of adequate avoidance measures is one of the key question, various such measures (mechanical, physical and chemical) have been compared.     

Safe biotechnology. Part 6. Safety assessment,in respect of human health,of microorganisms used in biotechnology

The assessment of microorganisms in respect to human health is an important step for the introduction of new natural and genetically modified production strains to biotechnology. This report outlines the potential hazards posed by industrial microorganisms, important considerations related to pathogenicity, such as routes and portals of entry into the human body, mechanisms of spread of biological material and a definition of pathogenicity.Furthermore the most important steps in the assessment of pathogenicity of unknown strains are described. A short overview on characterization and in vitro and in vivo tests is presented. The hazard related to allergens and toxic metabolites is reviewed and the choice of methods and the handling of strains with unknown potential are discussed.     

Prosopis juliflora pollen allergen induced hypersensitivity and anaphylaxis studies in guinea pigs

In vivo and in vitro allergenic activities of Prosopis juliflora pollen allergens were measured in guinea pigs. Intracutaneous skin test showed an early wheal flare response and a late erythema-redness, sensitized with various concentrations (100, 50, 25, 5 and 1.5 micrograms/ml) of Prosopis juliflora pollen extract after administration of a challenging dose. A 50 micrograms/ml sensitizing dose of Prosopis juliflora pollen allergen gave optimum skin response as both early and late effects. The nature of immunochemical reactivity between pollen allergens and reaginic antibodies were further characterized by histamine release test, gel diffusion test, radioallergosorbent test and passive cutaneous anaphylaxis test. These tests confirm allergenicity caused by Prosopis juliflora pollen allergens and showed the binding of allergens with reaginic antibody and its regulation in guinea pigs.