Methods For Detection Of Allergens Research Articles

Commercialized rapid immunoanalytical tests for determination of allergenic food proteins: an overview

Food allergies have become an important health issue especially in industrialized countries. Undeclared allergenic ingredients or the presence of "hidden" allergens because of contamination during the food production process pose great health risks to sensitised individuals. The EU directive for food labelling lists allergenic foods that have to be declared on food products by the manufacturers. The list includes gluten-containing cereals, crustaceans, eggs, fish, peanuts, soybeans, milk, various nuts (e.g. almond, hazelnut, and walnut, etc.), celery, mustard, sesame seeds, lupin, and molluscs. Reliable methods for detection and quantification of food allergens are needed that can be applied in a fast and easy-to-use manner, are portable, and need only limited technical equipment. This review focuses on the latest developments in food allergen analysis with special emphasis on fast immunoanalytical methods such as rapid enzyme-linked immunosorbent assays (ELISA), lateral-flow immunochromatographic assays (LFA) and dipstick tests. Emerging technologies such as immunochemical microarrays and biosensors are also discussed and their application to food allergen analysis is reviewed. Finally, a comprehensive overview of rapid immunochemical test kits that are currently available commercially is given in tabular form.

Soybean allergen detection methods – A comparison study

Soybean containing products are widely consumed, thus reliable methods for detection of soy in foods are needed in order to make appropriate risk assessment studies to adequately protect soy allergic patients. Six methods were compared using eight food products with a declared content of soy: a direct sandwich ELISA based on polyclonal rabbit antibody (ab) to raw soy flakes, a commercial and an in-house competitive ELISA both based on ab to denatured, 'renatured' soy protein, an enzyme-allergosorbent test (EAST) inhibition based on two sera from soy allergic patients, histamine release (HR) using basophils passively sensitized with patient serum and a PCR method detecting soy DNA. Eight food products were selected as model foods to test the performance of the methods. There was an overall good agreement between the methods in terms of ranks of soy content but not the quantity. The sandwich ELISA aimed at native soy proteins had the lowest detection limit of 0.05 ppm, but only identified soy in 5/8 products, and generally in lower amounts compared to other methods. The competitive ELISA had a higher detection limit of 21 ppm, but seemed more successful in detecting processed soy. Only HR, EAST inhibition and PCR detected soy in all eight products. In spite of a general good correlation in terms of ranks of soy content, more than a single method may be necessary to confirm the presence of soy in foods.

Antibody binding to hazelnut (Corylus avellana) proteins: the effects of extraction procedure and hazelnut source

The quality of an applied protein extract is important in both serological and in vivo diagnosis of allergy, and for allergen detection methods. In the present study the effects of the extraction procedure and hazelnut source on antibody binding to hazelnut (Corylus avellana) proteins were investigated. An overnight extraction procedure in tris–glycine buffer at 45°C was compared to a quick, vigorous extraction in a tris buffer at room temperature (RT). Antibody binding was studied by the use of polyclonal antiserum, a monoclonal antibody against the pollen-unrelated allergen Cor a 9, and human IgE from hazelnut allergic individuals. The extraction procedure, as well as the storage time and degree of processing of hazelnuts affected the protein constitution of the extract, as visualised by SDS–PAGE. Fresh and roasted (10 min at 170°C) hazelnuts generally contained a higher amount of intact hazelnut proteins than elder (>6 months) raw hazelnuts stored at RT. The time of storage of raw hazelnuts was inversely related to the amounts of Cor a 9 and IgE-binding proteins. In general, a rapid extraction procedure increased the efficacy of protein extraction and antibody-binding capacity to the proteins compared to an overnight extraction protocol. The present study indicates that for the production of hazelnut protein extracts, the use of fresh hazelnuts is important. A quick, vigorous extraction in a tris buffer might contribute positively, at least for extraction of Cor a 9.

Optical Resonance-Enhanced Absorption-Based Near-Field Immunochip Biosensor for Allergen Detection

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.

Development of a method for the quantification of whey allergen traces in mixed-fruit juices based on liquid chromatography with mass spectrometric detection

The availability of accurate and sensitive detection methods for food allergens is crucial for the food industry to ensure the correct labelling of their products in order to protect allergic consumers. For this purpose a method using solid-phase extraction and liquid chromatography coupled to mass spectrometry was developed to detect traces of three allergenic cow milk proteins (lactalbumin, lactoglobulins A and B) in mixed-fruit juice samples. Different sample pre-treatments were compared and the best recoveries were obtained with a method employing a solid-phase extraction cartridge. Recoveries ranging from 68% to 79% were achieved for 5 and 20 μg/ml tested and the limit of detection was set at 1 μg/ml. Both full scan and multiple ion monitoring acquisition modes were investigated and compared. The method was utilized to analyse 15 mixed-fruit juices collected from the market and was found to be capable of positively identifying all three milk proteins. The developed method enables the unambiguous determination of allergenic whey proteins in mixed-fruit juices and can assist in the protection of milk allergic individuals.

Signal enhancement of surface plasmon resonance-based immunoassays for the allergen detection

A surface plasmon resonance (SPR) biosensor was used to determine the recombinant group 1 house dust mite allergen (rDer f1) in both HBS-EP buffer and fetal bovine serum (FBS). The monoclonal antibody was immobilized onto the CM5 sensor chip surface using an amine coupling method. The procedures of antibody immobilization and the subsequent primary and enhanced immunoassay were monitored in real time. The sensitivity for rDer f1 detection was remarkably improved by using intact polyclonal antibody as signal amplifying agent. Using this signal enhanced SPR immunosensor, rDer f1 in HBS-EP buffer and FBS was detected at a concentration of 15.4 and 32.1 ng/ml, respectively. The result demonstrates that SPR biosensor is a simple and reliable method for allergen detection.

Food allergen detection methods and the challenge to protect food-allergic consumers

The detection of allergenic ingredients in food products has received increased attention from the food industry and legislative and regulatory agencies over recent years. This has resulted in the improvement of measures aimed at the protection of food-allergic consumers. The controlled production of food products and control activities executed by food inspection agencies rely on the availability of methods capable of detecting traces of allergenic ingredients. The development of such methods faces a multitude of analytical challenges. Those challenges will be identified and discussed in this review. Furthermore, future developments and trends in analytical methodology as applied to the detection of food allergens are reported.

Detection of celery (Apium graveolens), mustard (Sinapis alba, Brassica juncea, Brassica nigra) and sesame (Sesamum indicum) in food by real-time PCR

Legislation requires labelling of foods containing allergic ingredients, amongst them celery, mustard and sesame. Here we present robust quantitative and sensitive methods for real-time PCR detection of celery, mustard (Sinapis alba and Brassica sp.) and sesame in food. The development of the DNA-based assays was part of an effort to generate alternative detection methods for allergens for which effective protein-based assays are lacking. The celery and sesame methods were specific for the celery mannitol dehydrogenase gene and the sesame allergen encoding 2S albumin gene, respectively, when tested against a range of plant materials. The mustard method was specific for the allergen encoding sinA gene and its homologues present in different Brassica sp. All primer probe pairs gave high amplification efficiency and sensitivities below approximately ten molecules of purified template DNA. These DNA-based detection methods will constitute supplementary and complementary methods to the traditional protein-based methods. Laboratories may choose different analysis formats depending on the food matrix, the availability of specific tests and the performance characteristics of the tests.

High-throughput fluorescent multiplex array for indoor allergen exposure assessment

Current enzyme immunoassay methods for detection of common indoor allergens in environmental dust samples are labor-intensive and time consuming. To develop and validate a fluorescent multiplex array to measure 6 (Der p 1, Der f 1, Der p 2, Der f 2, Fel d 1, and Can f 1) indoor allergen levels simultaneously. A multiplex array for 6 allergens, using mAbs covalently coupled to fluorescent microspheres, was developed using a single universal standard composed of purified natural allergens. The multiplex array was validated by comparing the measured dust mite, cat, and dog allergen levels in household dust samples to those obtained by standard ELISA methods. Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for dust mite, cat, and dog allergens: R(2) values ranging from 0.90 to 0.99 (P < .001). In addition, the sensitivity, limit of detection (<0.1 ng/mL), reproducibility, intra-assay coefficient of variance (<5%), and interassay coefficient of variance (<25%) of the fluorescent multiplex array were shown to be equal to or better than the ELISA method. A multiplex array has been developed to measure simultaneously 6 indoor allergens from a single sample. The array will facilitate epidemiologic studies and indoor air quality assessments and can, in principle, be expanded to include other allergens and biologics. The multiplex array lends itself to clinical studies, population-based environmental surveys, and allergen avoidance studies comparing allergen exposure in large populations over several time points.

Polymerase Chain Reaction Techniques for Food Allergen Detection

Food allergies represent an important health problem in industrialized countries. Undeclared allergenic foods as contaminants in food products pose a major risk for sensitized persons. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labeling and improve consumer protection. The methods currently used for the detection of potential allergens in foods are to target either the allergen itself or a marker that indicates the presence of the offending food. As markers for the presence of potentially allergenic foods or ingredients, specific proteins or DNA fragments are targeted. In routine food analysis, the enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR) in the form of a real-time PCR or in combination with an ELISA have been used. The availability, the characteristics, and some future aspects of DNA-based methods in the rapid and sensitive detection of potentially allergenic food constituents or contaminations are discussed in this review.

Regulatory and Compliance Activities to Protect Food-Allergic Consumers in Canada: Research in Support of Standard Setting and Consumer Protection

An overview is presented of the activities of Health Canada and the Canadian Food Inspection Agency (CFIA) in the area of food allergens. Since the 1990s, changes were made in the Food and Drug Regulations in order to better protect allergic consumers by imposing labeling requirements to clearly identify sources of priority food allergens in prepackaged foods. Policies of application as well as risk management strategies are discussed with some statistics on allergen-related food recalls in Canada for the years 1997--2001. Health Canada's allergen method development program is a pioneering research initiative that was developed in the early 1990s in support of the changing Canadian regulatory environment. The objectives and some of the accomplishments of this program are presented. The development of the Canadian Compendium of Allergen Methodologies under a Web-based application to compile data on evaluated allergen detection methods will provide further support to compliance activities nationally, as well as to the international analytical community in both government and the food industry. Some emerging techniques for the confirmation of results generated by enzyme-linked immunosorbent assays are also discussed.

Food Allergen Detection Methods: A Coordinated Approach

The levels (1-2%) and increasing severity of allergic responses to food in the adult population are well documented, as is the phenomenon of even higher (3-8%) and apparently increasing incidence in children, albeit that susceptibility decreases with age. Problematic foods include peanut, milk, eggs, tree nuts, and sesame, but the list is growing as awareness continues to rise. The amounts of such foods that can cause allergic reactions is difficult to gauge; however, the general consensus is that ingestion of low parts per million is sufficient to cause severe reactions in badly affected individuals. Symptoms can rapidly-within minutes-progress from minor discomfort to severe, even life-threatening anaphylactic shock in those worst affected. Given the combination of high incidence of atopy, potential severity of response, and apparently widespread instances of "hidden" allergens in the food supply, it is not surprising that this issue is increasingly subject to legislative and regulatory scrutiny. In order to assist in the control of allergen levels in foods to acceptable levels, analysts require a combination of test methods, each designed to produce accurate, timely, and cost-effective analytical information. Such information contributes significantly to Hazard Analysis Critical Control Point programs to determine food manufacturers' risk and improves the accuracy of monitoring and surveillance by food industry, commercial, and enforcement laboratories. Analysis thereby facilitates improvements in compliance with labeling laws with concomitant reductions in risks to atopic consumers. This article describes a combination of analytical approaches to fulfill the various needs of these 3 analytical communities.

Information provision for allergic consumers – where are we going with food allergen labelling?

As the current treatment for food allergy involves dietary exclusion of the problem food, information for food-allergic consumers provided on food labels about the nature of allergenic ingredients is important to the management of their condition. The members of an EU-funded networking project, InformAll, focusing on developing strategies for the provision of credible, reliable sources of information for food allergy sufferers, regulators and the food industry, have been considering these matters with respect to food labelling. This paper presents an overview of the genesis of the new EU directive on food labelling, its relevance to food-allergic consumers and the problems that might arise if precautionary labelling becomes more widespread in response to concerns regarding inadvertent allergen contamination in foods. International efforts to define threshold levels of allergens able to trigger a reaction coupled with validated allergen detection methods are essential if the food industry is to implement effective hazard control procedures and address the problems of cross-contact allergens without devaluing the information provided to consumers on food labels.

Effect of roasting history and buffer composition on peanut protein extraction efficiency

Peanut is a major allergenic food. Undeclared peanut (allergens) from mis-formulation or contamination during food processing pose a potential risk for sensitized individuals and must be avoided. Reliable detection and quantification methods for food allergens are necessary in order to ensure compliance with food labelling and to improve consumer protection. The extraction of proteins from allergenic foods and complex food products is an important step in any allergen detection method. In this study, the protein extraction efficiency of various buffers prepared in-house and some extraction buffers included in some commercial allergen enzyme-linked immunosorbent assay (ELISA) test kits for peanut determination in food products were tested. In addition, the effect of roasting history on the extractability of peanut protein was investigated by the biuret and the bicinchoninic acid (BCA) assays. Elevated roasting temperatures in food processing were found to have a major impact on protein extraction efficiency by reducing protein yields of oil and dry roasted peanuts by 50-75% and 75-80%, respectively, compared with the raw material. Extraction buffers operating in the higher pH range (pH 8-11) showed best yields.

Rapid Detection of Fish Major Allergen Parvalbumin by Surface Plasmon Resonance Biosensor

ABSTRACT: Seafood allergy is a common and major cause of food allergy in adults. In recent years, seafood allergy has become a serious problem with the increase of seafood consumption. To develop a rapid allergen detection method based on the affinity of antigen‐antibody interaction, fish major allergen, parvalbumin, was used for kinetic analysis by a surface plasmon resonance (SPR) biosensor. Anti‐parvalbumin murine monoclonal antibody (MAb) EG8 was immobilized onto a carboxymethyl dextran (CMD) surface. By the injection of various concentrations of purified carp parvalbumin (CPa), a standard curve and the affinity constants (KD and k*) for the MAb EG8‐CPa model system were determined. In addition, kinetic data were also obtained by the injection of serial dilutions of extracts from seafood products: sardine fish cake (tsumire) and dried skipjack tuna (katsuonut). Sardine tsumire and katsuonut contained 0.11 mg/kg and 0.39 mg/kg parvalbumins, respectively, where affinity constants KD and k* were almost similar among paralbumins from different sources. In the SPR system, the allergen can be detected only for 5 min according to the allergen‐MAb binding interaction. Consequently, by the use of a SPR biosensor, kinetic analysis based on the allergen specific MAb would be a rapid and powerful tool for allergen detection and quantification.

Methods for allergen analysis in food: a review

Food allergies represent an important health problem in industrialized countries. Undeclared allergens as contaminants in food products pose a major risk for sensitized persons. A proposal to amend the European Food Labelling Directive requires that all ingredients intentionally added to food products will have to be included on the label. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labelling and to improve consumer protection. Methods available so far are based on protein or DNA detection. This review presents an up-to-date picture of the characteristics of the major food allergens and collects published methods for the determination of food allergens or the presence of potentially allergenic constituents in food products. A summary of the current availability of commercial allergen detection kits is given. One part of the paper describes various methods that have been generally employed in the detection of allergens in food; their advantages and drawbacks are discussed in brief. The main part of this review, however, focuses on specific food allergens and appropriate methods for their detection in food products. Special emphasis is given to allergenic foods explicitly mentioned in the Amendment to the European Food Labelling Directive that pose a potential risk for allergic individuals, namely celery, cereals containing gluten (including wheat, rye and barley) crustaceans, eggs, fish, peanuts, soybeans, milk and dairy products, mustard, tree-nuts, sesame seeds, and sulphite at concentrations of at least 10 mg kg−1. Sulphites, however, are not discussed.

Comparison of commercially available ELISA kits with human sera-based detection methods for peanut allergens in foods

Undeclared peanut allergens as contaminants in foodstuffs represent a major health problem for sensitized persons. Various immunochemical techniques are employed to detect and quantify peanut allergens. There is an urgent need to compare and standardize those test systems to enable comparable allergen analyses of foodstuffs, comparable studies, and consequent and consistent measures against the presence of hidden peanut allergens. The present study compared commercially available peanut ELISA kits with human sera-based immunoassay techniques (dot blotting and Western blotting), enabling semiquantitative and quantitative detection, and identification of peanut contaminants in foodstuffs. Additionally, the effect of conventional roasting conditions on the detection and quantification of peanut with the selected methods was investigated.

Is biosensor a viable method for food allergen detection?

The emerging health issue of food-induced allergic reactions presents an important challenge to the food industry. Unfortunately, methods for food allergen detection are still mostly unavailable. Surface plasmon resonance (SPR) immunoassays are based on the biological input of antibody–allergen binding, while the measurable output is either a resonance angle or refractive index value. The feasibility of applying the experimental SPREETA™ evaluation kit as a commercially available biosensor to detect peanut allergens was examined. Preliminary results achieved a detection limit 0.7 μg/ml. This miniature biosensor technology may offer the food industry with a tool to allow for immediate, on-line detection and quantification of inadvertent allergens in food — during the food’s production — not after. Such a tool will be of great benefit to the food processor — enabling management decisions to be made early in the production cycle.