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.     

Allergen immunoassays—considerations for use of naturally incurred standards

The enzyme-linked immunosorbent assay (ELISA) offers many advantages for the detection of potentially hazardous allergenic food residues that might become adventitious components of other foods during the course of food production and processing. ELISAs detect proteins, and food allergens are proteins. ELISAs are sufficiently sensitive and specific for detection of food allergen residues. ELISAs can also be produced in formats that are compatible with the industrial food processing environment. However, ELISAs also have disadvantages that should be carefully evaluated and widely recognized. Various food-processing operations can have profound effects on the detectability of allergenic food residues. ELISAs detect intact proteins but protein hydrolysates evade detection in some ELISA formats. The residual proteins present in some ingredients derived from commonly allergenic sources may also not be easily detected with ELISAs because of the nature of the protein residues remaining, e.g. lipophilic. Processing operations can dramatically lower the solubility of proteins. In some food formulations, heat processing, in particular, induces chemical modifications that can affect antibody binding to epitopes in the ELISA. The use of naturally incurred standards where allergenic food residues are incorporated into various representative food matrices and then processed in a manner similar to “real-world” food processing can reveal some of the limitations of allergen ELISAs. Methods for the preparation of naturally incurred standards in chocolate, cookie, muffin, ice cream, pasta, frankfurter, and cream of potato soup are provided as examples.     

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

Challenges and trends in the determination of selected chemical contaminants and allergens in food

This article covers challenges and trends in the determination of some major food chemical contaminants and allergens, which-among others-are being monitored by Health Canada's Food Directorate and for which background levels in food and human exposure are being analyzed and calculated. Eleven different contaminants/contaminant groups and allergens have been selected for detailed discussion in this paper. They occur in foods as a result of: use as a food additive or ingredient; processing-induced reactions; food packaging migration; deliberate adulteration; and/or presence as a chemical contaminant or natural toxin in the environment. Examples include acrylamide as a food-processing-induced contaminant, bisphenol A as a food packaging-derived chemical, melamine and related compounds as food adulterants and persistent organic pollutants, and perchlorate as an environmental contaminant. Ochratoxin A, fumonisins, and paralytic shellfish poisoning toxins are examples of naturally occurring toxins whereas sulfites, peanuts, and milk exemplify common allergenic food additives/ingredients. To deal with the increasing number of sample matrices and analytes of interest, two analytical approaches have become increasingly prevalent. The first has been the development of rapid screening methods for a variety of analytes based on immunochemical techniques, utilizing ELISA or surface plasmon resonance technology. The second is the development of highly sophisticated multi-analyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry for identification and simultaneous quantification of a wide range of contaminants, often with much less requirement for tedious cleanup procedures. Whereas rapid screening methods enable testing of large numbers of samples, the multi analyte mass spectrometric methods enable full quantification with confirmation of the analytes of interest. Both approaches are useful when gathering surveillance data to determine occurrence and background levels of both recognized and newly identified contaminants in foods in order to estimate human daily intake for health risk assessment.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one‐dimensional gel electrophoresis (D1‐DE) as an alternative to the 2‐DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one‐dimensional combination of IEF and SDS‐PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1‐DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one‐dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin‐based protein‐protein interactions. Therefore, D1‐DE could be used in routine work as a convenient alternative to 2‐DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.     

Application of current allergy assessment guidelines to next-generation biotechnology-derived crops

In any single day, our immune systems are exposed to thousands of different proteins from the environment and the food we eat. In a portion of the human population, some of those proteins will stimulate the immune systems to synthesize immunoglobulin E in an allergenic response. The discrepancy between the vast numbers of proteins we encounter and the limited number of proteins that actually become allergens have led scientists on a quest to discover what unique features exist that make proteins destined to be allergens. The information gained from these studies has led to an allergy assessment strategy that characterizes the potential allergenicity of biotechnology products prior to their commercialization. This testing strategy appears to be effective as shown by the fact that there have been no clinically documented food allergic reactions to any of the biotechnology proteins introduced into food crops, to date. The next generation of biotechnology products will most likely contain more complex traits, including nutritionally enhanced food crops, and the question arises as to whether the current allergy assessment strategy will be sufficient to protect the health of the consuming public. In this paper, we discuss general allergen characteristics in order to better understand how proteins become allergens, summarize the current allergy assessment process, evaluate the different aspects of this process for their adequacy in determining the allergenic potential of engineered functional foods, and, finally, we assess the possibility of new technologies having a positive impact on the allergy assessment of nutritionally enhanced crops.     

Quantitative methods for food allergens: a review

The quantitative detection of allergens in the food chain is a strategic health objective as the prevalence of allergy continues to rise. Food allergenicity is caused by proteins either in their native form or in forms resulting from food processing. Progress in mass spectrometry greatly opened up the field of proteomics. These advances are now available for the detection and the quantification of traces of allergenic proteins in complex mixtures, and complete the set of biological tests used until now, such as ELISA or PCR. We review methods classified according to their ability to simultaneously quantify and identify allergenic proteins and underline major advances in the mass-spectrometric methods. Stéphanie Kirsch and Séverine Fourdrilis contributed equally to this paper.     

In Vitro Gastric and Intestinal Digestions of Pulsed Light-Treated Shrimp Extracts

Pulsed ultraviolet light (PUV), a novel technology most commonly used for microbial inactivation, has recently been employed to effectively mitigate food allergens in peanuts, soybean, shrimp, and almond. Putative mechanisms for the efficacy of PUV in reducing allergen reactivity include photothermal, photochemical, and photophysical effects. To date, there are no published data highlighting the effects of in vitro simulated gastric and intestinal digestion on the stability of PUV reduced allergen reactivity of food. In this study, PUV-treated shrimp extracts were subjected to simulated gastric fluid containing pepsin and simulated intestinal fluid containing trypsin and chymotrypsin, and then tested for changes in allergen potency. SDS-PAGE showed no major band deviation between undigested and digested PUV-treated shrimp extracts. IgE binding to tropomyosin remained markedly decreased as seen in Western blot analysis. Total shrimp allergen reactivity remained unchanged following in vitro peptic digestion and was markedly reduced following in vitro intestinal digestion as illustrated in indirect ELISA. The PUV reduced shrimp allergens remained at a low level under the in vitro simulated digestive conditions. The results inferred that PUV could be a potential method to create less allergenic shrimp products that would remain at a low allergen level under human gastric and intestinal digestive conditions.     

The frontiers of mass spectrometry-based techniques in food allergenomics

In the last years proteomic science has started to provide an important contribution to the disclosure of basic aspects of food-related diseases. Among these, the identification of proteins involved in food allergy and their mechanism of activation of toxicity. Elucidation of these key issues requires the integration of clinical, immunological, genomic and proteomic approaches. These combined research efforts are aimed to obtain structural and functional information to assist the development of novel, more reliable and powerful diagnostic protocols alternative to the currently available procedures, mainly based on food challenge tests. Another crucial aspect related to food allergy is the need for methods to detect trace amounts of allergenic proteins in foods. Mass spectrometry is the only non-immunological method for high-specificity and high-sensitivity detection of allergens in foods. Nowadays, once provided the appropriate sample handling and the correct operative conditions, qualitative and quantitative determination of allergens in foods and ingredients can be efficiently obtained by MALDI-TOF-MS and LC-MS/MS methods, with limits of detection and quantification in the low-ppb range. The availability of accurate and fast alternatives to immunological ELISA tests may also enable the development of novel therapeutic strategies and food processing technologies to aid patients with food allergy or intolerance, and to support allergen labelling and certification processes, all issues where the role of proteomic science is emerging.     

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.     

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.     

Assessing the allergenicity of proteins introduced into genetically modified crops using specific human IgE assays

Global commercial production of genetically modified (GM) crops has grown to over 67 million hectares annually, primarily of herbicide-tolerant and insect protection crop varieties. GM crops are produced by the insertion of specific genes that either encode a protein, or a regulatory RNA sequence. A comprehensive safety evaluation is conducted for each new commercial GM crop, including an assessment of the potential allergenicity of any newly introduced protein. If the gene was derived from an allergenic organism, or the protein sequence is highly similar to a known allergen, immunoassays, e.g., Western blot assays and enzyme-linked immunosorbent assay tests, are performed to identify protein-specific IgE binding by sera of individuals allergic to the gene source, or the source of the sequence-matched allergen. Although such assays are commonly used to identify previously unknown allergens, criteria have not been established to demonstrate that a protein is unlikely to cause allergic reactions. This review discusses factors that affect the predictive value of these tests, including clinical selection criteria for serum donors, selection of blocking reagents to reduce nonspecific antibody binding, inhibition assays to verify specificity of binding, and scientifically justified limits of detection (sensitivity) in the absence of information regarding biological thresholds.     

Maize food allergy: lipid-transfer proteins,endochitinases, and alpha-zein precursor are relevant maize allergens in double-blind placebo-controlled maize-challenge-positive patients

Italian patients with maize anaphylaxis have been shown to have IgE toward two major maize allergens: an alpha-amylase inhibitor and a 9-kDa LTP. A complete study on maize food allergens in patients with positive maize double-blind, placebo-controlled food challenge (DBPCFC) is lacking. The objective was to utilize the three maize protein fractions to identify and characterize the most relevant IgE-binding proteins recognized by the sera of Italian and Swiss patients with either a positive maize-DBPCFC or a history of maize-induced anaphylaxis. Osborne’s protein fractions of maize were extracted to obtain water-soluble, total zein, and total protein fractions. Protein IgE-binding capacity was investigated by SDS-PAGE immunoblotting using the sera from DBPCFC-positive patients and from patients with maize-induced anaphylaxis. Purified maize LTP was used to inhibit the IgE immunoblotting of the three protein fractions. IgE immunoblotting demonstrated that the 9-kDa LTP was recognized by all the Italian patients and by none of the Swiss patients. Other allergens were: 14-kDa α-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-β precursor, and 26 kDa zein-α precursor; a newly described allergen, the globulin-2 precursor, identified in the total protein fraction. It is noteworthy that maize LTP and endochitinase were cross-reactive with grape LTP and one grape endochitinase. LTP was found to be the only major allergen in Italian patients with either positive maize challenge or a history of maize-induced anaphylaxis. We have identified other maize allergens in subjects with maize food allergy, as grape cross-reactive endochitinase, however, the clinical significance of these proteins needs to be investigated in larger groups of patients with allergy to these food items.     

Determination of allergens in foods

Food allergy presents an emerging challenge to food safety and mandatory labelling of relevant food allergens is still in debate. To date there are only a few validated detection methods available for a limited number of food allergens. Immunological detection of food allergens can involve either human serum IgE or animal antibodies. Enzyme-linked immunosorbent assays are currently the method of choice to determine allergens in various food products. Polymerase chain reaction assays are promising tools for the future. Performance parameters of the methods such as sensitivity, specificity, limit of detection, recovery and reproducibility are reviewed in detail.     

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Abstract  

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.     

Effects of chemical, physical, and technological processes on the nature of food allergens

A review is presented of studies of different processing techniques and their effect on the allergenicity and antigenicity of certain allergenic foods. An overview of investigated technologies is given with regard to their impact on the protein structure and their potential application in the production of hypoallergenic foods. The use of physical processes (such as heating, high pressure, microparticulation, ultrafiltration, and irradiation), chemical processes (such as proteolysis, fermentation, and refining by extraction), and biotechnological approaches, as well as the effects of these processes on individual allergenic foods, are included. Additionally, the implications of food processing for food allergen analysis with respect to food safety assessment and industrial quality control are briefly discussed.     

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Abstract  Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically. Graphical abstract        

Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: new data from the EuroPrevall project

A large number of food allergens able to induce allergic symptoms in predisposed individuals, including severe, even life-threatening reactions, have been identified and characterized. However, proteins able to cause such IgE-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their 3D structures, biological activity and stability, will help to improve diagnosis of food allergy, avoid unnecessary exclusion diets and assess the risk of cross-reactive allergies to other food sources. This review is dedicated to summarizing current knowledge about the most important food allergen protein families and to presenting data from the EuroPrevall allergen library, a proof-of-concept collection of highly purified, characterized and authenticated food allergens from animal and plant food sources to facilitate improved diagnosis of food allergies. Relevant food allergen sources     

First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry

The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is described. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. Detected concentrations ranged from 10 to 1000 μg/g, demonstrating that the mass spectrometric based method is a useful tool for allergen screening.