Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.

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Zusammenfassung. Allergene Lebensmittelbestandteile stellen für viele Nahrungsmittelallergiker in industrialisierten L?ndern ein reales Gesundheitsrisiko dar, wenn sie als nicht deklarierte Zutaten oder unerkannte Kontaminationen in Lebensmitteln enthalten sind. Diese so genannten versteckten Allergene k?nnen, teilweise in geringsten Mengen, schwere Reaktion bei sensibilisierten Personen auszul?sen, manchmal auch mit t?dlichem Ausgang. Zum Nachweis allergener Lebensmittelbestandteile wurden bereits verschiedene analytische Methoden, wie z. B. ELISA- und PCR-Verfahren, entwickelt. Allerdings sind diese Methoden nicht in der Lage, das allergene Potenzial solcher Bestandteile zu erfassen. Um jedoch die biologische Aktivit?t von Allergenen messen zu k?nnen wurde ein Testsystem entwickelt, das auf dem Mechanismus der Typ-I Allergie basiert. Dazu wurden basophile Leuk?miezellen der Ratte mit den Genen des humanen hochaffinen Rezeptors für IgE transfiziert. Die daraus resultierende Zelllinie exprimiert einen chim?ren Rezeptor (mit der IgE-bindenden humanen α-Kette) stabil auf ihrer Oberfl?che und ist somit in der Lage, auch allergenspezifisches IgE aus Allergikerseren zu binden, was mit der Ausgangszelllinie nicht m?glich ist. Die Vernetzung des rezeptorgebundenen IgEs durch das Allergen führt in der Folge zur Freisetzung entzündungsausl?sender Mediatoren, die im Zellkulturüberstand gemessen werden k?nnen. Diese transfizierte Zelllinie wurde zur Analyse einer Vielzahl von Allergenextrakten eingesetzt, darunter auch Extrakte von Lebensmitteln, die Haselnuss- oder Erdnussallergene enthielten. Der Vergleich des biologischen Testsystems mit den ELISA- und PCR-Verfahren für Hasel- und Erdnuss ergab eine ?hnliche Sensitivit?t und Spezifit?t. Die etablierte Zelllinie ist ein neues, wertvolles Hilfsmittel für den Nachweis von Allergenen in komplexen, zusammengesetzten Lebensmitteln und im Besonderen zur Erfassung des allergenen Potenzials solcher Nahrungsmittelbestandteile. Somit kann dieses neue Nachweisverfahren helfen, zus?tzliche wichtige Informationen für die Risikobewertung von Lebensmitteln zu gewinnen.

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Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure

 Stability under digestion is thought to be an important prerequisite determining the allergenicity of food proteins. To test this hypothesis, two important allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts were digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the two digests were investigated by analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins, as shown by electrophoresis. These results were also underlined by immunblot data. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands  1 30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from four patients and sera from three additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are superior to immunoblotting for the immunologic testing of digests. In accordance with clinical observations, the allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions. Received: 6 July 1998     

Food allergy—towards predictive testing for novel foods

The risks associated with IgE-mediated food allergy highlight the need for methods to screen for potential food allergens. Clinical and immunological tests are available for the diagnosis of food allergy to known food allergens, but this does not extend to the evaluation, or prediction of allergenicity in novel foods. This category includes foods produced using novel processes, genetically modified (GM) foods, and foods that might be used as alternatives to traditional foods. Through the collation and analysis of the protein sequences of known allergens and their epitopes, it is possible to identify related groups which correlate with observed clinical cross-reactivities. 3-D modelling extends the use of sequence data and can be used to display eptiopes on the surface of a molecule. Experimental models support sequence analysis and 3-D modelling. Observed crossreactivities can be examined by Western blots prepared from native 2-D gels of a whole food preparation (e.g. hazelnut, peanut), and common proteins identified. IgEs to novel proteins can be raised in Brown Norway rat (a high IgE responder strain), and the proteins tested in simulated digest to determine epitope stability. Using the CSL serum bank, epitope binding can be examined through the ability of an allergen to cross-link the high affinity IgE receptor and thereby release mediators using in vitro cell-based models. This range of methods, in combination with data mining, provides a variety of screening options for testing the potential of a novel food to be allergenic, which does not involve prior exposure to the consumer.     

A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

花生过敏原检测方法研究进展

花生是重要食物过敏原之一,能引起严重的过敏反应。目前尚没有花生过敏的特效疗法,严格避免食入含花生的食物是花生过敏患者的最佳选择,所以食物中花生的检测就显得尤为重要。本文对花生主要过敏原及其过敏反应研究进展进行简要介绍,重点介绍花生过敏原的主要检测方法的新发展,包括传统的酶联免疫吸附实验(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,ELISA和免疫印迹法都是应用较为普遍的检验方法,PCR方法应用较少,但它能从DNA/RNA方面检测花生过敏原的存在;新兴检测方法主要包括生物传感器和质谱法,这两种方法在花生检测方面具有可观的前景。未来的这些检测方法将朝着快速、灵敏、简便的方向发展。     

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.     

Mitigation of Major Peanut Allergens by Pulsed Ultraviolet Light

Peanut allergy represents one of the most severe IgE-mediated reactions with food, but to date, the only effective way to prevent peanut allergy is total avoidance. If allergens could be mitigated during food processing before a product reaches the consumer, this would substantially lessen the food allergy problem. The efficacy of pulsed ultraviolet light (PUV), a novel food processing technology, on reducing peanut allergens, was examined. This study revealed for the first time that PUV was also capable of deactivating Ara h 2, the most potent allergenic protein of peanut. Protein extracts from raw and roasted peanuts were treated for 2, 4, and 6?min and peanut butter slurry was treated for 1, 2, and 3?min in a Xenon Steripulse XL 3000? PUV system. The distance from the central axis of the lamp was varied at 10.8, 14.6, and 18.2?cm. The SDS?CPAGE showed a reduction in the protein band intensity for Ara h 1, Ara h 2, and Ara h 3 at the energy levels ranging from 111.6 to 223.2?J/cm². Reduction of the protein band intensity for peanut allergens increased with treatment time but decreased with increased distance from the PUV lamp. The ELISA for peanut extracts and peanut butter slurry showed a reduction in IgE binding of up to 12.9- and 6.7-folds, respectively, compared to control.     

Reducing the allergenic capacity of peanut extracts and liquid peanut butter by phenolic compounds

Phenolic compounds are known to form soluble and insoluble complexes with proteins. The objective of this study was to determine if phenolics such as caffeic, chlorogenic and ferulic acids form insoluble and irreversible complexes with major peanut allergens, and if such complexation reduces immunoglobulin E (IgE) binding. After adding each of the phenolics to peanut extracts and liquid peanut butter, the soluble materials were analysed by SDS–PAGE and inhibition ELISA. Results showed that addition of the phenolics precipitated most of the major peanut allergens, Ara h 1 and Ara h 2, and that complexation was irreversible. IgE binding was reduced approximately 10- to 16-fold. We concluded that reducing IgE binding by phenolics is feasible. The research, if proven by clinical studies, could lead to the development of less allergenic liquid peanut-based products.     

Polyphenol oxidase/caffeic acid may reduce the allergenic properties of peanut allergens

Polyphenol oxidase (PPO) catalyzes the oxidation of tyrosine residues of proteins and, therefore, their cross‐linking. Previously we demonstrated that cross‐links produced by peroxidase (POD), which also catalyzes tyrosine oxidation, led to a reduction in the allergenic properties of peanut allergens. 11 We postulated in this study that PPO can also reduce the allergenic properties by cross‐linking the allergens. Because caffeic acid, a phenolic compound, can cross‐link proteins, its effect on peanut allergens was also examined. In the experiments, peanut extracts were treated with and without PPO, PPO/caffeic (pH 8, 37 °C for 1 h) and caffeic acid (pH 10.5, overnight), respectively. The samples were then analyzed for cross‐links and IgE binding by SDS‐PAGE, Western blots, and competitive inhibition ELISA. Results showed that, in all cases, cross‐links and a decrease of the levels of two peanut major allergens, Ara h 1 and Ara h 2, were observed. Of the three treatments, PPO/caffeic was the most effective in reducing IgE binding or the allergenic properties of peanut allergens. The availability of tyrosine residues was also demonstrated in a POD‐treated system, using a monoclonal antibody against dityrosine. We concluded that PPO/caffeic acid reduced the allergenic properties of Ara h 1 and Ara h 2 by cross‐linking and decreasing the levels of allergens. Copyright © 2005 Society of Chemical Industry     

Peanut and hazelnut traces in cookies and chocolates: Relationship between analytical results and declaration of food allergens on product labels

Accidental exposure to hazelnut or peanut constitutes a real threat to the health of allergic consumers. Correct information regarding food product ingredients is of paramount importance for the consumer, thereby reducing exposure to food allergens. In this study, 569 cookies and chocolates on the European market were purchased. All products were analysed to determine peanut and hazelnut content, allowing a comparison of the analytical results with information provided on the product label. Compared to cookies, chocolates are more likely to contain undeclared allergens, while, in both food categories, hazelnut traces were detected at higher frequencies than peanut. The presence of a precautionary label was found to be related to a higher frequency of positive test results. The majority of chocolates carrying a precautionary label tested positive for hazelnut, whereas peanut traces were not be detected in 75% of the cookies carrying a precautionary label.     

Development and validation of two dipstick type immunoassays for determination of trace amounts of peanut and hazelnut in processed foods

Two dipstick-type sandwich-ELISAs were developed allowing the detection of trace amounts of peanut and hazelnut in processed foods. The detection limit of both assays was about 10 ng/ml of hazelnut or peanut protein, equivalent to 1 ppm protein in a food sample. The dipstick format allows a fast and cost-effective screening of foods because no specific instrumentation, such as microplate reader and washer, is necessary. The tests can be performed within 3 and 4 hours respectively. Various commercial food samples were tested and the results were compared with previously described and validated quantitative ELISAs for peanut and hazelnut. Results of the dipstick assays and the corresponding microplate ELISAs were in complete concordance. Our results indicated that the dipstick format could be a useful tool for fast and convenient monitoring of food production with regard to the labelling of allergens in food products. This would contribute to improve the protection of consumers from severe allergic reactions.     

食品中花生过敏原及其检测方法的研究进展

花生是常见的过敏原之一,能够引起严重的过敏反应。由于缺乏明确的治疗花生过敏的方法,只能让花生过敏患者尽量避免食入含有花生的食物。但在实际的生产过程中,食品加工往往需要经过复杂的生产工艺,会造成食品之间的交叉污染,部分食品难以准确判断是否含有花生过敏原。因此对于食品中花生过敏原的检测方法的开发就显得尤为重要。本文主要对花生中过敏原的种类及其检测方法的研究进展进行了综述,主要对以下几种方法做了介绍,包括酶联免疫吸附法(ELISA)、免疫印迹法、聚合酶链式反应(PCR)等,以及新兴的生物传感器和质谱法,并对检测方法的未来发展趋势进行了展望。     

Effects of pulsed UV-light on peanut allergens in extracts and liquid peanut butter

ABSTRACT:  Pulsed ultraviolet (PUV) light, a nonthermal technology, was used to treat both the peanut extracts and liquid peanut butter. The objective was to determine if such treatment would lead to a reduction in the allergenic properties of the peanut extract and butter. Peanut samples were PUV treated using a Xenon RS-3000C under the following conditions: 3 pulses/s, 14.6 cm from the central axis of the lamp, 4 min (extract) or 3 min (liquid peanut butter). After the treatment, the peanut samples were centrifuged and the supernatants analyzed by SDS-PAGE and competitive inhibition enzyme-linked immunosorbent assay (ciELISA). For comparison, boiling treatments were also performed. SDS-PAGE showed that while boiling treatment had little effect on the peanut allergens, PUV-light-treated samples displayed a reduced solubility or level of peanut allergens (63 kDa). Solubility of another allergen (18 to 20 kDa) was unaffected. Insoluble aggregates formed were responsible for the reduced level of allergens in PUV-light-treated samples. ciELISA showed that untreated samples exhibited an IgE binding 7-fold higher than the PUV-treated samples. It was concluded that PUV light was effective in reducing IgE binding of peanut extracts and liquid peanut butter. The current study provides an approach to the development of a possibly less allergenic peanut product. However, the reduction in actual allergenicity needs to be confirmed by clinical studies.     

Evaluation of immunochromatographic test kits for food allergens using processed food models

It has been mandatory to label five allergenic substances (AS; egg, milk, wheat, buckwheat and peanut) in all processed foods, since April 2002 in Japan. Two kinds of ELISA kits have been provided as screening test kits for the Japanese official method. The kits have many advantages but some disadvantages, i.e., the kits are not necessarily suitable for daily monitoring in food manufacturing plants, because they require various analytical equipments and the use of complicated procedures. To overcome these drawbacks, we have developed other diagnostic kits based on immunochromatography that should enable more rapid and simple screening for food allergens. Then we examined the performance of these immunochromatographic test kits (IC kits) in terms of sensitivity, repeatability and cross-reactivity to AS proteins in 11 kinds of food models with various heating conditions and physical properties. We also examined processed food models including AS protein of constant concentration, using the IC kits and ELISA kits, and compared the results. The IC kits detected AS proteins at 5 microg/g in the extracts from processed food models, and provided highly reproducible results. Cross-reactivity among the AS proteins was not observed. The results obtained using the IC kits showed performance equivalent to that of the ELISA kits we examined in unheating processed food models including AS proteins of constant concentration. The IC kits should be more suitable for daily monitoring in food manufacturing plants.     

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Hazelnuts are widely used in the food industry, especially confectionary foods. Nevertheless, these nuts contain several allergenic proteins that may be unexpectedly present as contaminants in various foods and may pose a serious threat to allergic consumers. The enzyme-linked immunosorbent assay (ELISA) is the preferred method to assess the level of hazelnut protein contamination. It is commonly used by both the food industry and enforcement agencies. Several ELISA kits are commercially available. However, protein detectability by ELISA may be affected by severe changes that proteins undergo during processing. The aim of this study is therefore to investigate the impact of processing on the ability to detect hazelnut protein by four commercial ELISA kits. Hazelnut proteins in the presence or absence of soluble wheat proteins were modified with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affected the hazelnut protein detection by the commercial kits. The observed impact was highly dependent on the type of ELISA kit used.     

Mitigation of peanut allergenic reactivity by combined processing: Pressured heating and enzymatic hydrolysis

Among food allergens, peanut is one of the most critical. This study evaluates peanut allergenic features after the combination of heat, pressure, and enzymatic digestion under sonication, by immunodetection using serum IgE of sensitized patients and mass-spectroscopy. In the studied population, there was a predominance of patients with sensitization to Ara h 9 (nsLTP) followed by sensitization to seed storage proteins (Sprot, Ara h 1, 2, 3, and 6). The Sprot sensitized patients showed higher reactivity. The enzyme E5 was efficient for inducing protein fragmentation and allergenic reactivity reduction when it was used combined with pressured heating treatments such as autoclave and Controlled Instantaneous Depressurization (DIC). Only a few Ara h 1 and Ara h 3 peptides were identified after enzymatic digestion of DIC peanut samples. The combination of pressured heating treatments and enzymatic hydrolysis was the most efficient method to strongly mitigate or even eliminate the allergenic potential of peanut. Our findings set a possibility for a group of patients in which their allergy could be treated with a processed less-allergenic peanut and consequently less risky, more easy and quicker desensitization treatment.Industrial relevanceThe findings identify innovative thermal, pressure and enzymatic processing conditions highly effective to mitigate or even abolish the allergenic potency of peanut, which may be relevant for consumers, clinicians, regulatory agencies and the food industry. The applications of processed peanut with reduced IgE binding potency for tolerance induction might be a convenient strategy.     

IDENTIFICATION AND QUANTIFICATION OF MAJOR PEANUT ALLERGENS (IN CHINA) WITH IGE-BINDING PROPERTY

Peanut allergens have not been studied in China. This study aimed to investigate (1) whether there are differences in the relative amounts of major peanut allergens between the Chinese peanut varieties and the American, and (2) the effect of cooking methods on peanut allergenicity. The allergenic property of raw peanuts and peanut preparations was assessed by immunoblotting and enzyme-linked immunosorbent assay. The relative contents of the major peanut allergens were quantified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. For results, Ara h 1 and Ara h 3 were major peanut allergens in China. The amounts of Ara h 1 and Ara h 3 in peanut varieties differ significantly and were both lower than the American varieties. The immunoglobulin E (IgE)-binding ability of different processed peanuts to IgE was not significantly different. Therefore, peanut varieties may induce different amounts of allergens. The relative lower contents of Ara h 1 and Ara h 3 may lead to the lower prevalence of peanut allergy.

PRACTICAL APPLICATIONS

Because of its nutritional and rheological properties, peanut is used in a wide range of different foods, and peanut allergy represents an important health problem. It is essential to identify the compounds of peanut allergens and to study their characteristics in order to explore approaches for the therapies and to breed the nonallergenic peanut seed.     

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 µg g^-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.     

Commercial soybean lecithins: a source of hidden allergens?

 Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb 025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20 ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14 kDa and 94 kDa. A heat-stable allergen of 39 kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer. Received: 22 January 1998     

Detection of sesame seed DNA in foods using real-time PCR

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.