Proteolytic processing of the peanut allergen Ara h 3.

The allergen Ara h 3 has been purified recently from peanuts. In contrast to recombinant Ara h 3, a 60 kDa single-chain polypeptide, the allergen isolated from its native source is extensively proteolytically processed. The characteristic proteolytic processing for 11S plant storage proteins of the glycinin family is observed for Ara h 3 yielding an acidic and a basic subunit, bound by a disulfide bridge. In addition to this, proteolytic truncation is observed for the acidic subunit but not for the basic subunit of Ara h 3. A series of Ara h 3 polypeptides ranging from 13-45 kDa was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and each band was digested by trypsin. Peptides related to the bands were identified and a scheme positioning the different polypeptides in the Ara h 3 sequence has been constructed. Peptide analysis showed sequence heterogeneity at two positions indicating the presence of multiple genes encoding variant, but highly homologous Ara h 3 proteins. The pool of Ara h 3 polypeptides from its native source illustrated that allergen from the peanut is much more complex than the recombinant protein used for epitope mapping experiments. From several Ara h 3 truncation products one or more immunoglobulin E (IgE) binding sites had been removed. Characterization of the allergenicity of Ara h 3 should therefore also include IgE-binding studies with peanut-derived Ara h 3, providing the high degree of variation in the Ara h 3 protein structure, as this is what peanut-allergic individuals are confronted with.

Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin.

The high resistance of Brazil nut 2S albumin, previously identified as an allergen, against proteolysis by pepsin was examined in this work. Although the denaturation temperature of this protein exceeds the 110 degrees C at neutral pH, at low pH a fully reversible thermal denaturation was observed at approximately 82 degrees C. The poor digestibility of the protein by pepsin illustrates the tight globular packing. Chemical processing (i.e., subsequent reduction and alkylation of the protein) was used to destabilize the globular fold. Far-UV circular dichroism and infrared spectroscopy showed that the reduced and alkylated form had lost its beta-structures, whereas the alpha-helix content was conserved. The free energy of stabilization of the globular fold of the processed protein as assessed by a guanidine titration study was only 30-40% of that of the native form. Size exclusion chromatography indicated that the heavy chain lost its globular character once separated from the native 2S albumin. The consequences of these changes in structural stability for degradation by pepsin were analyzed using gel electrophoresis and mass spectrometry. Whereas native 2S albumin was digested slowly in 1 h, the reduced and alkylated protein was digested completely within 30 s. These results are discussed in view of the potential allergenicity of Brazil nut 2S albumin.

Genetically engineered foods: implications for food allergy.

The products of agricultural biotechnology, including such common foods as corn and soybeans, are already reaching the consumer marketplace. Consumer exposure to such foods is already fairly significant, particularly in the USA. Thus far, no reports exist regarding allergic reactions to the crops that have been approved for introduction into the food supply. These crops have been modified to only a minor extent by comparison with their traditional counterparts, and the level of expression of new and novel proteins is quite low. Thus, consumer exposure to these novel proteins is very low and unlikely to result in allergic sensitization. Nevertheless, foods produced through agricultural biotechnology must be assessed for safety, including their potential allergenicity, before they may be approved by worldwide regulatory agencies for entry into the food supply. However, the adequacy of the current approach to the assessment of the potential allergenicity of foods produced through agricultural biotechnology has been the subject of considerable scientific and regulatory debate.

A novel approach for the detection of potentially hazardous pepsin stable hazelnut proteins as contaminants in chocolate-based food.

Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.

A randomized, double-blinded, placebo-controlled oral challenge study to evaluate the allergenicity of commercial, food-grade fish gelatin.

BACKGROUND: Recent interest in the labeling of foods and food proteins derived from allergenic sources necessitates determination of the potential allergenicity of such food ingredients. Fish gelatin is extracted from the skin of fish species known to elicit allergic reactions in sensitized individuals. OBJECTIVE: To determine the allergenicity of fish gelatin by double-blinded, placebo-controlled food challenges (DBPCFC) in clinically fish-allergic individuals. METHODS: Thirty fish-allergic patients diagnosed according to the EAACI Guidelines were included (age 9-50 years). Skin prick tests (SPT) and Histamine Release tests (HR) were performed with fish gelatin and codfish, and codfish-specific IgE was measured. All patients underwent DBPCFC with a cumulative dose of 14.61 g fish gelatin. RESULTS: In all 30 patients SPT, HR, and specific IgE to codfish were positive. SPT and HR with fish gelatin were positive in 3/30 and 7/30, respectively. One patient showed mild reaction to placebo and no reaction to the active challenge. Two patients reported mild subjective reactions to active challenge. Upon re-challenge one of them described subjective symptoms again to the active challenge (7.61 g cumulated dose of fish gelatin) with no reaction to placebo, while the other experienced very mild subjective symptoms to placebo and nothing to the active. The proportion of truly sensitive patients was estimated to 0.03 in the total study group. CONCLUSION: None of 30 fish allergic patients reacted adversely to the ingestion of 3.61 g cumulative dose of fish gelatin. In this study fish gelatin presents no risk to fish-allergic patients at the doses typically used. Statistically, these results indicate that there is 95% certainty that 90% of fish-allergic consumers will not react to ingestion of a 3.61 g cumulative dose of fish gelatin.

Validated sandwich enzyme-linked immunosorbent assay for casein and its application to retail and milk-allergic complaint foods.

Cows' milk is a commonly allergenic food. Cross-contamination of milk proteins into nondairy, kosher-pareve foods prepared on shared processing equipment can cause severe, life-threatening reactions in milk-allergic individuals. A sandwich-type enzyme-linked immunosorbent assay (ELISA; 96-well plate format) was developed for the detection of undeclared casein in foods. Rabbit anti-casein antibodies were used as the capture reagent. Food samples and standards were ground, extracted in 0.01 M phosphate-buffered saline, clarified by centrifugation, and added to the wells. Goat anti-casein antibodies were employed as the detector antibody, and the amount of antibody bound was determined with a commercial rabbit anti-goat immunoglobulin conjugated to alkaline phosphatase, with subsequent substrate reaction. Antibodies developed were specific to casein, with no cross-reaction observed with 30 foods and food ingredients. Non-milk-containing products such as fruit juices, fruit juice bars, sorbets, and dark and pareve-labeled chocolate were purchased from June 2002 through June 2003. In addition, samples allegedly causing eight milk-allergic consumer complaints were analyzed. The ELISA had a detection limit of less than 0.5 ppm of casein. The casein content in the analyzed foods ranged from less than 0.5 ppm to more than 40,000 ppm casein; undeclared casein residues were found in all of the samples implicated in allergic reactions. The levels of milk contamination in some of the other surveyed products could also be hazardous for milk-allergic consumers. This ELISA method provides a useful quality control tool for the food industry and could also be used as a validation of kosher-pareve status.

Detection of soy proteins in processed foods: literature overview and new experimental work.

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.

Detection of walnut residues in foods using an enzyme-linked immunosorbent assay.

Tree nuts, including walnuts, can be responsible for allergic reactions. Food manufacturers have the responsibility to declare the presence of walnuts on packaged foods even when trace residues may be present from the use of shared equipment or the adventitious contamination of ingredients. The aim of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) method for the detection of walnut protein residues. Mixtures of raw and roasted English walnuts of several varieties were defatted, powdered, and used as separate antigens in sheep and New Zealand white rabbits. An ELISA was developed using the sheep antiroasted walnut serum as the capture reagent and rabbit antiroasted walnut serum as the detector reagent followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. The performance of the ELISA was validated by testing known amounts of walnut (0 to 100 ppm) either spiked into or manufactured into milk chocolate, cookies, muffins, or ice cream. Recoveries of 1 to 100 ppm walnut-in-chocolate ranged from 71.6% to 119%+/- 7% to 16.5%. The walnut ELISA has a detection limit of 1 ppm (1 microg/g) walnut in several food matrices. Substantial cross-reactivity was observed with pecan while minimal cross-reactivity was noted for hazelnut, mustard, mace, and poppy seed among almost 100 foods and food ingredients tested. This walnut ELISA can be used to detect undeclared walnut residues in foods and ingredients and as a tool to validate the effectiveness of allergen control programs for walnuts.

Consumer attitudes and risks associated with packaged foods having advisory labeling regarding the presence of peanuts.

BACKGROUND: Foods with advisory labeling (eg, "may contain") are increasingly prevalent. Consumers with food allergies might ignore advisory labeling advice. OBJECTIVE: We sought to determine whether consumers with food allergy heeded advisory labels and whether products with advisory labels contained detectable peanut allergen. METHODS: Surveys (n = 625 in 2003 and n = 645 in 2006) were conducted at Food Allergy & Anaphylaxis Network patient conferences. Food products bearing advisory statements regarding peanuts were analyzed for the presence of peanut. RESULTS: Consumers were less likely to heed advisory labeling in 2006 (75%) compared with in 2003 (85%, P < .01); behavior varied significantly according to the form of the statement. Peanut protein was detected in 10% (20/200) of total food products bearing advisory statements, although clinically significant levels of peanut (>1 mg of peanut or >0.25 mg of peanut protein) were detected in only 13 of 200 such products. CONCLUSION: Consumers with food allergy are increasingly ignoring advisory labeling. Because food products with advisory labeling do contain detectable levels of peanuts, a risk exists to consumers choosing to eat such foods. The format of the labeling statement did not influence the likelihood of finding detectable peanut, except for products listing peanuts as a minor ingredient, but did influence the choices of consumers with food allergy. CLINICAL IMPLICATIONS: Allergic patients are taking risks by increasingly disregarding advisory labeling.

Determination of no-observed-adverse-effect levels and eliciting doses in a representative group of peanut-sensitized children.

BACKGROUND: Current labeling practices for allergenic foods like peanut can be inadequate. For future regulatory and industry guidelines, information on no-observed-adverse-effect levels (NOAELs) and eliciting doses (EDs) for allergenic foods is necessary. OBJECTIVE: To determine NOAEL and ED in a representative group of peanut-sensitized children, relate these data to history and sensitization, and evaluate the outcome of dietary management. METHODS: From an overall eligible group of 96 peanut-sensitized children, a representative group of 27 was evaluated by questionnaires, skin prick test, determination of specific IgE, and double-blind placebo-controlled food challenge (DBPCFC) with peanut according to the international consensus protocol, with 9 doses ranging from 10 microg to 3 g peanut flour. Dietary management was evaluated over a 12-month period. RESULTS: Twenty-two children (81%) had a positive DBPCFC. The NOAEL in this group was 1 mg peanut flour, corresponding to 2 mg whole peanut. The ED for subjective symptoms (10 mg to 3 g) was significantly lower than for objective symptoms (100 mg to 3 g; P = .002). Severe reactions occurred only at high doses. EDs were not correlated to previous reactions by history, skin prick test, or specific IgE levels. All patients with a positive DBPCFC were advised to follow a strict diet. During the follow-up period, 10 patients had a less strict diet likely containing traces of peanut. In 3 cases, a mild reaction occurred with food products labeled "may contain peanut." CONCLUSION: The NOAEL in a representative group of children with peanut allergy was 2 mg. Dietary compliance in half of this group was inadequate.

Assessing genetically modified crops to minimize the risk of increased food allergy: a review.

The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the current understanding of food allergens and knowledge regarding the prediction of allergenic activity. Although there have been refinements, the key aspects of the evaluation have not changed. The allergenic properties of the gene donor and the host (recipient) organisms are considered in determining the appropriate testing strategy. The amino acid sequence of the encoded protein is compared to all known allergens to determine whether the protein is a known allergen or is sufficiently similar to any known allergen to indicate an increased probability of allergic cross-reactivity. Stability of the protein in the presence of acid with the stomach protease pepsin is tested as a risk factor for food allergenicity. In vitro or in vivo human IgE binding are tested when appropriate, if the gene donor is an allergen or the sequence of the protein is similar to an allergen. Serum donors and skin test subjects are selected based on their proven allergic responses to the gene donor or to material containing the allergen that was matched in sequence. While some scientists and regulators have suggested using animal models, performing broadly targeted serum IgE testing or extensive pre- or post-market clinical tests, current evidence does not support these tests as being predictive or practical. Based on the evidence to date, the current assessment process has worked well to prevent the unintended introduction of allergens in commercial GM crops.

IgE binding to pepsin-digested food extracts.

BACKGROUND: Pepsin resistance of allergens like lipid transfer protein and 2S albumin has been suggested as explanation for the severity of symptoms often induced by these allergens. Component-resolved diagnosis with purified labile and stable allergens has therefore been proposed to better characterize the risk involved in a positive in vitro IgE test. However, for many foods, purified allergens are not (yet) available. OBJECTIVE: It was the aim of this study to evaluate the potential of pepsin-digested whole-food extracts to distinguish between IgE responses to stable (potentially severe) and labile (mild) allergens. METHODS: Sera (n = 143) from Italian, Spanish and Dutch patients with hazelnut and/or apple ingestion-related symptoms were analyzed for residual IgE binding to pepsin-resistant hazelnut and/or apple allergens. Control and pepsin-digested hazelnut and apple extracts were used for radioallergosorbent test analysis and immunoblot analysis. RESULTS: Pepsin digestion of food extracts, like from hazelnut and apple used for in vitro diagnostic tests, provides a way to distinguish sensitization to pepsin-resistant allergens from that to pepsin-susceptible allergens. In this selected group of patients, IgE reactivity to pepsin-digested extracts correlated with sensitization to the stable allergen lipid transfer protein. The analysis further revealed that the use of soluble pepsin can result in false-positive in vitro tests (2/143). CONCLUSION: Pepsin-digested food extracts are a convenient tool to identify patients with IgE antibodies against potentially dangerous stable allergens, in particular for those foods where the relevant stable allergens have not yet been identified. This can increase the clinical prognostic value of food allergy serology.

How much food is too much? Threshold doses for allergenic foods.

This review summarizes recent findings and controversies in the area of threshold doses for allergenic foods. Over the years, there have been many clinical reports that ingestion of small amounts of food can elicit IgE-mediated allergic reactions. In exquisitely allergic individuals, the threshold dose for elicitation of such reactions is often considered to be zero. However, some food-allergic patients report that they can tolerate small quantities of allergenic food. Are very low quantities hazardous to food-allergic consumers? How much of the offending food is too much? Why is the concept of a threshold level important? There have been very few studies to date on threshold doses for allergenic foods, and more research is needed in this important area.

Food allergy and the food industry.

Although most food-allergic reactions occur after ingestion of nonpackaged food products, the food industry has been subjected to increasing scrutiny of their allergen controls; the resulting impact on the industry has been remarkable. Undeclared food allergens have been responsible for many food product recalls during the past 13 years, and the food industry has made significant investment, effort, and improvements in allergen control during this time. Recently, tests for some allergenic foods have been commercialized, and proven useful to the industry in controlling allergens and helpful for regulatory agencies investigating food-allergic consumer complaints. However, testing methods still do not exist for some of the common allergenic foods. Labeling initiatives have been pursued to make ingredient listings more easily understood by food-allergic consumers, but further improvements could still be made. Additional research to determine eliciting doses for allergenic foods is needed to enable science-based risk assessment and risk management.

A Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantitation of Selected Peanut Proteins in Foods.

A sandwich-type, enzyme-linked immunosorbent assay (ELISA) was developed for the detection of selected peanut proteins in foods. Monoclonal antibodies against a series of allergenic peanut proteins were used as the capture antibody. Food sample extracts were then added, and polyclonal rabbit antibodies directed against roasted peanut proteins were employed as secondary antibodies. The amount of allergen bound to the solid-phase was determined by a biotin and streptavidin-peroxidase system. Radioallergosorbent assay (RAST) inhibition studies of the food extracts were done as a comparison. The coefficient of determination for the ELISA and RAST assays was 0.85. Selected food samples were tested by RAST inhibition at another laboratory for comparison. Skin tests were done with selected samples in peanut-allergic adults, and the results correlated to the ELISA and RAST inhibition results. In other studies, defatted peanut protein (0.01 to 5.0%) were added to vanilla ice cream, then extracted and analyzed using ELISA and skin tests. The sensitivity of the ELISA in ice cream was approximately 40 µg/ml. In six of seven peanut-sensitive adults tested, the lowest level of added peanut protein (0.01%, 21 µg/ml) still caused a positive skin test reaction.

In vitro and in vivo characterization of hazelnut skin prick test extracts.

RATIONALE: Hazelnut allergy ranks among the most frequently observed food allergies. Clinical symptoms range from the oral allergy syndrome to life threatening anaphylaxis. Diagnosis of hazelnut allergy partially relies on in vivo testing by means of skin prick testing (SPT). The aim of this study was to characterize hazelnut SPT extracts both in vitro and in vivo. METHODS: Hazelnut SPT extracts were investigated for protein concentration and composition. The major hazelnut allergen Cor a 1, lipid transfer protein (LTP) and thaumatin-like-protein (TLP) were monitored by competitive RIA and immunoblotting. SPT extracts (n = 6) were analyzed for skin reactivity and the correlation between the SPT extract protein concentration and the mean skin reactivity (HEIC) was determined in a group of hazelnut-allergic patients (n = 30). For one SPT extract, the threshold level for Cor a 1 was determined in Cor a 1-monosensitized patients (n = 5). RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. Although some proteins were present in most extracts (bands at 10, 22-28, 32 and around 48 kDa), clear differences in composition were observed (both intra- and inter-variability). The concentration of the major hazelnut allergen Cor a 1 differed up to a factor 50 (0.6-32 micrograms/ml). LTP was virtually absent in 3/9 SPT extracts and variable quantities of TLP were detected by immunoblotting. Some patients (6/30) had a false-negative SPT with 3/6 SPT extracts. There was a clear correlation between the protein concentration and the mean HEIC (RPearson = 0.87). The threshold level for Cor a 1 was +/- 3.2 ng/ml as assessed with one of the products investigated. CONCLUSIONS: Heterogeneous protein concentration/composition of SPT extracts results in variable skin test responses. The absence of potentially severe allergens like LTP may lead to false-negative SPT results that jeopardize a patient's safety. From these results it can be concluded that there is a strong need for standardization of products for SPT.

Bioinformatic methods for allergenicity assessment using a comprehensive allergen database.

BACKGROUND: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. METHODS: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. RESULTS: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared >or=35% identity in an overlap of >or=80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. CONCLUSIONS: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

The distribution of individual threshold doses eliciting allergic reactions in a population with peanut allergy.

BACKGROUND: Hidden peanut in consumer products can endanger patients with peanut allergy. Individual threshold doses for eliciting allergic reactions need to be elucidated to assess the risks for development of allergic reactions after accidental ingestion of peanut in a population with peanut allergy. OBJECTIVE: We sought to determine the distribution of individual threshold doses in a population with peanut allergy and to correlate these thresholds to the severity of peanut-induced symptoms. METHODS: Twenty-six adult patients with a convincing history of peanut-related symptoms, a specific IgE level of 0.7 kU/L or greater, or a positive skin prick test response of 2+ or greater to peanut were included. These patients underwent double-blind, placebo-controlled food challenges with increasing doses of peanut. A threshold dose could be established when objective or repetitive subjective reactions occurred after active doses. RESULTS: All patients had subjective oral symptoms (n = 26), prior subjective gastrointestinal symptoms (n = 14), or objective symptoms (n = 5). Reactions started within 30 minutes after ingestion of peanut, but in 2 patients additional symptoms were delayed by 1 to 2 hours. Threshold doses for allergic reactions ranged from a dose as low as 100 microg up to 1 g of peanut protein. Fifty percent of the study population (95% CI, 30%-70%) already had an allergic reaction after ingestion of 3 mg of peanut protein. Patients with severe symptoms had lower threshold doses compared with those of patients with mild symptoms (P =.027). CONCLUSIONS: A substantial part of a population with peanut allergy will react to very low amounts of peanut, requiring accurate declaration of peanut content in consumer products. This is even more important because patients with severe reactions react to lower doses than patients with mild symptoms.

Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy.

BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8. METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.