Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: a comparative in vitro study and partial characterization of digestion-resistant peptides.

SCOPE: There are differences in stability to pepsin between the major allergens in peanut; however, data are from different reports using different digestion models. This study provides a comprehensive comparison of the digestibility of the major peanut allergens. METHODS AND RESULTS: Peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were incubated with pepsin to mimic the effect of gastric digestion. Samples were analyzed using SDS-PAGE. To further investigate resistance to digestion, Ara h 2 was additionally subjected to digestion with trypsin and residual peptides were characterized. Ara h 1 and Ara h 3 were rapidly hydrolyzed by pepsin. On the contrary, Ara h 2 and Ara h 6 were resistant to pepsin digestion, even at very high concentrations of pepsin. In fact, limited proteolysis could only be demonstrated by SDS-PAGE performed under reducing conditions, indicating an important role for the disulfide bridges in maintaining the quaternary structure of Ara h 2 and Ara h 6. Trypsin digestion of Ara h 2 similarly resulted in large residual peptides and these were identified. CONCLUSION: Ara h 2 and Ara h 6 are considerably more stable towards digestion than Ara h 1 and Ara h 3.

Lupine allergy: not simply cross-reactivity with peanut or soy.

BACKGROUND: Reports of lupine allergy are increasing as its use in food products increases. Lupine allergy might be the consequence of cross-reactivity after sensitization to peanut or other legumes or de novo sensitization. Lupine allergens have not been completely characterized. OBJECTIVES: We sought to identify allergens associated with lupine allergy, evaluate potential cross-reactivity with peanut, and determine eliciting doses (EDs) for lupine allergy by using double-blind, placebo-controlled food challenges. METHODS: Six patients with a history of allergic reactions to lupine flour were evaluated by using skin prick tests, CAP tests, and double-blind, placebo-controlled food challenges. Three of these patients were also allergic to peanut. Lupine allergens were characterized by means of IgE immunoblotting and peptide sequencing. RESULTS: In all 6 patients the ED for lupine flour was 3 mg or less for subjective symptoms and 300 mg or more for objective symptoms. The low ED and moderate-to-severe historical symptoms indicate significant allergenicity of lupine flour. Two patients allergic to lupine but not to peanut displayed IgE binding predominantly to approximately 66-kd proteins and weak binding to 14- and 24-kd proteins, whereas patients with peanut allergy and lupine allergy showed weak binding to lupine proteins of about 14 to 21 or 66 kd. Inhibition of binding was primarily species specific. CONCLUSION: Lupine allergy can occur either separately or together with peanut allergy, as demonstrated by 3 patients who are cosensitized to peanut and lupine. CLINICAL IMPLICATIONS: Lupine flour is allergenic and potentially cross-reactive with peanut allergen, thus posing some risk if used as a replacement for soy flour.

Detecting fish parvalbumin with commercial mouse monoclonal anti-frog parvalbumin IgG.

Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians. It is the major cross-reactive allergen associated with both fish and frog allergy. We used two-dimensional electrophoretic and immunoblotting techniques to investigate the utility of a commercial monoclonal anti-frog parvalbumin IgG for detecting parvalbumin present in some commonly consumed fish species. The 2D electrophoresis and immunoblots revealed species-specific differences in proteins that appear to represent various numbers of isoforms of parvalbumin in carp (5), catfish (3), cod (1) and tilapia (2). No parvalbumin was detected in yellowfin tuna. Based on minor differences in relative intensities of protein staining and immunodetection, parvalbumin isoforms may have slight differences in the epitope region recognized by the anti-frog parvalbumin antibody. These results suggest that the frog anti-parvalbumin antibody can be used as a valuable tool to detect parvalbumins from the fish tested in this study, except yellowfin tuna.

Food allergen labeling in the USA and Europe.

PURPOSE OF REVIEW: The ingredient statement on the label of packaged foods is an important source of information for food-allergic consumers. New legislation in the USA and European Union will increase the amount of information available to food-allergic consumers. RECENT FINDINGS: The USA has implemented the Food Allergen Labeling & Consumer Protection Act, which mandates use of clear labeling and source labeling of ingredients derived from commonly allergenic sources. Similarly, the European Union is implementing new regulations that mandate the source labeling of ingredients derived from commonly allergenic foods and fuller disclosure of allergenic food components, even when present in small amounts. The food industry is providing consumers with information relating to the presence of allergenic residues by adding voluntary advisory statements, such as 'may contain peanuts'. SUMMARY: Food-allergic consumers are advised to follow specific strict avoidance diets that exclude allergenic foods and ingredients derived from these foods. Thus individuals must avoid potentially hazardous residues of the allergenic food that might occur as the result of common practices in the food industry. The ingredient statement on packaged food labels now contains more information than ever before.

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library.

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.

Gaining perspective on the allergenicity assessment of genetically modified food crops.

Genetically modified plants are created by the insertion of foreign genes into plant cells followed by the generation of reproductively stable stock plants for rapid and precise improvements in agricultural crops. Current products provide resistance to insect pests, plant viruses or herbicides. Future products include nutritionally enhanced crops, salt and draught tolerant crops and plant produced industrial enzymes or pharmaceuticals. The risk that a newly expressed protein might cause serious allergic reactions is real, but the probability is relatively small. Regulatory agencies require a premarket evaluation of the genetically modified crop to reduce the potential for increased risks of food allergy. While absolute proof of safety is not possible, the major risk - transfer of a potent major allergen or nearly identical crossreactive protein - is minimized by allergen-specific serum immunoglobulin E tests that evaluate proteins taken from major allergenic sources or proteins with sequences highly similar to any allergen. Other tests are performed to identify proteins that are likely to sensitize consumers. Experience indicates the current assessment process is working effectively. However, further guidance on bioinformatics and immunoglobulin E assays could increase the reliability of the assessment. Further development of alternative assays may be needed to assess the next generation of products.

How accurate and safe is the diagnosis of hazelnut allergy by means of commercial skin prick test reagents?

BACKGROUND: Allergy to tree nuts, like hazelnuts, ranks among the most frequently observed food allergies. These allergies can start at early childhood and are, in contrast to other food allergies, not always outgrown by the patient. Tree nut allergy is frequently associated with severe reactions. Diagnosis partially relies on in vivo testing by means of a skin prick test (SPT) using commercially available SPT reagents. METHODS: Protein and allergen composition of nine commercial SPT solutions was evaluated using standard protein detection methods and specific immunoassays for measurement of five individual allergens. Diagnostic performance was assessed by SPT in 30 hazelnut-allergic subjects, of which 15 were provocation proven. RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. SDS-PAGE/silver staining revealed clear differences in protein composition. The major allergen Cor a 1 was present in all extracts but concentrations differed up to a factor 50. An allergen associated with severe symptoms, Cor a 8 (lipid transfer protein), was not detected on immunoblot in three products, and concentrations varied by more than a factor 100 as was shown by RAST inhibition. Similar observations were made for profilin, thaumatin-like protein and a not fully characterized 38-kD allergen. Ratios of individual allergens were variable among the nine extracts. SPT showed significant difference, and 6/30 patients displayed false-negative results using 3/9 products. CONCLUSION: Variability in the composition of products for the diagnosis of hazelnut allergy is extreme. Sometimes, allergens implicated in severe anaphylaxis are not detected by immunoblotting. These shortcomings in standardisation and quality control can potentially cause a false-negative diagnosis in subjects at risk of severe reactions to hazelnuts.