RAST Inhibition Research Articles

The effect of thermal processing on the IgE reactivity of the non‐specific lipid transfer protein from apple, Mal d 3

Non-specific lipid transfer proteins (LTPs) are involved in allergy to fresh and processed fruits. We have investigated the effect of thermal treatment and glycation on the physico-chemical and IgE-binding properties of the LTP from apple (Mal d 3). Mal d 3 was purified from apple peel and the effect of heating in the absence and presence of glucose investigated by CD spectroscopy, electrospray and MALDI-TOF mass spectrometry. IgE reactivity was determined by RAST and immunoblot inhibition, SPT and basophil histamine release test. The identity and IgE reactivity of purified Mal d 3 was confirmed. Mild heat treatment (90 degrees C, 20 min) in the absence or presence of glucose did not alter its IgE reactivity. More severe heat treatment (100 degrees C, 2 h) induced minor changes in protein structure, but a significant decrease in IgE-binding (30-fold) and biological activity (100- to 1000-fold). Addition of glucose resulted in up to four glucose residues attached to Mal d 3 and only a 2- and 10-fold decrease of IgE-binding and biological activity, respectively. Only severe heat treatment caused a significant decrease in the allergenicity of Mal d 3 but glycation had a protective effect. The presence of sugars in fruits may contribute to the thermostability of the allergenic activity of LTP in heat-processed foods.

Lipid Transfer Proteins from Fruit: Cloning, Expression and Quantification

Background: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. Methods: cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. Results: The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. Conclusion: rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.

The Major Grass Pollen Group 5 Allergen from Dactylis glomerata and Its C-Terminal Split Product Both Behave as Dimers: Implications for Allergen Standardization

Background: On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M_r) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M_r. The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. Methods: Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. Results: The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD^L/_AGY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. Conclusions: These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.

The effects of gastric digestion on codfish allergenicity

In a recent murine study, we showed that impaired gastric digestion supports the induction of fish allergy by protecting the digestion-sensitive major allergen parvalbumin and thus enhancing its sensitizing properties. The aim of the present study was to investigate whether impairment of peptic degradation might also play a role in the effector phase of codfish allergy. The resistance of cod proteins to digestion by simulated gastric fluid was assessed in vitro . Gastric solutions with pH values ranging from 1.25 to 5.0 were prepared, and the influence of the pH on protein degradation was evaluated by means of SDS-PAGE and IgE immunoblotting. The allergenic potency of digested and undigested cod extract was further characterized in RAST inhibition and basophil histamine release experiments. The digestion experiments revealed that codfish proteins were degraded within 1 minute under physiologic gastric conditions. An only marginal pH shift from 2.5 to 2.75 abrogated completely the digestion of cod allergens. In RAST inhibition experiments digested cod extracts showed a reduced IgE-binding capability that was dependent on the digestion time. Moreover, peptic fragments expressed a 10,000 times reduced allergenic potency, as evaluated on the basis of histamine release from human basophils. Codfish allergens have a grossly reduced ability to trigger an intestinal allergic reaction when they are physiologically degraded. Impairment of the physiologic digestion might thus lower the threshold levels of a food allergen in sensitized patients.

Exposición y sensibilización a Tyrophagus putrescentiae en una población de alérgicos a Dermatophagoides pteronyssinus en Huelva

Background In this work we analyzed the allergological importance of Tyrophagus putrescentiae (Tp) in Huelva (SE Spain). We studied the level of exposition and the grade of sensitization to Tp in a group of patients sensitized to Dermatophagoides pteronyssinus (Dpt). The allergenic cross-reactivity between Dpt and Tp was determined by RAST inhibition. Methods and results We analyzed house dust samples from the dwellings of allergic patients with documented Dpt sensitization. Skin test (ST), conjunctival provocation (CP) and/or specific IgE (RAST) to Tp were performed when Tp was identificated in the house dust sample of the patient. Among the 136 dust samples studied, Dpt was the most frequently identified mite species (94,8 %) and Tp was found in third position (41,1 %) after Glycyphagus domesticus (54,4 %). Among the 45 patients studied, 23 (51,1 %) presented, at least, two positive tests, 18 (40 %) were not sensitized to Tp and 4 (8,8 %) showed contradictory results. 26 patients (57,7 %) inhabited in urban areas and 19 (42,2 %) in rural regions. We determined specific IgE (RAST) to Tp in 25 patients, and the results were positive in 12, with only 7 with values greater than 2 kU/L. No significant correlation were found between IgE-antibody levels to Dpt and Tp. The RAST inhibition studies confirmed the low cross-reactivity between these mites and only in one patient Dpt partially inhibited the IgE-binding to Dpt extract. Conclusions Tp was the second more frequent storage mite in the house dust samples from patients allergic to Dpt in Huelva. However, only half of the exposed patients were sensitized to Tp and the majority inhabited in urban areas. No significant correlation were found between IgE-antibody levels to Dpt and Tp. The RAST inhibition studies confirmed the low cross-reactivity between these mites.

Comparison of the allergenic potency of spores and mycelium of Cladosporium

The allergenic potency of spore and mycelium extracts of Cladosporium was estimated by RAST, RAST inhibition and PCA tests. Spores contained a concentration of allergens higher than mycelia. Results of PCA tests suggested that spores contained specific allergens. However, in a comparative study of extracts from different species of Cladosporium animal and human models gave different estimates of the allergenic potency of the different species. In spite of these variations it was shown that extracts from spores of Cladosporium contained the highest amount of Cladosporium allergens.

The Helix aspersa (Brown Garden Snail) Allergen Repertoire

Background: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. Patients and Methods: In 60 atopicpatients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. Results: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. Conclusions: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.

Monoclonal Antibody-Based ELISA to Quantify the Major Allergen of Cynodon dactylon (Bermuda Grass) Pollen, Cyn d 1

Background: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31–32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. Methods:After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. Results: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1–9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. Conclusions: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.

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

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. 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. 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. 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. 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.

Characterization of natural Dac g 1 variants: An alternative to recombinant group 1 allergens

Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.

Cross-reactivity to eel, eelpout and ocean pout in codfish-allergic patients.

Fish allergy is one of the most common food allergies in both children and adults and patients with allergic reactions to one fish species have in many cases been given the advice to avoid all fish, without further evaluation. The possible common reactivity between different fish species is not well studied. Because of this and a possible exploitation of fish species hitherto not much used in the Scandinavian diet ocean pout, eelpout and eel were evaluated. We examined the serological and biological cross-reactivity of these species in double-blind challenged-confirmed codfish-allergic patients using CAP, Maxisorp-radio allergosorbent test (RAST) inhibition, western blot, skin prick test (SPT) and histamine release (HR). All 18 codfish allergic patients had specific IgE to ocean pout, eelpout and eel determined by Maxisorp-RAST. All four fish species could induce basophil HR using blood from 16 of 18 patients and all patients tested reacted in SPT. This study demonstrates that patients with a verified clinical allergy to codfish in a high frequency express biological cross-reactivity to other fish species. By RAST inhibition this common reactivity was shown to be a true cross-reactivity.

Chemically modified recombinant Fel d 1: A good alternative to hypoallergenic mutants

Rationale Hypoallergenic mutants of recombinant major allergens are proposed to make immunotherapy safer and more efficient. Their development, however, is laborious and requires detailed structural information. The aim of this study was to develop chemically-modified recombinant Fel d 1 as an alternative to a hypoallergenic mutant. Methods Fel d 1 was cloned and expressed in Pichia pastoris as a hybrid molecule of both chains of the allergen. The resulting recombinant (r)Fel d 1 was compared to purified natural (n)Fel d 1 by CD and mass spectroscopy, RAST(-inhibition), competitive RIA, ELISA, immunoblot, basophil histamine release (BHR) and T-cell proliferation assays. Chemical modifocation was carried out using glutaraldehyde. Hypoallergenicity was assessed by RAST-inhibition (RI) and BHR. Results rFel d 1 was expressed at ∼500 mg/L and was shown to be glycosylated by the yeast. Physico-chemical and immunological comparison with nFel d 1 showed that the recombinant molecule is well-folded and that the immune-reactivity of both molecules is very similar. Deglycosylation did not influence immune-reactivity. Chemical modification resulted in at least a 200-fold decrease in IgE reactivity (RI) and a 1000-fold decrease in biological activity (BHR). Conclusions Chemically modification of recombinant major allergens is a good and easy alternative to the development of hypoallergenic mutants.

The Ficus-fruit-syndrome is a distinct entity not related to natural rubber latex allergy

Rationale Ornamental Ficus benjamina (FB) is a new indoor allergen potentially associated with hypersensitivity to fig fruit ( Ficus carica). We evaluated the prevalence of sensitization and studied the cross-reactivity with other fruits and with Hevea latex. Methods A prick test solution from FB latex was tested in 4930 consecutive patients with suspect inhalant allergy. Patients positive to FB were further skin tested with exotic fruits by the prick-to-prick method. RAST inhibition using fig fruit as solid phase was performed in 11 patients with clinical fruit allergy. Results Among 2662 patients reacting to any aeroallergen, 66 (2.5%) reacted to FB. Ten patients showed isolated sensitization. Only 10.6% of FB-positive subjects were also positive to Hevea latex. Sensitization to FB was specifically associated with positive skin tests to fresh fig (83%), dried fig (37%), kiwi (28%), papaya (22%), avocado (19%), banana (15%), and pineapple (10%) (n=54). Clinical reactions were reported mainly from fresh and dried fig and kiwi (47, 60, and 64% of skin test-positive patients). Fig fruit RAST was inhibited 87% by FB, 89% by fresh fig, 80% by dried fig, 38% by kiwi, and 59 and 44% by the thiolproteases ficin and papain, respectively. No inhibition was obtained with Hevea latex and rHev b 6.02 (hevein). Conclusions Sensitization to FB latex is found in 2.5% of atopics and is largely independent of Hevea latex allergy. Sensitization is commonly associated with allergy to figs and other tropical fruits (“Ficus-fruit syndrome”). This cross-reactivity is mediated, at least in part, by thiolproteases but not by hevein-like allergens.

Latex allergy and cross-reactivity to manioc: Report of 2 cases

Rationale Allergy to natural latex is frequently associated with hypersensitivity to fruits and recently, a case of cross-reactivity to manioc was reported. Objective The authors present the cases of two female patients sensitized to latex who have developed anaphylactic reaction with urticaria and bronchospasm immediately after eating boiled manioc. Method Skin prick test to common aeroallergens, latex, manioc and other foods were performed. Serum latex specific IgE levels were measured with CAP-RAST method and RAST inhibition tests were carried out with manioc and latex. Results Skin prick tests to common aeroallergens were negative. Skin prick tests to commercial latex extract were positive in both patients as well as prick tests with raw and boiled manioc. They both had positive skin tests to fresh papaya and one of them to pineapple. Tests were negative to banana, kiwi and chestnut. They both presented elevated serum latex specific IgE. RAST inhibition assays revealed cross-reactivity between latex and manioc. Conclusion These two cases support the previously reported cross-reactivity between latex and manioc, definitely adding it to the list of foods cross-reacting with latex.

Antigenic latex protein from rubber bands

Rationale To determine if there is extractable antigenic latex protein (ALP) in commercially available rubber bands. Methods Packages of commercially available rubber bands were purchased from different retail office supply stores. Sample A was manufactured in the United States and Samples B and C were manufactured in Thailand. The rubber bands were placed in plastic bags and mailed to the laboratory for testing protein content by ASTM Lowry assay (LOWRY) and the Bradford method and for ALP content by latex RAST inhibition (RI) and four monoclonal-based ELISA tests (Hev b5, Hev b 6.02, Hev b1, and Hev b3). Results With the LOWRY and Bradford methods, the protein values for Sample B and C were less than the threshold limit of the assays and no ALP was detectable with any of the RI or ELISA assays in all samples. Sample A had detectable a protein content by LOWRY and Bradford methods of 51.2 μg/gm though the content was barely over the threshold limit of 50 μg/gm. Conclusion These assays were unable to detect significant levels of protein or ALP in any of the extracts from the three lots of rubber bands. The small amount of protein detected in Sample A was either an assay artifact or possibly a contaminant from casein which historically was added during the manufacturing process for stabilization of the rubber compound. There is little scientific evidence to suggest that these commercial products would elicit allergic reactions in latex allergic patients.

Cross reactivity between rat and mouse urinary allergens

Rationale Laboratory animal workers who are sensitized to rats are highly likely also to be sensitized to mice. In a recent study of 100 individuals with specific IgE to rat urine, 62% were also IgE positive to mouse urine. Conversely 95% of mouse-sensitized individuals were also rat sensitized. Rat and mouse urinary proteins both belong to the lipocalin group of proteins and share 66% sequence homology. It is likely that rat and mouse urinary allergens share common B cell epitopes. We have therefore investigated the degree of cross reactivity between rat and mouse urinary allergens. Methods Six individuals with a RAST binding to rat and mouse of greater than 10% were selected for RAST inhibition experiments. Individual sera were incubated overnight with 1mg, 0.1mg, 0.01mg or 0.001mg rat and mouse urine. RAST to rat and mouse urinary protein were carried out the following day using the inhibited sera. Results All individuals exhibited very different inhibition profiles, irrespective of their specific IgE levels to rat and mouse urinary allergens. In general rat urine inhibited the binding of IgE to mouse urine (55-95% inhibition) more strongly than mouse urine was capable of inhibiting specific IgE to rat urine (30–80% inhibition). There was almost complete inhibition with homologous urinary protein. Conclusions These finding confirm immunologically that rat and mouse urinary allergens have common and some unique B cell epitopes which result in varying inhibitions profiles.

Occupational Asthma Due to Grain Pests Eurygaster and Ephestia

Background: Workers occupationally exposed to grain dust have a high prevalence of asthma. The pathogenesis of their respiratory symptoms remains obscure when sensitization to cereal allergens has not been proved. Given the ubiquity of arthropods in stored vegetable products, we have studied the allergenic potential of two very prevalent grain pests, Eurygaster and Ephestia, as a cause of occupational asthma. We have also studied the allergenic relationship between Anisakis simplex (AS)and these pests. Methods: We selected 15 asthmatic workers exposed to cereal dust, in whom sensitization to cereal allergens was not clear. As controls, we selected a patient who suffered from anaphylaxis after the ingestion of cereals, 6 patients sensitized to different arthropods, 1 patient who suffered from asthma after inhaling fish flour contaminated with AS, and a pool of 40 asthmatic patients with different ethiologies not due to arthropods or cereals. We performed prick tests with these pests, AS, and pure and parasitized flours, bronchial challenges, specific IgE determination, and RAST inhibition. Results: All of the 15 cereal‐exposed workers were sensitized to Eurygaster and Ephestia. Only 2 had detectable levels of serum IgE to these pests, but 13 presented a positive prick test result and IgE to AS without problems after eating fish. Bronchial challenges were positive to Eurygaster in 7 patients and to Ephestia in 2 patients. Twelve patients had positive prick tests to parasitized flour but not to pure flour. The patient with cereal anaplylaxis presented sensitization to both flours but not to AS. In the control group, the patient who suffered from asthma after the inhalation of AS‐parasitized fish flour presented a positive prick test result and high levels of specific IgE to these pests. The RAST inhibition showed cross‐reactivity between Eurygaster and AS in this patient, but the inhibition was not as evident with the sera of other workers. The same tests in the group of 40 asthmatic patients were negative. Conclusions: The clinical importance of pests as a cause of baker's asthma should not be underestimated. Sensitization to Eurygaster and Ephestia could be important in the asthma episodes suffered by our patients, and the parasitized wheat was found to have a higher allergenic potential than pure cereal flour.

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.

Fast ω-gliadin is a major allergen in wheat-dependent exercise-induced anaphylaxis

Background: Wheat-dependent exercise-induced anaphylaxis is an anaphylaxy induced by physical exercise after ingestion of wheat. An immediate-type hypersensitivity to water/salt-insoluble fraction of wheat proteins (gluten) has been considered to underlie in this disease. Objective: The aim of the study is to determine the major allergen in Japanese patients with wheat-dependent exercise-induced anaphylaxis by using a panel of purified wheat gliadins and glutenins. Methods: Water/salt-insoluble wheat proteins, α-gliadin, β-gliadin, γ-gliadin, fast ω-gliadin, slow ω-gliadin, high molecular weight glutenin and low molecular weight glutenin, were purified, and five patients with wheat-dependent exercise-induced anaphylaxis, whose diagnose had been determined by positive-challenge test, were evaluated for skin prick test, dot-blotting test and CAP–RAST inhibition test by using these purified wheat proteins. Results: The fast ω-gliadin was the most potent allergen among these water/salt-insoluble proteins when evaluated by skin prick test and dot-blotting test. Fast and slow ω-gliadin, and γ-gliadin caused dose-dependent inhibition of the serum IgE-binding to solid-phase gluten in the patients. The incubation with fast ω-gliadin of the patient's serum caused dose-dependent inhibition in the IgE-binding to γ-gliadin as well as slow ω-gliadin, indicating a cross-reactivity of these proteins in IgE-binding. Conclusion: We concluded that fast ω-gliadin is a major allergen among these water/salt-insoluble proteins for wheat-dependent exercise-induced anaphylaxis in Japanese patients, and IgE against fast ω-gliadin cross-reacts to γ-gliadin and slow ω-gliadin.

Cross-reactivity between Ficus benjamina latex and fig fruit in patients with clinical fig allergy.

Anaphylactic reactions to fig fruits (Ficus carica) have been reported from subjects sensitized to Ficus benjamina (FB) latex allergens. Figs may also be involved in the latex-fruit syndrome. To study the immunologic relationship between fig fruit, Ficus benjamina, natural rubber latex (Hevea brasiliensis), and other tropical fruits. RAST inhibition and Western blotting with FB and fruit extracts was performed in five patients with oral allergy syndrome (OAS) or anaphylaxis after the ingestion of figs and one patient with symptoms from exposure to FB trees. Co-sensitization to rubber latex and tropical fruits (kiwi, banana, avocado, papaya, pineapple, mulberry) was studied by skin testing. RAST to FB was inhibited >95% by FB extracts and 16-65% (mean 49%) by extracts from fresh fig. RAST to fig fruit was inhibited >95% by FB and fresh fig, 63-97% (mean 86%) by dried fig, and 0-84% (mean 35.5%) by kiwi fruit. FB and fig extracts lost most of their allergenicity when denatured by heat (95 degrees C) or reduced by dithiothreitol. Western blotting after non-reducing gel electrophoresis revealed IgE binding to proteins of 22 and 28-34 kDa in FB latex; however, no corresponding allergens could be detected in fig extracts. Positive skin tests were obtained most often with kiwi fruit, papaya, and avocado. Sensitization to rubber latex could not be demonstrated in any of the patients. RAST to papain was positive in three of five patients. Allergic reactions to fresh or dried figs can present as a consequence of primary sensitization to airborne FB allergens independent of sensitization to rubber latex allergens. Kiwi fruit, papaya, and avocado as well as pineapple and banana may be other fruits associated with sensitization to Ficus allergens.