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- Materials and methods
Background: Wheat is a potent allergen source and can cause baker’s asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker’s asthma, wheat-induced food allergy and grass pollen allergy.
Methods: We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker’s asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts.
Results: We identified recombinant wheat flour allergens, which are specifically recognized by patients suffering from baker’s asthma, but not from patients with food allergy to wheat or pollen allergy. rPhl p 1 and rPhl p 5 were identified as marker allergens specific for grass pollen allergy. They can be used to replace grass pollen extracts for allergy diagnosis and to identify grass pollen allergic patients among patients suffering from baker’s asthma and wheat-induced food allergy. Profilin was identified as a cross-reactive allergen recognized by patients suffering from baker’s asthma, food and pollen allergy.
Conclusions: Our results indicate that it will be possible to design serological tests based on micro-arrayed recombinant wheat seed and grass pollen allergens for the discrimination of baker’s asthma, wheat-induced food allergy and grass pollen allergy.
Triticum aestivum (bread wheat) can cause three distinct immunoglobulin-E (IgE)-mediated allergies, respiratory allergy caused by inhalation of wheat flour, food allergy by ingestion of wheat products and wheat (i.e. grass) pollen allergy. Respiratory allergy to wheat flour is one of the most frequent causes of occupational asthma. Approximately 1–10% of bakery workers are affected by baker’s asthma (1). Wheat-induced food allergy is common and affects up to 8% of young children (≤3 years) and 2% of adolescents and adults (2–5). Triticum aestivum is an important member of the grass family (6). Up to 40% of all allergic individuals show IgE reactivity to grass pollen allergens (7). Several studies have reported cross-reactivity between wheat flour and grass pollen due to common IgE epitopes in wheat flour and grass pollen proteins (8–10). Furthermore, diagnosis based on wheat flour extract does not allow discriminating between patients suffering from respiratory allergy and those suffering from food allergy to wheat. Therefore, precise diagnosis still relies on specific inhalation challenge in case of respiratory allergy to wheat flour (11) and double-blind, placebo-controlled food challenge (DBPCFC) in the case of suspected food allergy (12). To improve IgE-serological tests for the discrimination between patients suffering from baker’s asthma, wheat-induced food allergy and grass pollen allergy, we have developed an allergen micro-array on the basis of purified allergen molecules. We investigated the IgE reactivity profile to recombinant wheat allergens, timothy grass pollen allergens and compared the test results with clinical results and serological tests based on allergen extracts. The results from the micro-arrays show that recombinant wheat allergens can be identified, which are highly specific for baker’s asthma, whereas others represent cross-reactive allergens in pollen and food allergy. The IgE reactivity profile of patients to the recombinant wheat and grass pollen allergens may allow differentiating between baker’s asthma, food allergy to wheat and grass pollen allergy.
- Top of page
- Materials and methods
Allergens from wheat (T. aestivum) can mediate baker’s asthma, food allergy and grass pollen allergy. Here, we investigated the usefulness of micro-arrayed recombinant wheat seed allergens and recombinant grass pollen allergens for the differential diagnosis of respiratory allergy to wheat flour, wheat-induced food allergy and grass pollen allergy. We compared IgE antibody reactivities to traditional allergen extracts and micro-arrayed recombinant allergens in three groups of clinically well-characterized patients.
The panel of six recombinant wheat seed allergens had been obtained by IgE immunoscreening of wheat seed cDNA library using sera from patients with baker’s asthma. Of the six wheat seed allergens described by us, only thioredoxin H has been identified earlier as wheat allergen together with an alpha amylase inhibitor, gliadins, a serine carboxypeptidase, a leucine-rich repeat protein, agglutinin, a putative high mobility group protein and a Phl p 1 homologue (24). Other earlier described wheat allergens comprise alpha amylase inhibitor, omega 5 gliadin, members of the alpha amylase/trypsin inhibitor family, glutenin, lipid transfer protein, peroxidase, serpin, a lipid binding protein and glycogen/starch synthase (25–28).
Using four of the recombinant allergens isolated by us, we were able to differentiate between respiratory forms of wheat allergy and food allergy to wheat, which indicates that patients suffering from baker’s asthma are sensitized to allergens, which are different from those recognized by patients suffering from food allergy to wheat. The allergen panel is not yet complete and also other allergens may be involved in baker’s asthma because only 64% of the patients suffering from respiratory allergies to wheat were diagnosed, but our results indicate that it may be possible to develop recombinant allergen-based tests for the discrimination of the two types of wheat-induced allergies.
Except profilin, which occurs as cross-reactive structure in grass pollen and wheat seeds, the other five recombinant wheat seed allergens were specifically recognized by patients suffering from allergies to wheat seed products, but not by grass pollen allergic patients. The recombinant wheat seed allergens should therefore also be useful to discriminate pollen and seed-induced forms of allergies. The latter aspect seems to be of particular importance because we found that half of the wheat food allergic patients with positive IgE reactivity to crude timothy grass pollen extract showed no evidence for a clinically relevant grass pollen allergy. Moreover, 65% of the patients with grass pollen allergy exhibited false positive IgE test results to crude wheat seed extracts, but none of these patients suffered from clinically relevant respiratory or food allergy to wheat seed products. These findings are in accordance with former studies, which have shown that a CAP-FEIA positive result to wheat flour extract does not correlate with clinical symptoms (9). Our results thus indicate that in vitro diagnosis of allergy to wheat may be improved by using micro-arrayed recombinant wheat seed allergens.
In agreement with former investigations, we found that the clinical condition of grass pollen allergy could be readily diagnosed with recombinant timothy grass pollen marker allergens Phl p 1 and Phl p 5, which therefore can be confirmed as marker allergens for a genuine sensitization to grass pollen (22, 23, 29). Moreover, it was possible to identify with micro-arrayed recombinant grass pollen allergens (e.g. Phl p 1, Phl p 5) those patients among the patients with wheat-induced food allergy and bakers asthma, who suffered also from grass pollen-related symptoms.
In addition to the species-specific marker allergens rPhl p 1 and rPhl p 5, the cross-reactive allergens rPhl p 7 and rPhl p 12 were included in the micro-array. We observed that all patients positive to wheat seed profilin also were positive to timothy grass pollen profilin (rPhl p 12), which can be explained by the high cross-reactivity between profilins from different allergen sources (30).
In conclusion, our study demonstrates that micro-arrays based on recombinant wheat seed allergens and grass pollen allergens may be useful to discriminate among three different clinical conditions of allergy caused by grasses, i.e. wheat flour-induced respiratory allergy, food allergy to wheat seeds and grass pollen allergy. These tests provide fast results regarding the IgE reactivities against large numbers of allergen molecules based on only minute amounts of serum and thus offer a large number of diagnostic informations to the allergologist, which should facilitate diagnosis and therapy of allergies.