Diagnosing wheat allergy remains challenging due to the use of oral wheat provocations, which implicate risks to patients and highlights the need for safer, non-invasive diagnostic methods. Here, we present the first direct comparison of a pediatric and adult cohort to study wheat allergy and wheat tolerance using an improved and validated basophil activation test (BAT). Blood samples from 24 children and 26 adults, clinically classified as facing oral food challenges, were analyzed using our bin-based BAT, enabling standardized data analysis and visualization. In children, the BAT showed significantly higher median basophil activation in wheat-allergic compared to wheat-tolerant individuals. Receiver operating characteristic analysis revealed that BAT responses to wheat, gluten, and gliadin extracts (area under the curve (AUC): 0.71–0.73) had greater diagnostic accuracies than extract-based wheat and gluten-specific immunoglobulin E (sIgE) measurements (AUC: 0.69, 0.70). However, Tri a 19-sIgE, showed the highest diagnostic performance (AUC: 0.97). In adults, BAT responses did not differ significantly between allergic and tolerant individuals. The bin-based BAT is a robust and reproducible diagnostic tool for wheat allergy diagnosis with automated data analysis capabilities. Significant differences were only evident in the pediatric cohort, indicating age-related immunological differences in basophil responsiveness or immune sensitization profiles. These differences could be linked to immune system maturation, variations in immunoglobulin E (IgE) avidity, or differential expression of the high-affinity IgE receptor (FcεRI) on basophils. While Tri a 19 sIgE was the best single predictor in children, its clinical utility remains controversial due to conflicting results in the scientific literature.

Background: Wheat allergy dependent on augmentation factors (WALDA) represents the most common form of IgE‐mediated wheat allergy in adults. Although ω5‐gliadin is the major allergen in WALDA, sIgE reactivity to non‐gluten wheat proteins remains poorly characterized. Methods: Serum samples from 54 adults with challenge‐confirmed WALDA and 21 tolerant controls (including 5 with positive sIgE to ω5‐gliadin) from two German centres were analysed for sIgE reactivity to 19 recombinant wheat proteins and the cross‐reactive carbohydrate determinant (CCD) MUXF3. Results: sIgE to α−/β‐gliadin (Tri a 21.0101) was highly specific for WALDA, present in 29/54 patients (53.7%) but absent in controls (100% specificity, p < 0.001). Receiver operating characteristic analysis revealed excellent discriminatory ability (area under the curve = 0.85) with an optimal cut‐off of 0.22 kU/l. Reactivity to non‐gluten wheat proteins was limited, with 24.1% of patients showing sIgE to thioredoxin, 11.3% to dehydrin, 9.3% to any α‐amylase/trypsin inhibitor subunit, and minimal sIgE to non‐specific lipid transfer proteins (Tri a 14), thiol reductase, triosephosphate isomerase, peroxiredoxin, serpin, glyceraldehyde‐3‐phosphate dehydrogenase, serine protease inhibitor‐like allergen, and peroxidase. Non‐gluten reactivity correlated strongly with MUXF3 CCD (ρ = 0.776), total IgE (ρ = 0.646), and wheat extract sIgE (ρ = 0.534; all p < 0.001). Grass pollen‐sensitized individuals showed increased non‐gluten protein reactivity ( p < 0.001). Controls demonstrated higher MUXF3 CCD reactivity (38.1% vs. 11.6%, p < 0.05). Conclusion: The data suggest that WALDA is characterized by highly specific reactivity to gliadin proteins, with limited sensitization to non‐gluten wheat components. sIgE to α−/β‐gliadin represents a promising diagnostic biomarker with superior specificity compared with ω5‐gliadin alone.