SPT is a simple and cost-effective method for diagnosing IgE-mediated wheat allergy with various wheat extracts widely commercially available. However, the effectiveness varies significantly depending on the type of extract used, which critically affects sensitivity and specificity. A study using glycerinated extract reported high sensitivity (81%) but low specificity (64%) [25] (Table 1). Expanding on this, another study compared the diagnostic capacity of an in-house Coca-10% EtOH extract with a commercial extract, demonstrating the superior accuracy of the in-house extract [26]. The increased accuracy likely results from the solubility of gliadin, the major wheat allergen, in alcohol, which enhances the diagnostic value of alcohol-based extracts. Due to the limitations of current diagnostic methods, gliadin and glutenin extracts were developed to improve diagnostic accuracy in wheat allergy. A study among 31 children with wheat allergy found that the gliadin extract with the optimal cut-off point of 2.5 mm demonstrated the highest diagnostic performance, with a sensitivity of 84.2%, specificity of 88.9%, and accuracy of 85.7%. In contrast to the gliadin extract, the commercial extract exhibited significantly lower sensitivity (55%) and accuracy (65.5%). Furthermore, the gliadin extract had the lowest negative likelihood ratio (-LR) among all extracts tested, indicating that a negative gliadin SPT result is highly reliable for ruling out wheat allergy. The stability of the gliadin extract was investigated using SPT and a cell degranulation assay measuring beta-hexosaminidase release [27]. The study found that the extract remained stable for up to 12 months when stored at 2–8 °C, supporting its practicality for long-term clinical use. In a systematic review and meta-analysis by Riggioni et al. [24] and the most recent AAAAI–EAACI PRACTALL guideline, Standardizing Oral Food Challenges—2024 Update [28], a commercial extract of wheat with a cut-off point of 3 mm (IQR 3–5) showed a sensitivity of 53% and a specificity of 72% for the diagnosis of IgE-mediated wheat allergy.

In WDEIA, few studies have evaluated the diagnostic accuracy of the SPT. One study compared the diagnostic performance of SPT using a commercial wheat extract with prick-to-prick (PTP) testing with wheat flour and gluten. All patients with WDEIA tested positive for PTP with gluten, while only 8 of 16 patients tested positive with the commercial extract in the SPT [29], demonstrating the superior performance of PTP testing. In addition, another study using the in-house Coca-10% EtOH extract for diagnosing WDEIA found that 12 of 14 patients tested positive in the SPT [10], indicating variability in the effectiveness of different extracts. This variability is attributed to the solubility of ω−5-gliadin and HMW glutenin, major allergens of WDEIA [30, 31] in alcohol, which explains why the alcohol-based extract showed higher sensitivity than the commercial wheat extract.

Overall, the lower sensitivity of commercial wheat extract for diagnosing IgE-mediated wheat allergy and WDEIA results from the water-insolubility of gliadin, requiring alcohol for proper dissolution.

Food-sIgE assays are widely available in clinical settings and are commonly used to diagnose wheat allergy, and they may help predict the severity of the reactions [32]. However, this must be interpreted with clinical history, as a positive result in the absence of clinical symptoms indicates sensitization without clinical significance. The ImmunoCAP assay, a fluorescent method, is currently standard for sIgE quantification. The assay’s various cut-off points offer different diagnostic capacities (Tables 2 and 3). For instance, a cut-off at 26 kUA/L provides 61% sensitivity, 92% specificity, and a negative predictive value (NPV) of 87% [33]. In comparison, a 0.35 kUA/L cut-off showed 96% sensitivity, 20% specificity, and an NPV of 97% [34]. Moreover, a study on Thai children with wheat anaphylaxis found that a 0.35 kUA/L cut-off achieved 100% sensitivity, 50% specificity, and 90% accuracy [26].

Component-resolved diagnosis (CRD) was developed to identify specific wheat allergens, such as ω−5-gliadin, and improve diagnostic accuracy. The fluorescence enzyme immunoassay (FEIA) ImmunoCAP® method identifies a limited range of wheat allergens, including gliadin, ω−5-gliadin (Tri a 19), and the non-specific lipid transfer protein (ns-LTP, Tri a 14). As a major allergen in IgE-mediated wheat allergy [31, 35], ω−5-gliadin-sIgE may provide a more precise diagnosis than wheat-sIgE [26, 36,37,38]. Diagnostic yield increases when adding ω−5-gliadin-sIgE, LMW- (Tri a 36), or HMW-glutenin (Tria 26) to wheat-sIgE in the tested panel [38]. In a study of 30 children with a history of IgE-mediated wheat allergy, a 0.35 kUA/L cut-off for ω−5-gliadin-sIgE alone yielded a diagnostic accuracy of 76.7%, whereas combining both tests increased the accuracy to 86.7% [26]. Although Makela et al. [18] found no significant difference in diagnostic capacity between wheat-sIgE and ω−5-gliadin-sIgE using ImmunoCAP, specific IgE measurement via microarray assay improved diagnostic yield, particularly when more than five components tested positive. The improved diagnostic yield may be due to the microarray assay including other wheat allergens, such as α-, β-, γ-gliadin, and water-soluble allergens like alpha-amylase inhibitors (AAI). Distinguishing between patients allergic to ω−5-gliadin and those allergic to other allergens remains challenging, so tests that cover multiple allergens may be more sensitive than single allergen analyses. Considering these findings, no definitive components or sIgE threshold reliably predicts positive wheat challenges. Therefore, allergists should confirm wheat allergy through oral challenges, especially when clinical history is unclear.

While high wheat-sIgE can predict wheat allergy severity [39], wheat anaphylaxis has been reported even in patients with low wheat-sIgE [40]. There were no differences in baseline characteristics between patients with high and low wheat-sIgE, except those with low wheat sIgE showed a more dispersed IgE immunoblot pattern than those with high sIgE levels. The likely allergens in patients with high wheat-sIgE levels were α-, β-, and γ-gliadin. In contrast, those with low wheat-sIgE levels were likely allergic to high molecular weight (HMW) glutenin [40].

In a recent systematic review and meta-analysis by Riggioni et al. [24] and the AAAAI–EAACI PRACTALL guideline Standardizing Oral Food Challenges—2024 Update [28], wheat-sIgE (cutpoint: 0.6 kUA/L, IQR 0.35–5.6) and ω−5-gliadin-sIgE (cutpoint: 0.3 kUA/L, IQR 0.1–0.6) demonstrated sensitivities of 72% and 79%, and specificities of 79% and 78%, respectively, for diagnosing IgE-mediated wheat allergy.

Because ω−5-gliadin and HMW glutenin are the major allergens in WDEIA, ω−5-gliadin-sIgE has better diagnostic capacity than wheat-sIgE [41]. A study of 14 patients with WDEIA found that the median ω−5-gliadin-sIgE level was higher than the wheat-sIgE level (3.8 kUA/L vs 1.3 kUA/L) [10]. In addition, a study of 16 patients with WDEIA found that 3 patients had wheat-specific IgE levels below 0.35 kUA/L, indicating that WDEIA diagnosis might be missed if based solely on wheat sIgE. In contrast, all patients had ω−5-gliadin-specific IgE levels above 0.35 kUA/L [29].

A new subtype of WDEIA has been reported in patients with a history of using soap-containing hydrolyzed wheat protein (HWP). HWP is prepared from the water-insoluble part of wheat protein, followed by enzymatic or acidic hydrolysis. When comparing HWP-WDEIA to conventional WDEIA (CO-WDEIA), HWP-WDEIA showed a higher positive rate of sIgE to wheat and gluten. In contrast, CO-WDEIA showed a higher positive rate of ω−5-gliadin-sIgE. This finding suggests that the hydrolysis process eliminated ω−5-gliadin in HWP.

The BAT is a flow cytometry assay that measures activation markers (e.g., CD63 and CD203c) on basophils after stimulation with specific allergens. BAT has demonstrated a high level of specificity but lower sensitivity compared to current SPT and sIgE tests, except for peanut and sesame allergies, where it shows moderate sensitivity. Therefore, current recommendations suggest using BAT in patients with an uncertain diagnosis of IgE-mediated peanut or sesame allergy to support the diagnosis [7, 28]. One major drawback of this assay is that approximately 10% of the population are non-responders, making interpretation impossible in these cases. Additionally, the assay requires fresh blood and should be conducted using validated methods and standardized conditions. Furthermore, the assay has a dose–response curve, and a standardized threshold has not yet been validated [16].

Currently, no large-scale studies have used BAT to diagnose IgE-mediated wheat allergy. However, a few small cohort studies have evaluated the usefulness of BAT in differentiating IgE-mediated wheat allergy or WDEIA from controls. Furthermore, BAT has been assessed in distinguishing WDEIA subtypes, such as CO-WDEIA and HWP-WDEIA (Table 4) [42,43,44,45,46,47,48]. Tokuda et al. [48] demonstrated that the CD203chigh% induced by native ω−5‐gliadin had a higher area under the curve (AUC) based on ROC analysis compared to wheat-sIgE (CAP-FEIA, Phadia, Tokyo, Japan) for distinguishing patients with IgE-mediated wheat allergy from wheat-tolerant patients. With a cut-off point of 14.4%, the CD203chigh% test showed 85.0% sensitivity, 77.2% specificity, 86.8% positive predictive value (PPV), and 70.8% negative predictive value (NPV). In comparison, wheat-sIgE with a cut-off of 4.1 UA/mL showed comparable sensitivity (81.4%) and PPV (74.5%) but had lower specificity (55.6%) and NPV (65.2%). BAT has been particularly useful in distinguishing WDEIA patients from controls. For instance, a study by Gabler et al. [45] found that measuring the basophil activation marker CD63 after stimulation with ω−5-gliadin and HMW glutenin effectively identified WDEIA patients. BAT also showed potential in distinguishing WDEIA subtypes. Chinuki et al. [43] demonstrated that patients with CO-WDEIA significantly enhanced CD203c expression in response to purified ω−5-gliadin. In contrast, patients with HWP-WDEIA exhibited CD203c activation in response to the hydrolyzed wheat protein component HWP-A (a component found in Japanese HWP soap manufactured by Katayama Chemical, Osaka, Japan) in a concentration-dependent manner.

Interestingly, other wheat proteins measured by basophil activation, including peroxidase-1 (35 kDa) and beta-glucosidase (60 kDa), were also reported to be associated with cases of grass pollen-related WDEIA. These patients tested negative for ω−5-gliadin-sIgE but had high levels of sweet vernal grass pollen-sIgE and timothy grass pollen-sIgE compared to those with CO-WDEIA or HWP-WDEIA [47]. Alpha/beta gliadin MM1 is another reported wheat allergen associated with WDEIA, and it has been suggested to include alpha/beta gliadin MM1 in allergen-sIgE tests to improve the sensitivity for diagnosing WDEIA [42].

MAT is an alternative in vitro diagnostic test similar to the BAT, but instead of using whole blood, the MAT uses plasma or serum to sensitize mast cells. In both tests, the expression of activation markers is measured following stimulation with a food allergen. The MAT has a similar specificity to BAT in diagnosing peanut allergy but exhibits lower sensitivity [17, 28]. For patients with wheat allergy, a study by Bodinier et al. [49] used the MAT to investigate different wheat-induced allergic conditions. The study demonstrated that, in patients with wheat-induced atopic eczema/dermatitis, sera from patients induced enhanced degranulation in response to albumins/globulins extract. In contrast, in cases of WDEIA or IgE-mediated wheat allergy, sera primarily showed degranulation with LMW glutenins, while the albumins/globulins fraction and LTP were rarely positive. Interestingly, in this study, ω−5-gliadin did not appear as a major allergen in the degranulation assays.

The Bead-Based Epitope Assay (BBEA) is a Luminex-based, high-throughput assay developed to simultaneously measure the levels of multiple epitope-specific antibodies, including the IgE, IgG, IgA, and IgD isotypes [50]. The analysis of epitope-specific antibody repertoires has provided novel insights into diagnosing clinical allergies to milk, egg, and peanuts. Additionally, it has been used to predict clinical reactivity, differentiate clinical phenotypes, and predict treatment outcomes in patients who have undergone oral immunotherapy [51,52,53,54,55,56,57,58]. BBEA has shown promising results and requires only a small amount of blood sample. However, the assay has several limitations, including its higher cost than SPT and food-sIgE. Additionally, the assay is only commercially available for peanuts in a limited number of centers.

Recently, this novel tool demonstrated its utility in diagnosing and differentiating the clinical phenotypes of children and adolescents with wheat allergy. The study evaluated 122 patients (83 wheat-allergic and 39 wheat-tolerant) using BBEA to measure epitope-specific (es)-IgE, es-IgG4, and es-IgG1 antibodies against 79 wheat peptides. These peptides were commercially synthesized from α-/β-gliadin, γ-gliadin, ω−5-gliadin, HMW glutenin, and LMW glutenin. Machine learning coupled with simulations identified wheat es-IgE, but not es-IgG4 or es-IgG1, as the most informative for diagnosing wheat allergy. Additionally, the level of es-IgE binding intensity correlated with the severity of wheat allergy phenotypes. Wheat anaphylaxis exhibited the highest es-IgE binding intensity compared to isolated cutaneous symptoms and WDEIA patients. Using a combination of four informative epitopes from ω−5-gliadin and γ-gliadin, BBEA achieved the highest AUC of 0.908, with 83.4% sensitivity and 88.4% specificity. In comparison, wheat-sIgE showed an AUC of 0.646, P < 0.001) [59].

The OFC remains the gold standard for confirming IgE-mediated wheat allergy when other in vivo and in vitro tests yield inconclusive results. There is no standardized protocol regarding dosing steps or total challenge dose. Additionally, the type of challenge food can vary based on cultural dietary habits, with options including bread, pasta, and udon. In 2020, the Adverse Reactions to Foods Committee of the American Academy of Allergy, Asthma & Immunology published updated guidelines for safely conducting an OFC in clinical settings [60]. For wheat challenges, a slice of bread containing 2–4 g of wheat protein can be used, with portion sizes ranging from ¼ to 2 slices, depending on age. Alternatively, pasta containing 3 g of wheat per cup may be used, with portion sizes ranging from ¼ to 1 cup. Dosing options include a 4-dose protocol (1/12, 1/6, 1/4, 1/2 of the total serving) or a 6-dose protocol (1%, 4%, 10%, 20%, 30%, 35% of the total serving), depending on the risk of severe reactions. Doses are typically administered 15–30 min apart. For WDEIA, the exercise–food challenge test is performed when clinical history, SPT, and/or wheat-sIgE, and wheat components-sIgE testing are insufficient for diagnosis. However, a key limitation of the exercise–food challenge test is its variable positivity rate, which ranges from 50 to 100%, depending on the protocol used [10]. Interestingly, the challenge dose required to elicit a positive reaction in WDEIA is typically higher than in standard OFCs for IgE-mediated wheat allergy, with reported doses ranging from 5.2 to 10.4 g of wheat protein. Optionally, cofactors such as aspirin and/or alcohol can be used to enhance the reaction during the exercise–food challenge test or OFC. Notably, anaphylactic responses in WDEIA tend to be more severe than those seen in typical IgE-mediated wheat anaphylaxis. This increased severity is often attributed to the greater involvement of cardiovascular symptoms (e.g., hypotension, syncope, loss of consciousness) along with respiratory symptoms (e.g., chest tightness, wheezing, cough, desaturation) [8,9,10, 61].