Abstract：
Screening for ω-5 gliadin specific IgE antibody (sIgE) has high diagnostic utility in cases of suspected wheat-dependent exercise-induced anaphylaxis (WDEIA); however, negative cases may require confirmatory tests, such as the oral challenge test. Thus, newly identified allergens that can be used for the serological diagnosis of WDEIA are needed. This study aimed to identify additional sIgE biomarkers of WDEIA.
Forty-two patients with WDEIA (5 negative/37 positive for ω-5 gliadin sIgE) were enrolled. For comparison, 8 patients with immediate-type wheat allergy without WDEIA and 20 healthy controls without wheat allergy were also enrolled. Extracted wheat proteins were separated by 2D-PAGE. Proteins that reacted with serum IgE antibody in 2D Western blotting (2D-WB) were identified using mass spectrometry. Recombinant proteins were synthesized in Escherichia coli, and the antigenicity was tested using ELISA and the basophil activation test.
In 2D-WB, nine proteins reacted with the serum IgE antibody from at least 60% of patients with WDEIA (n ≥ 25/42). ELISA revealed that alpha/beta gliadin MM1 exhibited the highest positive immunoreactivity in 23 of 26 patients who were positive for ω-5 gliadin sIgE (88%) and in 5 of 5 patients who were negative for ω-5 gliadin sIgE (100%). Alpha/beta gliadin MM1 exhibited significantly higher basophil activation in 14 patients with WDEIA when compared to 5 individuals without a wheat allergy.
Alpha/beta gliadin MM1 sIgE exhibited the highest seropositivity, even among patients who were negative for ω-5 gliadin sIgE. The inclusion of alpha/beta gliadin MM1 in allergen-sIgE tests may improve the sensitivity for diagnosing WDEIA.
Forty-two patients with WDEIA (5 negative/37 positive for ω-5 gliadin sIgE) were enrolled. For comparison, 8 patients with immediate-type wheat allergy without WDEIA and 20 healthy controls without wheat allergy were also enrolled. Extracted wheat proteins were separated by 2D-PAGE. Proteins that reacted with serum IgE antibody in 2D Western blotting (2D-WB) were identified using mass spectrometry. Recombinant proteins were synthesized in Escherichia coli, and the antigenicity was tested using ELISA and the basophil activation test.
In 2D-WB, nine proteins reacted with the serum IgE antibody from at least 60% of patients with WDEIA (n ≥ 25/42). ELISA revealed that alpha/beta gliadin MM1 exhibited the highest positive immunoreactivity in 23 of 26 patients who were positive for ω-5 gliadin sIgE (88%) and in 5 of 5 patients who were negative for ω-5 gliadin sIgE (100%). Alpha/beta gliadin MM1 exhibited significantly higher basophil activation in 14 patients with WDEIA when compared to 5 individuals without a wheat allergy.
Alpha/beta gliadin MM1 sIgE exhibited the highest seropositivity, even among patients who were negative for ω-5 gliadin sIgE. The inclusion of alpha/beta gliadin MM1 in allergen-sIgE tests may improve the sensitivity for diagnosing WDEIA.
摘要：
背景：在疑似小麦依赖性运动诱发的过敏反应（WDEIA）的情况下，筛选ω-5麦醇溶蛋白特异性IgE抗体（sIgE）具有很高的诊断效用；然而，阴性病例可能需要验证性测试，例如口头挑战测试。因此，需要新鉴定的可用于WDEIA血清学诊断的过敏原。本研究旨在鉴定WDEIA的其他sIgE生物标志物。
方法：纳入42例WDEIA患者（ω-5麦醇溶蛋白sIgE阴性/阳性37例）。为了比较，还招募了8名没有WDEIA的立即型小麦过敏患者和20名没有小麦过敏的健康对照。通过2D-PAGE分离提取的小麦蛋白。使用质谱鉴定在2DWestern印迹(2D-WB)中与血清IgE抗体反应的蛋白质。重组蛋白在大肠杆菌中合成，用ELISA和嗜碱性粒细胞活化试验检测抗原性。
结果：在2D-WB中，来自至少60%的WDEIA患者(n≥25/42)的9种蛋白质与血清IgE抗体发生反应.ELISA显示，α/β麦醇溶蛋白MM1在ω-5麦醇溶蛋白sIgE阳性的26例患者中有23例（88％）和ω-5麦醇溶蛋白sIgE阴性的5例患者中表现出最高的阳性免疫反应性（100％）。与5名没有小麦过敏的个体相比，14名WDEIA患者的α/β麦醇溶蛋白MM1表现出明显更高的嗜碱性粒细胞活化。
结论：α/β麦醇溶蛋白MM1sIgE表现出最高的血清阳性，甚至在ω-5麦醇溶蛋白sIgE阴性的患者中。在过敏原-sIgE测试中包含α/β麦醇溶蛋白MM1可能会提高诊断WDEIA的敏感性。
方法：纳入42例WDEIA患者（ω-5麦醇溶蛋白sIgE阴性/阳性37例）。为了比较，还招募了8名没有WDEIA的立即型小麦过敏患者和20名没有小麦过敏的健康对照。通过2D-PAGE分离提取的小麦蛋白。使用质谱鉴定在2DWestern印迹(2D-WB)中与血清IgE抗体反应的蛋白质。重组蛋白在大肠杆菌中合成，用ELISA和嗜碱性粒细胞活化试验检测抗原性。
结果：在2D-WB中，来自至少60%的WDEIA患者(n≥25/42)的9种蛋白质与血清IgE抗体发生反应.ELISA显示，α/β麦醇溶蛋白MM1在ω-5麦醇溶蛋白sIgE阳性的26例患者中有23例（88％）和ω-5麦醇溶蛋白sIgE阴性的5例患者中表现出最高的阳性免疫反应性（100％）。与5名没有小麦过敏的个体相比，14名WDEIA患者的α/β麦醇溶蛋白MM1表现出明显更高的嗜碱性粒细胞活化。
结论：α/β麦醇溶蛋白MM1sIgE表现出最高的血清阳性，甚至在ω-5麦醇溶蛋白sIgE阴性的患者中。在过敏原-sIgE测试中包含α/β麦醇溶蛋白MM1可能会提高诊断WDEIA的敏感性。
