Quantification of the ω5- and γ-gliadin content in wheat flour and rat plasma with an enzyme-linked immunosorbent assay using antibodies specific to their IgE-binding epitopes
Quantification of the ω5- and γ-gliadin content in wheat flour and rat plasma with an enzyme-linked immunosorbent assay using antibodies specific to their IgE-binding epitopes
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使用 IgE 结合表位特异性抗体，通过酶联免疫吸附测定对小麦粉和大鼠血浆中的 ω5- 和 γ-麦醇溶蛋白含量进行定量
DOI：
10.1016/j.alit.2018.04.012
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发表时间：
2019
影响因子：
6.8
通讯作者：
Matsuo H.
中科院分区：
文献类型：
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作者：
Yokooji T;Nouma H;Ogino R;Taogoshi T;Morita E;Matsuo H.
亲爱的编辑，u5-麦胶蛋白和g-麦胶蛋白分别被认为是小麦依赖运动诱导过敏反应（WDEIA）的常规和水解小麦蛋白型的致病性过敏原。1由于无小麦饮食对WDEIA患者至关重要，因此测量加工食品或低过敏性小麦中u5和g-麦胶蛋白的含量有助于确保患者的安全。2 e4 WDEIA的典型诊断是基于运动和/或阿司匹林摄入与小麦摄入的口腔激发试验阳性结果。先前的报告显示，血清麦胶蛋白监测与口腔激发试验相结合有助于排除WDEIA诊断中的假阴性结果。5,6因此，监测血浆中u5和g-麦胶蛋白的含量可以进一步提高口服激发试验诊断WDEIA的准确性。我们先前制备了兔多克隆抗体（Abs），特异性识别u5-麦胶蛋白的ige结合表位序列（KQQSPEQQQFPQQQIPQQQ）。在本研究中，我们还制备了特异性识别g-麦胶蛋白ige结合表位序列（QFLQPQQPFPQQPQ）的兔多克隆抗体。4,8此外，我们采用酶联免疫吸附法（ELISA）测定了小麦品种和大鼠摄入谷蛋白后血浆中u5和g-麦胶蛋白的含量。利用新西兰白兔制备了针对g-麦胶蛋白ige表位肽的多克隆抗体（IgGs），并利用变应原肽进行了亲和纯化。使用了先前研究中产生的针对u5-麦胶蛋白表位肽的igg。7采用夹心ELISA法测定样品中总u5-和g-麦胶蛋白的含量，采用抗麦胶蛋白Ab。本研究经广岛大学动物伦理委员会批准(批准号：a16 - 138)。所有细节见补充方法。我们首先使用ige结合表位抗体，用夹心ELISA法证实了对小麦蛋白的特异性检测。使用抗麦胶蛋白抗体的ELISA法可以检测到总麦胶蛋白（Tokyo Chemical Industry, Tokyo, Japan），但不能检测到大肠杆菌中产生的每个重组麦胶蛋白成分（补充图1A）。4使用抗u5-麦胶蛋白Ab的ELISA试剂盒特异性检测单独的u5-麦胶蛋白，而使用抗g-麦胶蛋白Ab的ELISA试剂盒与u1, 2-麦胶蛋白的反应比与g-麦胶蛋白的反应更强烈
Dear Editor, u5-Gliadin and g-gliadin are known as causative allergens for conventional and hydrolyzed wheat protein-type of wheatdependent exercise-induced anaphylaxis (WDEIA), respectively. 1 As a wheat-eliminating diet is crucial for patients with WDEIA, measuring the amounts of u5-and g-gliadins, in processed foods or hypoallergenic wheat is helpful to ensure the safety of patients. 2 e4 WDEIA is typically diagnosed based on the results of a positive-oral challenge test to exercise and/or aspirin intake in combination with wheat ingestion. Previous reports have shown that serum gliadin monitoring in combination with oral challenge tests was useful to exclude the false-negative results from diagnosis for WDEIA. 5, 6 Thus, monitoring of u5-and g-gliadins content in plasma can further improve the precision of diagnosis for WDEIA using oral challenge tests. We previously produced rabbit polyclonal antibodies (Abs) specifically recognizing the IgE-binding epitope sequences (KQQSPEQQQFPQQQIPQQQ) of u5-gliadin. 7 In this study, we also produced rabbit polyclonal Abs specifically recognizing the IgE-binding epitope sequences (QFLQPQQPFPQQPQ) of g-gliadin. 4, 8 Furthermore, we determined the content of u5-and g-gliadin in wheat cultivars and rat plasma after gluten ingestion using enzyme-linked immunosorbent assay (ELISA) with the derived Abs. Polyclonal Abs (IgGs) against IgE-epitope peptides of g-gliadin were produced using New Zealand white rabbits and affinity purified using allergen peptide. IgGs against the u5-gliadin epitope peptide produced in a previous study was used. 7 To determine the contents of total u5-and g-gliadin in samples, sandwich ELISA were performed using each anti-gliadin Ab. This study was approved by the animal ethics committee of Hiroshima University (approval No. A16-138). All details are shown in Supplementary Methods. We first confirmed the specific detection of wheat proteins with sandwich ELISA using the IgE-binding epitope Abs. The ELISA using the anti-gliadin Ab could detect total gliadin (Tokyo Chemical Industry, Tokyo, Japan) but not each recombinant gliadin component produced in Escherichia coli (Supplementary Fig. 1A). 4 The ELISA using anti-u5-gliadin Ab specifically detected u5-gliadin alone whereas the ELISA using anti-g-gliadin Ab reacted with u1, 2-gliadin more strongly rather than g-gliadins (Supplementary