Sotkovský Petr, Hubálek Martin, Hernychová Lenka, Novák Petr, Havranová Marie, Setinová Iva, Kitanovicová Andrea, Fuchs Martin, Stulík Jirí, Tucková Ludmila
Department of Immunology and Gnotobiology, Institute of Microbiology v.v.i, Czech Academy of Sciences, Prague, Czech Republic.
Proteomics. 2008 Apr;8(8):1677-91. doi: 10.1002/pmic.200700347.
Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.
小麦属于引发IgE介导的超敏反应的六大主要食物过敏原，可表现为皮肤、胃肠道和呼吸道症状。尽管谷物是大多数饮食中的主食，但仅鉴定出少数几种会引起超敏反应的小麦蛋白。为了鉴定小麦过敏原，采用一维电泳（1-DE）和二维电泳（2-DE）对盐溶性小麦提取物进行分离，并用摄入性小麦过敏患者的血清通过免疫印迹法检测IgE结合蛋白。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和四极杆飞行时间质谱（QTOF）对二维电泳中经常被IgE识别的蛋白质进行分析，并通过一维电泳和液相色谱-离子阱质谱联用（LCQ(DECA) nLC-MS/MS IT）技术完善其质谱图。运用这三种技术，我们鉴定出了19种潜在的小麦过敏原，如α-淀粉酶抑制剂、β-淀粉酶、肌动蛋白结合蛋白、丝氨酸蛋白酶抑制剂、β-D-葡聚糖外切酶和27K蛋白。采用新开发的酶联免疫吸附测定（ELISA）法，对针对舒拉米特小麦提取物以及1型和3型α-淀粉酶抑制剂的IgE抗体水平进行定量分析，结果显示，与健康对照者相比，过敏患者血清中的这些抗体水平显著升高。针对3型α-淀粉酶抑制剂的IgE抗体水平较低，在大多数患者血清中略高于临界值。我们的研究结果有助于小麦过敏原的鉴定，旨在提高血清IgE和细胞活化诊断检测的特异性。
