Electrophoretic and Immunochemical Characterization of Allergenic Proteins in Buckwheat
Electrophoretic and Immunochemical Characterization of Allergenic Proteins in Buckwheat
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荞麦过敏蛋白的电泳和免疫化学表征
DOI：
10.1159/000024431
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发表时间：
2000
影响因子：
2.8
通讯作者：
Y. Mine
中科院分区：
文献类型：
--
作者：
M. A. Yoshimasu;J. Zhang;S. Hayakawa;Y. Mine
背景：荞麦过敏并不常见，然而，它被认为是一种非常有效的过敏原。已发现摄入少量会产生过敏反应，特别是在儿童中。荞麦中主要过敏原的鉴定和表征目前正在进行中，然而，研究结果存在一些差异。方法：采用免疫印迹法对荞麦过敏患者血清进行主要过敏原的鉴定。一旦鉴定出过敏原蛋白，将其纯化，通过间接ELISA评估其IgE结合活性，并完成N-末端氨基酸序列。为了评估IgE结合表位的稳定性，将蛋白质级分暴露于各种处理并使用间接ELISA进行测定。最后，通过Western印迹和ELISA分析患者血清中抗荞麦IgG的存在。结果：检测到IgE结合蛋白质的分子量约为14和18 kDa。N-末端测序完成，发现共享一些同源性与大米过敏和交叉过敏性与荞麦蛋白质的大米蛋白。当水溶性蛋白部分被加热，暴露于酸性和碱性条件下，并完全变性，IgE结合活性降低。当通过尿素部分变性的馏分，IgE结合活性增加。此外，IgG结合活性仅在高于20 kDa区域的蛋白质中检测到。结论：分子量约为14和18 kDa的蛋白质被确定为在本研究中测试的荞麦过敏患者血清中的主要过敏原蛋白。结果还表明，这两种蛋白具有IgE结合能力。
Background: Buckwheat allergies are not common, however, it is considered to be a very potent allergen. Ingestion of small amounts has been found to produce anaphylactic reactions, particularly in children. Identification and characterization of the major allergen(s) in buckwheat are currently underway, however, there are some discrepancies in the findings. Methods: Identification of the major allergen(s) was determined through Western blotting using buckwheat-allergic patients’ sera. Once the allergenic proteins were identified, they were purified, their IgE-binding activity assessed through an indirect ELISA and the N-terminal amino acid sequence completed. To assess the stability of the IgE-binding epitopes, protein fractions were exposed to various treatments and assayed using an indirect ELISA. Lastly, the presence of anti-buckwheat IgG in the patients’ sera was analyzed through Western blotting and ELISA. Results: IgE binding was detected to proteins with molecular masses of approximately 14 and 18 kDa. N-terminal sequencing was completed and found to share some homology with rice proteins associated with rice allergies and cross-allergenicity with buckwheat proteins. When the water-soluble protein fraction was heated, exposed to acidic and alkaline conditions and fully denatured, IgE-binding activity was reduced. When the fraction was partially denatured through urea, IgE-binding activity increased. Furthermore, IgG-binding activity was detected with proteins only above the 20 kDa region. Conclusions: Proteins with molecular masses around 14 and 18 kDa were identified as the major allergenic proteins in the buckwheat-allergic patients’ sera tested in this study. Results also indicate that these two proteins possess IgE-binding capability.