Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays
Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays
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DOI：
10.1007/s00216-009-2842-5
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发表时间：
2009-09-01
影响因子：
4.3
通讯作者：
Wal, Jean-Michel
中科院分区：
文献类型：
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作者：
Bernard, Herve;Drumare, Marie-Francoise;Wal, Jean-Michel
2S白蛋白家族的蛋白质，如Ara h2和Ara h6，最常参与花生过敏。我们已经开发了一种反向酶过敏原吸附试验（EAST），其中总血清IgE抗体首先被固定化的抗人IgE单克隆抗体捕获，然后测量抗Ara h2和抗Ara h6特异性IgE与相应的标记过敏原的结合。该反向免疫测定用作直接EAST或EAST抑制测定，以研究全花生蛋白提取物和纯化的Ara h2和Ara h6与来自花生过敏患者的IgE抗体的相互作用。最后，我们确定了一些IgE结合表位上的阿糖胞苷h6使用的EAST的格式，其中蛋白质是固定在一个特定的，明确的，通过与特定的单克隆抗体（单克隆抗体）的微板上包被的方式。已经在表位水平上表征了这些mAb的精细特异性，并且它们与过敏原的结合因此掩盖了已知的特定表位并使其无法被IgE抗体识别。反向EAST增加了特异性信号/背景的比率。它避免了与竞争者如抗花生蛋白IgG抗体的干扰，并允许以比常规EAST更高的灵敏度和准确度研究IgE抗体与Ara h2或Ara h6之间相互作用的特异性和/或亲和力。当变应原由特异性mAb呈递时获得的EAST结果表明花生2S白蛋白中的同源分子结构域包含主要IgE表位并且强烈参与花生变应原性。
Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.