Redefining the Major Peanut Allergens

描述(申请人提供)：对花生的过敏反应是因为易感个体通过产生一种血浆蛋白，即IgE，与肥大细胞和嗜碱性细胞上的高亲和力受体FcepsilonRI结合，从而对花生产生过敏反应。这种IgE可以被特定的过敏原交联，导致肥大细胞和嗜碱性粒细胞的激活以及随后的过敏反应。根据三种主要的花生变应原与免疫印迹上的IgE结合的能力以及在RAST抑制试验中与IgE的相互作用，已详细描述了这三种过敏原。它们是ara h1、ara h2和ara h3。这些过敏原在多大程度上对花生粗提物中的活性起作用，从来没有文献记载过。我们建议使用体外功能分析来更好地定义主要的花生过敏原，并将使用花生过敏的小鼠模型在体内测试我们的体外发现。我们的初步数据表明，我们的大多数对花生过敏的患者对Ara H2的反应是2log或比Ara H1更敏感。作为这项建议的一部分，这些观察结果将与对免疫印迹这些血清的独立分析相关联。我们通过量化对纯化蛋白的反应性，并将其与花生粗提物的反应性进行比较，进一步评估了我们的患者对Ara H1和Ara H2的反应性。根据我们的初步数据，在我们检查的7名患者中，6名患者的Ara H1和Ara H2似乎都不是主要的功能性过敏原，而Ara H2可能在7名患者中的1名患者中起重要作用。在初步研究中，我们用阴离子交换层析分离了花生蛋白，并发现很大一部分功能性过敏活性层析在不含Ara H1或Ara H2的级分中。这些组分的含量尚不清楚，但将通过功能分析、2D凝胶、质谱学和IgE免疫印迹进行分析。因此，我们建议将功能分析与标准免疫印迹技术和蛋白质组学的能力相结合，以确定花生过敏原在花生全身反应患者中定量负责肥大细胞激活的分子细节。采用这种方法来定义新的、功能性的主要过敏原，有可能彻底改变我们的想法，即哪些花生过敏原是特定患者过敏反应中最重要的。

DESCRIPTION (provided by applicant): Allergic reactions to peanuts occur because susceptible individuals respond to exposure to peanuts by producing a plasma protein, IgE that binds to a high affinity receptor, FcepsilonRI on mast cells and basophils. This IgE can be cross-linked by specific allergens leading to activation of mast cells and basophils and subsequent allergic reactions. Three major peanut allergens have been described in detail based on their ability to bind IgE on Western blots and to interact with IgE in RAST-inhibition assays. These are Ara h1, Ara h2, and Ara h3. The degree to which these allergens contribute functionally to the activity in crude peanut extracts has never been documented. We propose to use in vitro functional assays to better define the major peanut allergens and will test our in vitro findings in vivo using a mouse model of peanut allergy. Our preliminary data suggests that most of our patients with hypersensitivity to peanuts react to Ara h2 at a 2 log or better sensitivity than to Ara h1. As part of this proposal, these observations will be correlated with independent analysis of these sera on immunoblot. We have further assessed the reactivity of our patients to Ara hl and Ara h2 by quantitating the reactivity to purified proteins and comparing that reactivity to the reactivity with crude peanut extracts. Based on our preliminary data, neither Ara hl nor Ara h2 appear to be major functional allergens in 6 of 7 patients we have examined, whereas Ara h2 may be of great importance in one of seven patients. In a preliminary study, we have separated peanut proteins by anion exchange chromatography and find that a significant portion of the functional allergic activity chromatographs in fractions that do not contain Ara hl or Ara h2. The contents of these fractions is unknown but will be analyzed by functional assays, 2d gels, mass spectroscopy, and IgE immunoblotting. Therefore, we propose to combine functional assays with standard immunoblotting techniques and the power of proteomics to define in molecular detail the peanut allergens quantitatively responsible for mast cell activation in patients with systemic reactions to peanuts. Employment of this approach to define novel, functional major allergens has the potential to completely change our thinking as to which peanut allergens are the most important in specific patients for allergic reactions.