Characterizing and optimizing IgE and IgG4 microarray peptide assays for Ara h 2

项目总结： 花生过敏是公认的最严重的食物过敏之一，因为它的流行， 坚持不懈，以及潜在的严重性。花生过敏反应从轻微到严重不等，范围从 从急性麻疹，严重的血管水肿，面部肿胀，支气管痉挛，过敏反应，甚至 死亡。据估计，美国每年为管理花生过敏而花费的医疗费用为数美元 十亿美元。因此，迫切需要开发改进的诊断方法。在17个国家中 已知的PN过敏原，高度同源的2S白蛋白，Ara h 2和Ara h 6是最多的 有效地诱导IgE介导的肥大细胞激活并检测特异性IgE Arah2或Arah6与临床相关的PN过敏的相关性最好。多肽微阵列已经 已经使用了十年，在筛选多肽和定性分析方面特别有用 免疫球蛋白结合。我们假设这些多肽微阵列有可能 更加量化并成为重要的诊断方法，但在以下方面存在重要差距 关于所分析的多肽的大小、如何处理样品、灵敏度、 测定的重复性和动态范围。我们会用一口井里的样品 口服免疫疗法的特征性临床试验，以检验以下假设：1)正常化 检测前根据Pn-sIgE(或IgG4)或Arah2-sIgE(或IgG4)的血清将提供更准确的结果 当血清以固定稀释度测定时，可以看到的与多肽的定量结合的量度 2)微阵列检测比BT-EL ISA或免疫CAP®更敏感，但具有 更受限制的动态范围。这些研究的结果将是加深对 微阵列的使用使这一重要工具可以应用于各种过敏症 条件。

Project Summary: Peanut allergy is recognized as one of the most severe food allergies due to its prevalence, persistence, and the potential severity. Peanut allergy reactions vary from mild to severe, ranging from acute hives, severe angioedema, swelling of the face, bronchospasm, anaphylaxis, to even death. The US annual healthcare cost of managing peanut allergy is estimated to be several billion dollars. Thus, there is an urgent need to develop improved diagnostics. Among the 17 known PN allergens, the highly homologous 2S albumins, Ara h 2 and Ara h 6, are the most potent for eliciting IgE-mediated mast cell activation and measurement of specific IgE to either Ara h 2 or Ara h 6 correlates best with clinically relevant PN allergy. Peptide microarrays have been in use for a decade and are particularly useful for screening peptides and qualitative analysis of immunoglobulin binding. We hypothesize that these peptide microarrays have the potential to be more quantitative and become important diagnostics but there are important gaps in knowledge in regards to the size of the peptides assayed, how samples are handled, sensitivity, reproducibility and the dynamic range of the assays. We will use samples from a well characterized clinical trial of oral immunotherapy to test the following hypotheses: 1) normalizing sera based on PN-sIgE (or IgG4) or Ara h 2-sIgE (or IgG4) prior to assay will give more accurate measure of quantitative binding to peptides that may be seen if sera are assayed at a fixed dilution and 2) microarray assays are more sensitive than either Bt-ELISA or ImmunoCap®, but have a more restricted dynamic range. The outcome of these studies will be an enhanced understanding of the use of microarrays so that this important tool can be applied to a variety of allergic conditions.