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

项目摘要： 花生过敏被认为是最严重的食物过敏之一，因为它的流行， 持久性和潜在的严重性。花生过敏反应从轻微到严重不等， 从急性荨麻疹，严重的血管性水肿，面部肿胀，支气管痉挛，过敏反应，甚至 死亡据估计，美国每年用于治疗花生过敏的医疗费用为 亿美元因此，迫切需要开发改进的诊断方法。的17 已知的PN过敏原，高度同源的2S白蛋白，Ara h 2和Ara h 6，是最常见的 有效地引发IgE介导的肥大细胞活化，并测量特异性IgE Ara h 2或Ara h 6与临床相关PN过敏相关性最好。肽微阵列具有 已经使用了十年，特别适用于筛选肽和定性分析 免疫球蛋白的结合。我们假设这些肽微阵列有潜力 更加量化并成为重要的诊断方法，但在 关于测定的肽的大小，如何处理样品，灵敏度， 测定的重现性和动态范围。我们将使用一口井里的样本 口服免疫疗法的特征性临床试验，以测试以下假设：1）正常化 在测定前基于PN-sIgE（或IgG 4）或Ara h 2-sIgE（或IgG 4）的血清将给出更准确的 如果在固定稀释度下测定血清，则可观察到的与肽的定量结合的量度 和2）微阵列测定比Bt-ELISA或ImmunoCap®更敏感，但具有 更严格的动态范围。这些研究的结果将是加深了解 微阵列的使用，使这一重要工具可以应用于各种过敏性 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.