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

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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.