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.
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