Protein structure plays a critical role in peanut allergen Ara h 2 stability and may determine immunodominant IgE binding epitopes

Rat basophilic leukemia (RBL)-SX38 cells (gift from J-P Kinet) express the alpha, beta, and gamma chains of the human high-affinity lgE receptor (Fc epsilon RI).Following sensitization with IgE from peanut-allergic human donors (total IgE> 100 kU per liter and/or peanut-specific IgE_>6 kU per liter), these cells can be triggered by exposure to anti-IgE or to very low concentrations of peanut allergens.We hypothesized that this assay would allow us to determine if individual sera from peanut-allergic patients react preferentially to Ara hl or Ara h2.We evaluated sera from 7 patients with severe reactions to peanuts.RBL-SX38 cells were labeled with 3H-serotonin and sensitized by overnight incubation with serum diluted 1:10 or greater and then washed.Anti-IgE or serial dilutions of purified Ara h I and Ara h2 were added to the sensitized cells and release of 3H-serotonin was measured.Degranulation by Ara h 1 or Ara h2 is expressed as net 3H-serotonin release relative to the release caused by anti-IgE.Compared to the release with anti-lgE (defined for this study as 100%), maximal signals with the purified peanut allergens were 71% _. + 14% for Ara h2 and 55% -+ 19% for Ara hi.Individual sera responded optimally at doses ranging from 0.25 to 250 ng/ml for Ara h2 and from 2.5 to 2500 ng/ml for Ara hi.Six of the seven serum samples reacted preferentially to Ara h2 compared to Ara hi.Cells sensitized with sera from two of these patients responded only to Ara h2, with no release to Ara h 1.With the other four sera.both Ara h I and Ara h2 caused degranulation, but the sensitized cells responded to Ara h2 at 10-100 fold lower concentrations than to Ara hi.Serum from one patient reacted similarly to both Ara hl and Ara h2 at 0.25 ng/ml (70% compared with anti-IgE).This functional assay suggests that for the majority of these peanut-allergic patients, Ara h2 is more potent than Arah 1.