Rationale-An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized.Methods-Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut allergic donors.Results-Following gel filtration, 75±7% of the applied protein and 76±16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85±2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ~20 kD and a range of 13-25 kD. When all the individual fractions were recombined, the measured activity was similar to that of the original extract (140±43% when measured with a pool of serum (n=2) and 66±7% when measured with individual sera (n=4)); when all individual fractions excluding the 20 kD fraction were recombined, the measured activity was only 8±2% (n=2) of the original extract when assayed with the serum pool and 10±4% (n=3) when assayed with the individual sera. Two dimensional gel electrophoresis of this biologically active fraction by revealed >60 protein spots . Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that greater than 97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins.Conclusions-Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

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