The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. SDS-PAGE of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5kD band and the identity of Ara h 6 was confirmed by LC-MS/MS. Ara h 6 showed a higher seroprevalence in chimeric-IgE ELISA (n=54), but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays, as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.
Ara h 6 has a reported seroprevalence similar to Ara h 2, a major peanut allergen. Both allergens have a similar molecular weight (14.5kD - Ara h 6; 17-18kD - Ara h 2) and share 55% identity in their amino acid sequences. Thus, it has been difficult to obtain natural Ara h 6 (nAra h 6) free of Ara h 2. Our study compares the IgE antibody reactivity of highly purified nAra h 6 to a natural Ara h 6 reference reagent (nAra h 6-R), recombinant Ara h 6 (rAra h 6) and natural Ara h 2 (nAra h 2). SDS-PAGE of the highly purified allergen revealed a single 14.5kD band and the identity of Ara h 6 was confirmed by tandem mass spectrometry (LC/MS-MS). The purified nAra h 6 contained <0.005% traces of nAra h 2 as assessed by ELISA. Natural Ara h 6 had a lower biological activity in basophil histamine release assays than nAra h 2. Chimeric ELISA showed that 70 and 75% of peanut allergic patients (n=57) had specific-IgE to nAra h 2 and nAra h 6 respectively. Natural Ara h 6-R and rAra h 6 displayed similar IgE antibody reactivity when compared to the nAra h 6. In conclusion, Ara h 6 is a major peanut allergen, with comparable immunoreactivity to Ara h 2. The highly purified Ara h 6, free of Ara h 2, will be useful for diagnostic IgE antibody assays, and for molecular and cellular studies to further investigate the immunological mechanisms of peanut allergy.
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