Anti-Ara h Western Blots
In order to determine whether the autoclaving process denatured specific allergenic proteins, following gel electrophoresis, we performed Western blot analyses on protein extracts of each processing condition using antibodies specific for peanut allergens.
Figure 2A shows a Western blot using an antibody selective for Ara h 1, a 64-kilodalton (kDa) 7S globulin. Both the raw and roasted peanut extracts appear to have the highest proportion of Ara h 1 as demonstrated by the greatest band intensity. The autoclaved extracts have very little to no detection of Ara h 1 via Western blot.
Ara h 2 is a 2S albumin protein of size 17 kDa and has been established as the most potent peanut allergen, being recognized by the serum IgE of over 90% of peanut-allergic patients28, 29. Moreover, recent literature suggests that Ara h 2 exists as two distinct isoforms in mature peanuts, differing by a stretch of 12 amino acids, and that the larger of the two isoforms may be a more potent allergen30, 31. In Figure 2B, we observe the presence of two distinct and intense bands in the roasted extract and to a lesser degree in the raw extract. In all autoclaved extracts, no distinct bands were observed for Ara h 2 but rather a general smear throughout the lanes.
In the case of Ara h 8, a plant panallergen of size 17 kDa, we observed a similarly high band intensity for both the raw and roasted peanut extracts, and very little to no detection in the autoclaved extracts (Figure 2C).
Roasting peanuts either before or after autoclaving (lanes 5 and 4, respectively) did not substantially alter the levels of allergen detection.