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Abstract
Immunoblotting was used to identify the allergenic proteins in various raw and processed almonds. Both denaturing and nondenaturing conditions were used to separate the proteins prior to electroblotting. Sera from each of the eight almond allergic individuals exhibited unique binding patterns with the almond proteins. As many as eight IgE-binding proteins were identified in immunoblots of SDS-PAGE separated almond proteins. Sera from four of the eight individuals reacted to a protein of approximate molecular weight of 70,000 daltons. Another protein, of approximately 45,000 to 50,000 molecular weight was also recognized by four individuals. The three different varieties of almonds that were studied exhibited identical IgE-binding patterns. Processing reduced the allergenicity as was evidenced by reduced IgE-binding in immunoblots of blanched almonds and roasted blanched almonds.
Fewer IgE-binding proteins were identified in immunoblots of almond proteins separated using nondenaturing conditions. As many as four IgE-binding proteins were exhibited in raw almonds. One of these proteins was recognized by the same four individuals that reacted to the 70,000 dalton protein in the SDS-PAGE immunoblots. Fewer IgE-binding proteins were exhibited in extracts of processed almonds separated under nondenaturing conditions as well.
The common IgE-binding protein in the nondenaturing immunoblots was purified using conventional chromatography. This protein (Pru a Bd 68K) had an apparent molecular weight of approximately 68,000 daltons. The IgE-binding activity of Pru a Bd 68K was eliminated by heating at 100-121$\sp\circ$C and was absent in immunoblots of almond butter and roasted blanched almond. While this protein exhibited IgE-binding activity by immunoblotting, skin tests with three almond allergic individuals have failed to give positive results. Peptide mapping and amino acid sequencing were initiated but further studies await confirmation of in vivo allergenicity.
Five commercially prepared allergen extracts were also studied and compared using immunoblotting with sera from the same eight almond allergic individuals. The different extracts varied widely in quantity and quality of protein. While IgE-binding patterns differed between extracts, all exhibited some reactivity.
