Methods: Sera (n=7, mean age: 42.1 yrs, range=16-74 yrs) were tested for IgE binding to soybean extract. Total protein was separated by 2-dimensional (2D)-PAGE. Diluted serum samples were analyzed by Western blotting using 2D-PAGE (2D-WB) with HRP-conjugated anti-human IgE antibody. PVDF membranes were stained with Direct Blue 71 and spots coincident with IgE binding were digested with lysyl endopeptidase and analyzed using TripleTOF 6600 liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Results: Fifteen IgE-reactive proteins were identified in the 2D-WB and LC-MS/MS, of which ten had been previously unidentified as allergenic components of soybean. The soybean positive sera of five patients reacted with three major known allergens (Gly m 5, 6 and 8). Interestingly, the sera of all patients reacted with one or more of the previously unidentified proteins. IgE reactivity to the soybean extract was found to be significantly reduced in an inhibition assay.
Conclusions: With the immunoproteomics approach applied, three major allergens Gly m 5, 6, 8 and multiple previously unknown proteins were identified in patients with soybean allergy.