Detection of Alternaria allergens by Western blotting

The technique of Western blotting (immunoblotting) is being increasingly recognized as a means of identifying and characterizing allergens in complex mixtures. Because the allergens are immobilized on nitrocellulose paper, they can be probed for binding of IgE from patients' sera. In addition, the blotting technique provides useful biochemical information, such as the molecular weight of the allergen being studied. In this study we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate antigens in a crude Alternaria preparation, electrophoretically transferred these antigens to nitrocellulose, and incubated the nitrocellulose strips with individual serum and pooled sera from 13 Alternaria-sensitive subjects, sera from three nonallergic individuals, and sera from three individuals allergic to ragweed but not Alternaria. The strips were then washed, incubated with 125I-labeled antihuman IgE, washed again, and exposed to autoradiography film. We observed IgE binding to 20 allergens. Five or more sera from Alternaria-sensitive patients recognized allergens in four molecular weight regions: 55,000 to 60,000, 40,000 to 45,000, 35,000 to 40,000, and 15,000 to 20,000. With pooled sera, the most intense IgE binding was directed at allergens with molecular weights above 40,000. No IgE binding was observed with sera from individuals who were not sensitive to Alternaria. We conclude that Western blotting is an effective method for detecting allergens in complex fungal mixtures.