Allergens in Aspergillus fumigatus. I. Characterization of two different allergen extracts and evaluation of their stability and the importance of carbohydrate for IgE binding.

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4°C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract. When the same experiment was performed on samples in a 50% glycerol solution, the results strongly indicated that glycerol had a stabilizing effect on allergens in both extracts. The enzyme content, estimated by the API ZYM-test, showed that both extracts contained several protein- and carbohydrate-degrading enzymes. The presence of these enzymes may explain the lability of the extracts.