ALLERGENS IN ASPERGILLUS-FUMIGATUS .1. CHARACTERIZATION OF 2 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 extr acted, and the extracts were analyzed separately. The submerged extrac t contained 31.9% protein and 8.3% carbohydrate, while the correspondi ng values were 17.0% and 33.3% for the surface material. With individu al sera from patients with allergic asthma, SDS-PAGE combined with imm unoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in add ition to several weak to medium IgE-binding components. The surface ex tract contained about the same number of IgE-binding components, but o nly one gave a strong reaction (20 kDa). The allergens present were sh own to have pi between 4.5 and 5.6 as demonstrated by isoelectric focu sing (IEF) combined with immunoblotting. For identification of A. fumi gatus glycoprotein allergens, both extracts were treated with periodat e under mild conditions. Two allergens of the submerged extract (90 an d 38 kDa) partly lost their IgE-binding ability by this treatment, ind icating that these components are glycoproteins and that the carbohydr ate 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 ke pt at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerg ed 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 experim ent was performed on samples in a 50% glycerol solution, the results s trongly indicated that glycerol had a stabilizing effect on allergens in both extracts. The enzyme content, estimated by the API ZYM-test, s howed that both extracts contained several protein- and carbohydrate-d egrading enzymes. The presence of these enzymes may explain the labili ty of the extracts.