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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of a 20 kD allergen from Aspergillus fumigatus.

Sera from patients sensitized to Aspergillus fumigatus (A.f.) were screened for specific IgE using sodiumdodecylsulphate-gradient-polyacrylamidegelelectrophoresis (SDSgPAGE) followed by immunoblotting to nitrocellulose. Approximately 25 IgE-binding components were detected. The components of molecular weight 20, 31, 44, 50, 53, 77 and 90 kD were reacting with more than 50% of the patients. The 90, 77 and 20-kD components showed up as the strongest IgE-binding bands. The 20-kD component, called Ag 20 kD, was purified and further characterized. Ag 20 kD was purified to apparent homogeneity. using a combination of size-exclusion chromatography on a Sephacryl S-200 column, preparative isoelectric focusing in a pI 2.5-6.5 gradient, and a Sephadex G-50 Superfine column. Fractions were characterized with protein and carbohydrate analyses, RAST and SDSgPAGE followed by immunoblotting to nitrocellulose. Ag 20 kD was found to be a glycoprotein as it stained with both Coomassie Brilliant Blue and PAS. However, it did not bind Con A, and thus, did probably not contain any terminal alpha-D mannopyranosyl end groups. The relation between mannose, galactose and glucose was found to be 2:1:0.5. The isoelectric point was heterogeneous within pH range 5-6, and the molecular weight was estimated to approximately 20 kD. An increased RAST response was shown for the purified component compared with crude extract using patient sera reacting with Ag 20. The antigen was shown not to be identical with the previously described Ag 3. Neither did it fit the description of Ag 5, 7 or 13 earlier described by the same group. The antigen is going to be used for further immunochemical and clinical investigations, and coupling to other systems for antigen characterization.

Methodological considerations when using nitrocellulose immunoblotting from polyacrylamide gels to study the mould allergens Aspergillus fumigatus and Alternaria alternata.

The effects of sample treatments, separation conditions, and the possible presence of antibody interference phenomena in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) followed by nitrocellulose immunoblotting (IB) were studied using allergens from Alternaria alternata (Alt.a.) and Aspergillus fumigatus (Asp.f.) as model systems. In order to obtain good resolution in the IB method the mould allergens were separated in gradient gels under dissociating conditions (SDSgPAGE) including sample treatment with boiling, SDS and 2-mercaptoethanol (ME). These treatments all reduced the IgE binding capacities of the Asp.f. and Alt.a. extracts studied. Nevertheless, a great variety of IgE-binding components were detected after IB, and this could probably be explained by refolding of allergens during the blotting procedure where SDS was partly removed. A comparison of IgG-enriched and IgG-reduced serum fractions in IB revealed small differences in the IgE-binding patterns suggesting that IgG interference is of minor importance in the system studied. The IB method must be individually optimized for each new allergen studied using well characterized sera, preferably monospecific for the major components. Taking these facts into consideration, the IB technique is a valuable complement to other methods in the study of mould allergens.

Specific IgE antibodies to 16 widespread mold genera in patients with suspected mold allergy.

A panel of 55 sera from patients with suspected mold allergy from two different geographic areas, A and B, was investigated for specific IgE antibodies to 16 individual molds using Phadebas RAST technique. The molds investigated were Alternaria, Cladosporium, Aspergillus, Penicillium, Mucor, and Candida from the former RAST panel and ten additional new mold genera. The RAST screening revealed that 73% of patients had specific IgE antibodies to at least one of the 16 molds. Eighteen percent were negative to the six molds of the previous RAST panel but had specific IgE antibodies to at least one of the ten new molds. The highest frequency of IgE antibodies in the A group could be assigned to Cladosporium, followed by Botrytis and Helminthosporium. In group B, sensitivity to Botrytis and Phoma showed the highest frequencies. This study revealed that the relative importance of mold genera may vary greatly between patient groups from different geographic areas. Some of the species of the expanded RAST panel seem to be at least as important as Alternaria and Cladosporium. Multiple sensitivities seem to be caused by sensitization by many mold species rather than by cross-reactivity.