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.
Samuelsen
H
H
Institute of Pharmacy, University of Oslo, Norway.
Karlsson-Borgå
A
A
Paulsen
B S
BS
Wold
J K
JK
Rolfsen
W
W
D000485:Allergens / Q000302:isolation & purification*; D001232:Aspergillus fumigatus / Q000276:immunology*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000302:isolation & purification; D008970:Molecular Weight
D000485:Allergens; D007073:Immunoglobulin E