Fumigatus Proteins Research Articles

Molecular cloning and expression of a recombinant Aspergillus fumigatus protein Asp f II with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis

The cDNA of Aspergillus fumigatus encoding an allergen was cloned and expressed in Escherichia coli. The 987 bp long cDNA clone expressed a recombinant protein Asp f II of 34 kd. This protein exhibited binding to immunoglobulin E (IgE) in the serum samples from patients with allergic bronchopulmonary aspergillosis (ABPA). The patients with ABPA and central bronchiectasis demonstrated high levels of serum IgE antibodies, whereas patients with ABPA without central bronchiectasis, patients with asthma and Aspergillus skin test reactivity but no evidence of ABPA, and patients with aspergilloma showed only low levels of IgE antibody to Asp f II. In two-dimensional electrophoresis, a native antigen electroeluted from an A. fumigatus culture filtrate antigen preparation showed an isoelectric point and molecular weight similar to that of Asp f II. In a competitive enzyme-linked immunosorbent assay (ELISA), the IgE antibody reactivity of Asp f II with patient serum samples could be significantly inhibited by culture filtrate antigens of A. fumigatus. These results indicate that Asp f II has immunologic reactivities comparable to those of native A. fumigatus antigens. The recombinant Asp f II can be expressed in E. coli in large quantities and should prove useful as a standardized allergen for sensitive and specific immunodiagnosis of ABPA, especially in patients with central bronchiectasis.

Immunoblot Analyses of the Serologic Response to Aspergillus fumigatus Antigens in Experimental Invasive Aspergillosis

The antibody response to Aspergillus fumigatus proteins was studied by the immunoblot technique in a rabbit model of invasive aspergillosis. Components of an A. fumigatus mycelial homogenate homogenate unbound by concanavalin A-Sepharose 4B chromatography were fractionated using SDS-PAGE and transferred to nitrocellulose papers. The protein fraction was probed with serial sera obtained from immunosuppressed or nonimmunosuppressed rabbits inoculated intravenously with saline or graded inocula of A. fumigatus conidia. Seroconversion against antigens of 41, 54, and 71 kDa was demonstrated in 7 (50%), 3 (21%), and 3 (21%) of 14 infected animals that survived greater than or equal to 10 days. Antibodies against the three antigens were detected in 4 (12.5%), 19 (59%), and 14 (44%) of 32 rabbits before immunosuppression or infection. Two-dimensional immunoblotting revealed that the 41-, 54-, and 71-kDa antigens were derived in denaturing conditions from a single component resolved in nondenaturing polyacrylamide gel electrophoresis.

Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus.

ELISA has emerged as a useful alternative to other more costly and complex tests. Polystyrene microhaemagglutination plates have been used as solid phase to absorb Aspergillus fumigatus protein and polysaccharide components for detection of specific antibodies in patients with various forms of pulmonary aspergillosis. IgG and IgE antibodies to the polysaccharide as well as the protein allergens have been found. For the IgE test a double antibody technique has been developed, which is more sensitive than the conventional indirect ELISA.

The uptake of Aspergillus fumigatus protein by serum IgG antibody from patients with pulmonary aspergillosis

A method is presented for measuring the uptake of Aspergillus fumigatus protein by IgG antibodies from human serum. The human IgG is first isolated with an anti-IgG conjugated immunosorbent and then incubated with radio-iodinated A. fumigatus protein. A group of twenty-three control sera gave the same background levels as tests without sera. The highest uptake of A. fumigatus protein was given by the sera of patients with aspergilloma, and lower values were obtained with six out of thirteen sera from patient's with asthma and twenty-five out of twenty-eight sera from patients with asthma and pulmonary eosinophilia, i.e. allergic bronchopulmonary aspergillosis. These results were in agreement with precipitin tests, Inhibition studies with unrelated antigens showed that the reactions were specific for the A. fumigatus protein, of which more was bound by test sera than with a crude extract.