Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients

Abstract

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.

FIG. 1.

Western blot analysis of Afmp1p in the culture supernatant of A. fumigatus. Lane 1, 10 μl of a concentrated culture supernatant; lane 2, 20 μg of a cell lysate.

FIG. 2.

Afmp1p is specific for A. fumigtus cells. Approximately 20 μg of each of the cell lysates from A. fumigatus (lane 1), A. flavus (lane 2), A. niger (lane 3), A. terreus (lane 4), P. marneffei (lane 5), C. albicans (lane 6), C. neoformans (lane 7), B. dermatitidis (lane 8), and H. capsulatum (lane 9) was loaded into each lane for Western blot analysis with a specific rabbit anti-Afmp1p antibody.

FIG. 3.

Standard curve for Afmp1p determined with a purified recombinant Afmp1p protein.

FIG. 4.

Afmp1p antigen ELISA is specific for A. fumigatus. Cell culture supernatants were obtained from cultures of A. fumigatus, A. flavus, A. niger, A. terreus, P. marneffei, C. albicans, C. neoformans, B. dermatitidis, and H. capsulatum grown to densities of about 1 × 10⁶ to 2 × 10⁶ cells/ml and were subjected to the Afmp1p antigen ELISA. The positive ELISA values are restricted to the supernatants of A. fumigatus cells. Notice that the largest dilution for the A. fumigatus culture supernatant with a positive OD₄₀₅ is 1:20.

FIG. 5.

Evaluation of sensitivity and specificity of the A. fumigatus antigen test for the detection of aspergillosis in patients.