Isolation, purification, and radiolabeling of a novel 120-kD surface protein on Blastomyces dermatitidis yeasts to detect antibody in infected patients.

Abstract

No well-defined Blastomyces-specific antigens are currently available. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify immunologically active molecules in the cell wall of B. dermatitidis. A major immunoreactive 120-kD protein (WI-1) was present in all five strains studied and comprised 5% of the protein in the cell wall extract obtained after freezing and thawing yeast cells. WI-1 was recognized by serum from all 10 patients with blastomycosis but by none of those from 5 patients with histoplasmosis. It was purified by electroelution, radiolabeled with 1II, and incorporated into a radioimmunoassay (RIA) for serodiagnosis of blastomycosis. Antibody to WI-1 was detected in 58 (85%)

Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis.

“…Bacteria on the filters were lysed and the filters processed for immunoscreening as described (6). Rabbit antiserum specific for WI-I (anti-WI-I titer, 1:10,240, as measured by radioimmunoassay [1]) was used to screen a portion ofthe expression library. The serum was preabsorbed with E. coli lysate (Promega Biotec, Madison, WI) overnight at 4°C.…”

“…Human antisera used in these experiments were from blastomycosis patients in whom antibody responses to WI-i had previously been demonstrated by RIA (1), and from seronegative control subjects with other fungal diseases or no illness. Sera had been stored at -70°C for up to 8 y prior to testing.…”

“…We recently described a novel 120-kD surface protein abundantly expressed on B. dermatitidis yeasts (1) and have demonstrated that the antigen is a target of antibody (1) and cell-mediated immune responses in infected humans (2). Based on these observations, we developed a radioimmunoassay to reliably diagnose infection by detecting antibody against the protein and have characterized T cells directed against processed forms of the protein in the course of human disease.…”

Comparison of antibody detection with <i>Blastomycesdermatitidis</i> yeast lysate antigens in serum specimens from immunized rabbits and infected dogs

“…Blastomycesdermatitidisis a thermally dimorphic fungus that exists in a mycelial mold-like form in nature at approximately 25˚C, but when incubated at 37˚C, it survives in the yeast form [1,2]. The organism exists in the environment as a spore-producing mold and the spores can be inhaled into the lungs where they develop into budding yeast cells [3].…”

“…Current culture methods may be effective, but very time consuming. Therefore researchers have focused on the development of immunodiagnostic assays in attempts to provide improved methods for the clinical diagnosis of blastomycosis in humans and animals [2,10,11,[14][15][16][17][18]. Our laboratory has developed various B. dermatitidisyeast phase lysate preparations and utilized these antigens in the ELISA for the detection of antibodies in serum specimens from immunized or infected animals [10,11,13,15].…”