Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis

Abstract

The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28-and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band. * Corresponding author. t Present address: American Micro/Scan, West Sacramento, CA 95691.

Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum

“…Using a similar approach, Calhoun et al (1) demonstrated reactivity of a 45-to 48-kDa antigen with IDCF-positive sera of coccidioidomycosis patients. We do not yet know if the F antigen isolated in this study is similar or identical to the 48-kDa antigen reported by Zimmer and Pappagianis (21) or the 45-to 48-kDa antigen reported by Calhoun et al (1). Although we have used the technique of immunoblotting to assess the monospecificity of antiserum prepared against the F antigen, we do not believe that this technique is well suited for analyses of the F antigen.…”

“…Although we have used the technique of immunoblotting to assess the monospecificity of antiserum prepared against the F antigen, we do not believe that this technique is well suited for analyses of the F antigen. This conclusion is based upon our finding that the F antigen is labile to the conditions used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which, as pointed out by Zimmer and Pappagianis (21), are required for optimal resolution of CDN components in immunoblots. Although 2D-IEP is less sensitive than immunoblot analyses and is limited to detection of precipitinogens.…”

An arthroconidial-spherule antigen of Coccidioides immitis: differential expression during in vitro fungal development and evidence for humoral response in humans after infection or vaccination

“…The tube precipitin antigen, which frequently reacts with antibodies from patients with coccidioidomycosis, is also detectable on the spherule surface. It has been suggested for that antigen as well that its presentation to host cells may involve the sloughing component of the spherule envelope (9,41).…”

Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays

“…Diagnosis of coccidioidomycosis is often made on the basis of serological assays employing crude antigens prepared from filtrates or lysates of mycelial or spherule-endospore phases of Coccidioides immitis (25). Currently used qualitative tests include immunodiffusion (ID) for the detection of immunoglobulin M (IgM) (IDTP) or IgG (IDCF) and enzyme immunoassay (EIA) for the detection of these two major antibody responses (4,19).…”