Antigens currently used in immunologic studies of coccidioidomycosis are coccidioidin (CDN), an autolysate of mycelial-phase cells of Coccidioides immitis, and spherulin (SPH), an autolysate of the spherule/endospore phase. Although CDN and SPH have an acceptable level of sensitivity and specificity, both are heterogenous preparations containing multiple components, some of which are specific for C. immitis, while others are cross reactive with heterologous fungi. During the current grant period, we have reported the identification and partial purification of three clinically significant antigens from CDN using a combination of immunoaffinity, lectin affinity, and ion-exchange chromatography. One of these antigens is reactive with IgM tube precipitin (TP) antibody to C. immitis; a second antigen is reactive with IgG complement-fixing (CF) antibody; and a third is a heat-stable (HS) exoantigen which if demonstrated in culture extracts of fungal isolates establishes the mycologic identification of C. immitis. In addition, we have partially purified a fourth antigen, designated Antigen 2 (Ag2), which reacts with sera of coccidioidomycosis patients, but is distinct from the TP, CF, and HS antigens of C. immitis. In this renewal application, we propose to: (i) prepare monoclonal and polyclonal antibodies specific for these antigens for use in purifying the antigens from CDN using the technique of immunoaffinity chromatography; (ii) establish the homogeneity of the purified antigens using the techniques of two-dimensional immunoelectrophoresis (2D-IEP), polyacrylamide gel electrophoresis (PAGE), and immunoblotting; (iii) assess the reactivity of the purified antigens in humoral and cell-mediated immune assays of coccidioidomycosis patients and experimentally-infected animals; and (iv) assess the specificity of the purified antigens in immunologic assays of heterologously- infected animals. Successful completion of these studies will provide purified coccidioidal antigens and monospecific antibodies for use as immunodiagnostic probes and for functional analyses of host-parasite interaction in this fungal disease.
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