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To identify antigens important to the diagnosis or pathogenesis of coccidioidomycosis, we analyzed three coccidioidal extracts by immunoblotting techniques with sera from patients infected with Coccidioides immitis and from immunized rabbits. When denatured fungal extracts were used, sera from at least 9 of 10 patients reacted with antigens of 100, 60, and 45 kilodaltons (kDa). By agar diffusion, the 100-kDa antigen appeared to be identical to the conventional tube precipitin antigen. Five of the six sera from patients with pulmonary syndromes reacted with another antigen of 70 kDa, but sera from patients with disseminated disease did not react with this antigen. Both the 100-kDa and the 70-kDa antigens were present in the 70-130-kDa region of the nondenatured fungal extracts. Our results suggest that the 100-kDa antigen may be useful in future diagnostic tests and that the 70-kDa antigen may identify patients whose infection is confined to the chest.

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and >200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.

A 33-kDa protein antigen purified from spherules of Coccidioides immitis was analyzed for ultrastructural localization and for binding to serum antibodies from infected or immunized humans. By using colloidal gold detection of affinity-purified anti-33-kDa protein antibodies, electron photomicrographs showed binding to the inner cell wall of arthroconidia and spherules and to the septa and glycocalyx surrounding endospores. Enzyme immunoassay measurements also demonstrated that the antigen was most abundant in mature spherules. Of 37 patients with coccidioidomycosis but without concurrent human immunodeficiency virus infections, all but 2 demonstrated immunoglobulin M (IgM) (usually with early infection) or IgG antibodies for the 33-kDa antigen.In contrast, only one of four HIV-infected patients with active coccidioidal infections demonstrated antibody. On the other hand, 107 of 108 patients without evident coccidioidomycosis and 15 of 16 patients with histoplasmosis did not have similar antibodies, indicating a high degree of specificity. Immunization of humans with a spherule vaccine produced IgM responses to this antigen that were not evident in placebo recipients.The immune responses resulting from infection with Coc-

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