Previous studies in our laboratory have shown that C-ASWS, an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis mycelia (C-ASWS-M) and spherules (C-ASWS-S) is biologically active in: (i) detecting in vivo and in vitro cellular immune responses in experimentally-infected animals and patients with various stages of coccidioidomycosis; (ii) detecting IgM tube precipitin antibody to C. immitis; and (iii) protecting mice against challenge with viable Coccidioides arthroconidia. These studies have established that C-ASWS-M and C-ASWS-S exhibit the spectrum of biological activities that are associated with coccidioidin (CDN), and autolysate of C. immitis mycelia, and spherulin, an autolysate of spherules, with the single exception of reactivity with complement-fixing IgG antibody. More recent studies provide evidence that the C-ASWS extracts are antigenically similar, if not identical, to an antigen (designated Antigen 2) present in both CDN and spherulin. This evidence is based upon our finding that the C-ASWS extracts fuse with Antigen 2 in tandem two-dimensional immunoelectrophoresis (2D-IEP) of CDN against burro anti-CDN and that solid-phase immunoadsorption (SPIA) of CDN on a column containing goat antiserum to C-ASWS-M or C-ASWS-S results in the adsorption of Antigen 2. Desorption of Antigen 2 from SPIA columns yields a preparation which is homogeneous by 2D-IEP and biologically active in eliciting skin test responses in Coccidioides-infected guinea pigs and in detecting tube precipitin antibody in patients with active coccidioidomycosis. On the basis of these results, we propose a study with the following specific aims: (i) isolate Antigen 2 from CDN and spherulin by SPIA using goat antisera to the C-ASWS extracts; (ii) assess the homogeneity of Antigen 2 preparations using the techniques of 2D-IEP and electroblotting; (iii) utilize conventional methods for purifying Antigen 2 preparations; (iv) establish the sensitivity and specificity of Antigen 2 in humoral and cellular immune assays of Coccidioides-, Histoplasma-, and Blastomyces-infected animals; and (v) characterize the chemical composition of Antigen 2 and assess whether the biological activities are attributed to a protein moiety, polysaccharride moiety or both.
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