ENZYME IMMUNOASSAYS FOR THE DIAGNOSIS OF BLASTOMYCOSIS

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Blastomycosis, a systemic granulomatous disease caused by the dimorphic fungal organism Blastomyces dermatitidis, exists in southeastern, southcentral and midwestern areas of the United States. Following inhalation of the infectious particle a wide variety of pulmonary and extrapulmonary manifestations may result. The diagnosis of human and canine blastomycosis has presented a challenge to clinicians for many years. In some cases cultural or histologic studies allow for rapid isolation and identification of the agent, but in other instances the procedures may be time consuming and may not provide evidence necessary for diagnosis. Immunodiagnostic assays generally provide a more rapid diagnosis, but problems related to specificity and sensitivity have been attributed to the currently used complement fixation (CF) and immunodiffusion (ID) tests. Recent studies in our laboratory (supported by an NIH AREA grant) using the enzyme-linked immunosorbent assay (ELISA) to detect anti-b-dermatitidis antibodies in human and dogs have provided encouraging results with respect to continued development of the assay. We have developed a new yeast cell lysate method for the preparation of antigens from various strains of B. dermatitidis and the reagents so produced have provided excellent results in the ELISA that were superior to results obtained with commercial B. dermatitidis antigens. Our work has indicated a need for an extensive study to optimize the assay and will be aimed at performing comparative evaluations concerned with (1) antigen preparation and characterization methods and (2) the development of antibody and antigen detection methods. The broad, long term objectives are concerned with comparative identification, isolation and purification of the active antigenic components of B. dermatitidis and to subsequently use the antigens and antibodies produced from them in ELISA or other assays to detect antibodies and antigens in humans and animals with blastomycosis. Yeast and mycelial phase lysate antigens will be prepared from human and canine isolates and compared with commercial B. dermatitidis commercial reagents with respect to antigenic composition (reactive and cross- reactive components) as determined by electrophoretic and immunoblotting methods. The lysate antigens and commercial reagents will be compared in ELISA procedures (microplate and nitrocellulose membrane) as determined by electrophoretic and immunoblotting methods. The lysate antigens and commercial reagents will be compared in ELISA procedures (microplate and nitrocellulose membrane) and an optimal antigen will be developed for the detection of antibody (IgG,IgM,IgA) associated with human and canine disease. Monospecific polyclonal antibodies will be prepared from the optimal antigen and utilized as probes for antigen detection. The ultimate aim of this project is to develop sensitive and specific methods to detect antibodies and antigens in humans and animals with blastomycosis.