STANDARDIZED ANTIGENS FOR DIAGNOSIS OF ASPERGILLOSIS

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Aspergillus fumigatus, a ubiquitous fungus is associated with at least five different clinical forms of diseases in man, among which, allergic bronchopulmonary aspergillosis (ABPA) is the most frequently encountered. The diagnosis of ABPA depends on clinical and immunological findings. Demonstration of specific antibodies in the sera of patients is of considerable value to the diagnosis of the disease. Various antigenic preparations used in the serological tests include secretory metabolites (culture filtrate) and various extracts of the cell wall and cytoplasm. All these preparations are crude extracts and show considerable variation in their reactivity with patients' sera. These antigens also show false positive and false negative reactions in various antibody detection tests. Thus, the currently available serological tests using these antigens are neither dependable nor reliable. In order to have dependable and reliable diagnostic tests, pure and standardized antigens are necessary. The specific aims are to isolate relevant antigens and develop standardized sensitive immunological tests for diagnosis. Our approach will be to produce monoclonal antibodies (MAb) to relevant antigens of A. fumigatus utilizing hybridoma technology. At least 5 to 10 MAb against relevant antigens of A. fumigatus as determined from their reactivity with patient sera, will be produced. Using these MAb, we will isolate specific antigens through affinity chromatography. The pure antigens thus obtained will be used in immunodiagnostic methods including enzyme-linked immunosorbent assay (ELISA) and in skin testing of patients with Aspergillus-related diseases. A very sensitive biotin-avidin-linked immunosorbent assay (BALISA) will be used to detect specific IgG and IgE antibodies. With hybridomas, a continuous supply of all reagents needed for diagnostic testings can be maintained and these reagents can be easily standardized. It then will be possible to diagnose the disease more reliably by comparing the results of different laboratories. This, in turn, will help to study the well-diagnosed cases for the immunopathogenic mechanisms involved in the disease which will result in better prognosis. Our efforts will be directed toward the production of standardized reagents through the use of MAb in the diagnosis of ABPA.