RATIONALE: Stachybotrys chartarum is known to produce the hemolysin, stachylysin, and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study, we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the development of a sandwich enzyme-linked immunosorbent assay (ELISA) for its detection. METHODS: BALB/c mice were immunized with purified stachylysin and hybridomas were screened using an antigen-mediated ELISA. Spores of S. chartarum and 18 other fungi including several Alternaria, Cladosporium, Aspergillus and Penicillium species were grown at room temperature or at 37Â°C. Spores were extracted overnight at 37Â°C in phosphate-buffered saline containing 0.05% Tween 20. mAb reactivity was investigated by sandwich ELISA using affinity purified polyclonal antibodies to capture stachylysin. Two other purified hemolysins from Penicillium chrysogenum and Aspergillus niger were also tested. RESULTS: Four mAbs were produced and their sensitivity in the sandwich ELISA varied from 10 to 50 ng/ml of purified stachylysin. All mAbs strongly reacted with spore extracts of S. chartarum but not with extracts of the 18 other fungi or the other hemolysins. Overnight extraction of spores at 37Â°C, when compared to room temperature, was found to significantly increase the amount of stachylysin detected in the sandwich ELISA. CONCLUSIONS: The antibodies and the sandwich assay document the feasibility of specific mAb-based detection assays for stachylysin. Current research aims at testing other Stachybotrys species and closely related fungi for potential cross-reactivities and to optimize the assay for the detection of stachylysin in experimental animal and human serum samples.
Full conference title:
2006 American Academy of Allergy, Asthma, and Immunology Annual Meeting
- AAAAI 2006 (62nd)