Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus

Abstract

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.