A monoclonal antibody (mAb) exhibiting a high degree of species specificity for the yeast phase of the dimorphic fungus Histopfmma capsufatum was produced by a modification of the standard mAb production protocol. The technique for generating mAbs involved the use of the immunosuppressive drug cyclophosphamide to diminish the response in mice to immunodominant cross-reactive epitopes. This mAb exhibited clear specificity and did not react by ELISA with the closely related genera Bfmtomyces, Paracoccidioides and Sporothrix. In Western blots it recognized a linear determinant on a 70-75 kDa molecule in H. capsulatum antigen, with an extremely faint reactivity to antigens of identical molecular mass derived from Sporothrix and Paruoccidoide, and no reactivity against Bfmtomyces antigen.

A panel of monoclonal antibodies (Mabs) was raised against histoplasmin, the antigen derived from the mycelial phase of Histoplasma capsulatum var. capsulatum which contains the diagnostically useful H and M antigens. A number of Mabs were obtained which recognized a 70-75 kD component of an antigenic preparation of H. capsulatum vat. capsulatum by Western blotting. When reacted with histoplasmin by Western blotting the Mabs recognized a similar 70-75 kD band, together with a series of higher molecular mass bands at approximately 130, 190 and 230 kD, a pattern which correlates strongly with both the published relative molecular mass (Mr) of the M antigen and the known subunit structure of the enzyme catalase. These Mabs were also shown to recognize a commercial preparation of Aspergillus niger catalase by ELISA. Other dimorphic fungi were also reactive with these Mabs by Western blotting, indicating the presence of common epitopes on the catalase molecules of these species.The dimorphic fungus Histoplasma capsulatum var. capsulatum is the causative agent of classical histoplasmosis (Histoplasma capsulati), a disease in which the primary infection is usually asymptomatic but which may present as an acute or chronic progressive pulmonary disease or, alternatively, as a disseminated infection. Infections are characterized by the presence of small intracellular yeasts 3-4/~m in diameter. On primary isolation the organism grows as a mycelial fungus on unenriched media at room temperature. Both the mycelial and yeast phases produce a number of exoantigens in broth culture, the most characteristic of which have been named the H and M antigens which are the primary constituents of histoplasmin [ 14]. These antigens have been of particular use in the serodiagnosis of histoplasmosis by immunodiffusion and are typically identified as reference lines in double diffusion tests [16,17]. In addition, these antigens have been detected in serum and urine from severe cases of disseminated histoplasmosis [18]. So far only one attempt to raise monoclonal antibodies (Mabs) to the M antigen [11] has been published. These authors produced a Mab which defined the M antigen as having a molecular mass of 70-75 kD, although they did not go on to investigate the structure and function of the 479 Med Mycol Downloaded from informahealthcare.com by Queen's University on 01/02/15For personal use only.

A panel of four murine monoclonal antibodies showing species specificity for the yeast phase of the pathogenic dimorphic fungus Paracoccidioides brasiliensis was produced by using a modification of the standard monoclonal antibody technology. This involved the use of the immunosuppressive drug cyclophosphamide to suppress the immune response of test animals to fungi showing cross-reactivity, i.e., to Histoplasma capsulatum. One monoclonal antibody, P4, which had a high titer by enzyme-linked immunosorbent assay, was shown to recognize a linear antigenic epitope of P. brasiliensis at a molecular size of 70,000 to 75,000 daltons by Western blot (immunoblot) analysis. The potential use of these monoclonal antibodies, which are the first species-specific probes to P. brasiliensis that have been produced, in the field of serodiagnosis is discussed.

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin Gl subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoforinans, since in this group the present latex agglutination test has some limitations.

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