Production of specific monoclonal antibodies to Aspergillus species and their use in immunohistochemical identification of aspergillosis.

Two anti-Aspergillus murine monoclonal antibodies (MAbs), designated 164G and 611F, have been produced; both specifically recognize cytoplasmic antigens of A. fumigatus, A. flavus, and A. niger by enzyme-linked immunosorbent assay. The MAbs can identify Aspergillus spp. both in frozen sections by immunofluorescence and in paraffin-embedded clinical specimens by immunofluorescence and immunoperoxidase staining.

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen.

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 G1 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. neoformans, since in this group the present latex agglutination test has some limitations.

Murine monoclonal antibodies recognizing a non-capsular antigen that distinguishes between Cryptococcus neoformans var. neoformans and C. neoformans var. gattii.

A panel of 4 monoclonal antibodies (mabs) of the IgG1 subclass have been made against a cytoplasmic antigen of Cryptococcus neoformans. Mab 4E2 recognized isolates of C. neoformans var. gatti by enzyme-linked immunosorbent assay (ELISA), whilst the other antibodies did not recognize these antigens. By Western blot 4E2 recognized determinants at 110-125, 65-70, 45-50 and 36-38 kDa. Mabs 9E6, 7C7 and 5D9 recognized bands at 36-38 and approximately 30 kDa. All 4 mabs (4E2, 9E6, 7C7 and 5D9) recognized both non-encapsulated and encapsulated isolates of C. neoformans var. neoformans by ELISA, and in addition showed reactivity to only the cytoplasm and cell membrane of yeasts by immunofluorescence. Mab 7C7 recognized antigens of the closely related fungus Trichosporon beigelii by ELISA but did not recognize any other fungal antigens. The other 3 mabs showed no recognition of T. beigelii or any other fungal pathogens tested.

Preparation of murine monoclonal antibodies against the yeast phase of the dimorphic fungus Sporothrix schenckii.

Three murine monoclonal antibodies (Mabs) were raised against a cytoplasmic antigen of the yeast phase of the pathogenic fungus Sporothrix schenckii using a modification of standard hybridoma technology incorporating the immunosuppressive drug cyclophosphamide. When tested for species-specificity within the pathogenic dimorphic fungi one of these Mabs (S5) showed little cross-reactivity by enzyme-linked immunosorbent assay and Western blot, though there was some recognition of Paracoccidioides brasiliensis antigen. This Mab recognized a 70-75 kDa molecule on reduced Western blots of S. schenckii antigen. The other two Mabs (S12 and S15) showed cross-reactivity with all dimorphic fungal antigens tested, though they appeared to recognize a molecule of similar molecular weight. This is the first report of any attempt to raise species-specific Mabs against this important causative agent of dermatological disease.

Preparation of species-specific murine monoclonal antibodies against the yeast phase of Paracoccidioides brasiliensis.

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.

Preparation of monoclonal antibodies that differentiate between Histoplasma capsulatum variant capsulatum and H. capsulatum variant duboisii.

The immunosuppressive drug cyclophosphamide was used to facilitate the production of monoclonal antibodies (Mabs) which differentiated between the yeast phase of the two variants of the dimorphic fungus Histoplasma capsulatum by both enzyme-linked immunosorbent assay and Western blot. Two Mabs are described, identifying epitopes on a 70-75 kDa molecule, which are specific to H. capsulatum var. capsulatum and which do not identify epitopes of H. capsulatum var. duboisii. These Mabs have potential use in the epidemiology and serodiagnosis of histoplasmosis in areas where both classical and African forms of the disease occur.

A murine monoclonal antibody exhibiting high species specificity for Histoplasma capsulatum var. capsulatum.

A monoclonal antibody (mAb) exhibiting a high degree of species specificity for the yeast phase of the dimorphic fungus Histoplasma capsulatum 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 Blastomyces, 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 Paracoccidioides, and no reactivity against Blastomyces antigen.

Evidence that the M antigen of Histoplasma capsulatum var. capsulatum is a catalase which exhibits cross-reactivity with other dimorphic fungi.

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 var. 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.

Immunogold electron microscopical detection of tubulin and actin within mycelial phase Histoplasma capsulatum capsulatum and H. capsulatum duboisii.

Immunogold electron microscopy using LR Gold as a resin was undertaken to determine the distribution of actin and tubulin within the hyphae of Histoplasma capsulatum capsulatum and H. capsulatum duboisii. Both of these proteins were found throughout the cytoplasm when probed with the appropriate monoclonal antibodies.

Nebulized fenoterol in severe asthma: determinants of the dose response.

We assessed the bronchodilator response to cumulative doses of nebulised fenoterol in 16 patients with severe asthma to determine (1) the magnitude of the bronchodilator response, (2) the factors determining this response, and (3) the dose producing maximum bronchodilation. To reflect the degree to which ventilatory function returned to normal, individual bronchodilator responses were assessed as the change in FEV1 (post-fenoterol FEV1 - initial FEV1) and expressed as a percentage of the predicted maximum response (predicted FEV1 - initial FEV1). The bronchodilator response was extremely varied (about 55% of predicted maximum in six patients, 40% in three patients, and 20% in seven patients), as was the dose producing maximal bronchodilation (1 mg in three patients, 2 mg in six patients, and 2.5 or 3 mg in seven patients). In individual patients the maximum response could be predicted by the response to the initial dose of fenoterol and by the duration of the episode of severe asthma. The maximum response was not predicted by the initial severity of obstruction or the patient's usual treatment.