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

Hamilton, Andrew J.; Bartholomew, M.A.; Fenelon, Lynda; Figueroa, Jose I.; Hay, Roderick J.

doi:10.1099/00221287-136-2-331

Cited by 48 publications

(50 citation statements)

References 11 publications

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“…Purified samples of 87-kDa protein (5 g/ l), pretreated with loading buffer containing 2-mercaptoethanol and boiled for 3 min, were loaded onto an SDS-10% (vol/vol) PAGE gel (with 2 mM thioglycolic acid in the upper buffer chamber) and electrophoresed at 160 V for 1 h. The gel was then blotted onto a polyvinylidene difluoride membrane as previously described (17). The blot was stained for 1 min with 0.1% (wt/vol) Coomassie blue R and destained.…”

Section: Methodsmentioning

confidence: 99%

“…For analysis of the fractions obtained during protein purification and characterization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (using 10% [vol/vol] gels), ELISA, and Western immunoblotting (using polyvinylidene difluoride membranes [Immobilon P; Millipore Corporation, Watford, United Kingdom]) were used as previously described (10,17,19). MAbs P1B (14) and 69F (the latter being an anti-H. capsulatum hsp80 MAb [18]) were used in immunodevelopment.…”

Section: Methodsmentioning

confidence: 99%

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Paracoccidioides brasiliensis 87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry

et al. 2002

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The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Paracoccidioides brasiliensis 87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry

et al. 2002

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“…To evaluate the fractions obtained during the purification of both proteins, SDS-PAGE (using 10 and 12% [vol/vol] gels), ELISA, and Western immunoblotting using polyvinylidene difluoride membranes (Immobilon P; Millipore Corp., Watford, United Kingdom) were used as previously described (10,18,22). Pooled PCM patients' sera were used to detect the 27-kDa protein, and monoclonal antibody P1B was used to identify the 87-kDa protein (15).…”

Section: Vol 41 2003 P Brasiliensis Recombinant-diagnostic Antigenmentioning

confidence: 99%

Combined Use of Paracoccidioides brasiliensis Recombinant 27-Kilodalton and Purified 87-Kilodalton Antigens in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Paracoccidioidomycosis

et al. 2003

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The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, wellcharacterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzymelinked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.Paracoccidioidomycosis (PCM), one of the most prevalent systemic fungal mycoses in Latin America, is caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus (1, 36).

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“…Finally, an inhibition enzyme-linked immunosorbent assay (ELISA) based on a murine monoclonal antibody demonstrated sensitivity similar to that of the polyclonal assay for detecting antigen in serum but was less sensitive for urinary antigen detection (16,17). However, these monoclonal reagents were also never commercially released.…”

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confidence: 99%

Evaluation of Commercially Available Reagents for Diagnosis of Histoplasmosis Infection in Immunocompromised Patients

2013

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Urinary histoplasma antigen measurement can be useful for diagnosing systemic histoplasmosis and for monitoring treatment response, especially in immunocompromised patients. However, testing has traditionally been limited to specialized reference laboratories, as immunoassay reagents for the antigen were not widely available. Recently, a polyclonal-antibody-based in vitro diagnostic (IVD) kit for histoplasma antigen detection was released, as well as monoclonal-antibody reagents against the target. We evaluated the analytical and clinical performance of the two reagents. Both assays were capable of detecting histoplasma antigen in urine samples over a wide dynamic range, although the monoclonal assay showed improved precision and analytical sensitivity relative to the polyclonal IVD. In a test set of clinically characterized patient samples, the monoclonal laboratory-developed test (LDT) demonstrated 90.5% sensitivity and 96.3% specificity versus 61.9% sensitivity and 79.3% specificity for the polyclonal IVD, with areas under the curve (AUCs) of 0.987 and 0.754, respectively. The major differences between the two assays were higher background reactivity in healthy donors with the polyclonal assay and an increased signal response in positive samples for the monoclonal assay. The impact of these differences on monitoring treatment response was evaluated in a series of patients undergoing treatment for histoplasmosis. While all the assays gave similar qualitative estimates of treatment response, responses were more evident using the monoclonal assay. In summary, we conclude that while multiple assays are available for measuring histoplasma antigen in urine, a monoclonal-antibody-based assay appears to provide improved analytical performance for management of immunocompromised histoplasmosis patients.

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

Cited by 48 publications

References 11 publications

Paracoccidioides brasiliensis 87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry

Paracoccidioides brasiliensis 87-Kilodalton Antigen, a Heat Shock Protein Useful in Diagnosis: Characterization, Purification, and Detection in Biopsy Material via Immunohistochemistry

Combined Use of Paracoccidioides brasiliensis Recombinant 27-Kilodalton and Purified 87-Kilodalton Antigens in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Paracoccidioidomycosis

Evaluation of Commercially Available Reagents for Diagnosis of Histoplasmosis Infection in Immunocompromised Patients

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