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

Abstract

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.

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

“…However, the complexity of the antigens traditionally used meant that regardless of the methodology employed, it almost invariably resulted in problems associated with a lack of antigen standardization and specificity (17,36). Recent work using protein purification methods and recombinant protein technology has identified a series of candidate antigens (4,5,6,11,12,29,31,34,37) that may be used in place of complex antigenic extracts, thus offering a solution to such problems. However, the use of individual purified or recombinant antigens in diagnostic tests can itself be problematic when, for example, particular antigens are recognized by a relatively low percentage of the patients tested, resulting in low sensitivity.…”

“…P. brasiliensis CIB 339 was obtained from the culture collection of the Corporación para Investigaciones Biológicas. P. brasiliensis cytoplasmic yeast antigen (CYA) was prepared as previously described (6). Briefly, cells were subcultured on slants of synthetic McVeigh-Morton medium (35) and incubated at 36°C for 3 days.…”

“…These include a 22-to 25-kDa protein (11), a 58-kDa glycoprotein (12), and an 87-kDa protein that has been purified and subsequently characterized as a member of the HSP70 family (6). Initially, this glycoprotein was detected in the sera of patients with PCM by an inhibition enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (15,16).…”

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…Interestingly, the heat shock proteins (HSPs) have been frequently shown to be immunodominant in infection by diverse pathogenic fungi, which is against the common rule that the immune response is generally targeted against microbe-specific antigens (55,102,219,236,294,295,364,418,442,457). Candida albicans is a significant human fungal pathogen capable of disseminating through blood and colonizing almost all organs (514).…”

Detection of Paracoccidioides brasiliensis gp70 Circulating Antigen and Follow-Up of Patients Undergoing Antimycotic Therapy

“…Gómez et al (19,20) used the inh-ELISA to detect the 87-kDa molecule in the sera of patients with PCM for both diagnostic and follow-up purposes. Antibody detection systems have also been used to assay other molecules for the diagnosis of PCM, such as a 22-to 25-kDa protein (14), a 58-kDa glycoprotein (15), and an 87-kDa protein (8).…”