Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

Franco, Marcello; Bagagli, Eduardo; Cunha, Marino; Chamma, Luiz Gastão; Fecchio, Denise

doi:10.1007/bf00436570

Cited by 20 publications

(20 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The detection of antibody by serological methods is very useful in the diagnosis of paracoccidioidomycosis (2, 3). However, the lack of antigen standardization may be a limitation (8,17). Therefore, recombinant forms of purified proteins are required as an alternative reagent to replace the crude antigenic preparations.…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

Cunha

Zancopé-Oliveira

Sueli³

et al. 2002

Clin Vaccine Immunol

View full text Add to dashboard Cite

The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients.

show abstract

Section: Discussionmentioning

confidence: 99%

Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

Cunha

Zancopé-Oliveira

Sueli³

et al. 2002

Clin Vaccine Immunol

View full text Add to dashboard Cite

show abstract

“…Rabbit anti-P. brasiliensis was prepared as previously described. 27 The primary antibodies were a mixture of rabbit anti-P. brasiliensis (1:30 dilution in normal goat serum) and J150-12A3, a protein A-purified IgG1 mouse anti-MBP monoclonal antibody (100 g/ml) produced in our laboratory. As negative controls, we used a mixture of protein A-purified normal rabbit IgG (50 g/ ml) and MOPC-31, an IgG1 antibody from a mouse myeloma cell line (100 g/ml) (Sigma Chemical Co., St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

Wagner

Franco

Kephart

et al. 1998

The American Journal of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal antimajor basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

show abstract

“…In this situation, immunodiffusion results may yield a false-negative result. Franco et al (5) have already demonstrated that the same isolate presented variability in antigenic profile. It was our hypothesis that the Pb-339 isolate was composed of different clones, and during subculture these different clones might or might not express gp43.…”

mentioning

confidence: 92%

“…The occurrence of instability in the synthesis of antigenic components by the same P. brasiliensis isolate under controlled incubation conditions was first observed by Franco et al (5). When a specific isolate is used to produce antigens for immunodiffusion tests or Western blot assays, the gp43 molecule must be present in these preparations.…”

mentioning

confidence: 98%

Variable gp43 Secretion by Paracoccidioides brasiliensis Clones Obtained by Two Different Culture Methods

2005

View full text Add to dashboard Cite

The main objectives of this study were to obtain clones of Paracoccidioides brasiliensis by two methods (micromanipulation and plating assay) and to determine if the secretion of the 43-kDa glycoprotein (gp43) is dependent on the clonal culture. The results show that the secretion of gp43 is not dependent on clonal cultures. Clones that originally were secretors of this molecule, after subculturing, lost this characteristic; on the other hand, clones that originally did not secrete gp43 began to secrete gp43 after subculturing.Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis. P. brasiliensis secretes and excretes various proteins and glycoproteins, and the 43-kDa molecule (gp43) is the immunodominant antigen and the main antigenic component, useful for diagnosis of paracoccidioidomycosis.The occurrence of instability in the synthesis of antigenic components by the same P. brasiliensis isolate under controlled incubation conditions was first observed by Franco et al. (5). When a specific isolate is used to produce antigens for immunodiffusion tests or Western blot assays, the gp43 molecule must be present in these preparations. However, this condition is not reproducible, and sometimes the final preparation does not contain gp43. Our hypothesis was that a culture is composed of different clones and for some unknown reason only some of them express gp43. A more reasonable approach to obtain a homogenous antigen preparation is to work with clonal cultures in which all cells contain the same genetic information.In the present study, we compared two ways to isolate single cells (clones) of P. brasiliensis, i.e., by a micromanipulation technique and by plating of single cells, and determined which medium is better in order to obtain clonal cultures. In addition, these clonal cultures were tested for their potential to secrete gp43 in order to confirm or reject our hypothesis about the relationship between gp43 secretion and clonal cultures. Also, clonal cultures were analyzed by rapidly amplified polymorphic DNA to verify possible polymorphism at the DNA level.

show abstract

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

Cited by 20 publications

References 11 publications

Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

Variable gp43 Secretion by Paracoccidioides brasiliensis Clones Obtained by Two Different Culture Methods

Contact Info

Product

Resources

About