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.     

Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry

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.     

Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.     

A novel 95-kilodalton antigen of Wuchereria bancrofti infective larvae identified by species-specific monoclonal antibodies.

CBA and BALB/c mice produced polyspecific and monospecific polyclonal antibody responses, respectively, following immunization with Wuchereria bancrofti stage-3 larvae. Two monoclonal antibodies (MAbs) were produced from the immunized BALB/c mouse. These MAbs (both isotype M) recognized a previously undescribed highly expressed W. bancrofti antigen present in stage-3 larvae. The epitopes bound by the MAbs appear to be species specific for W. bancrofti since the MAbs did not bind to antigens of either nine other nematode species or two vector species in Western blots (immunoblots). Phosphorylcholine epitopes, responsible for immunological cross-reactivity among nematodes, were identified only on a 200-kDa antigen and not on the 95-kDa molecule. The targets of these immunoglobulin M MAbs are not carbohydrate epitopes.     

Isolation and antigenicity of a 45-kilodalton Paracoccidioides brasiliensis immunodominant antigen.

In the present study, we analyzed human antibody responses to Paracoccidioides brasiliensis cellular antigens by the immunoblot technique to identify specific cellular components and to investigate the existence of antigen profile differences among serological responses of paracoccidioidomycosis (PCM) patients. Among the 64 PCM serum samples analyzed, a relatively homogeneous immunoglobulin G response to P. brasiliensis antigens was observed. The polypeptide with a mass of 45 kDa was the most clinically important, since antibody to this antigen was detectable in 90.6% of PCM patients studied and the six individuals who did not produce antibody were either at the end of treatment or in the posttherapy period and had shown clinical recovery. These facts suggested that the presence of this antibody may be an indicator of active disease. The 45-kDa antigen was also the most specific antigen of the PCM humoral immune response, since it reacted with only 2 of 79 (2.5%) heterologous serum samples tested: 1 histoplasmosis case and 1 tuberculosis case. This polypeptide was isolated from gels by electroelution and, when tested by an immunoradiometric assay and immunoblotting, maintained its reactivity with PCM sera and also with anti-P. brasiliensis polyclonal antibodies raised in rabbits at the same sensitivity levels as those obtained in immunoblotting with a crude antigen. Since in our assays the 45-kDa polypeptide was the major P. brasiliensis antigen and seemed to be specific for PCM, its use in alternative diagnostic methods is promising, especially in patients suspected of having the juvenile clinical form of PCM often associated with negative double-immunodiffusion results.     

Characterization of gp70 and anti-gp70 monoclonal antibodies in Paracoccidioides brasiliensis pathogenesis

Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis. In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa. A cell surface glycoprotein of 43 kDa is the major antigenic component of P. brasiliensis. Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation. Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM. Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant. Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability. Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43. gp70 inhibits NO and H(2)O(2) liberation by peritoneal macrophages in vitro, as does gp43. Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections. To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P. brasiliensis using anti-gp70 MAbs. This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.     

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.     

Immunohistochemical characterization of a set of monoclonal antibodies to human neuron-specific enolase.

This paper describes the immunohistochemical staining properties of four monoclonal antibodies (MAbs) (CF, EB, AD, and KB) which had been previously shown to be specific for purified neuron-specific enolase (NSE) by a solid-phase radioimmunoassay. In this study, the authors immunostained a spectrum of normal and neoplastic neuronal, "neuroendocrine," and nonneuronal tissues fixed in formalin and embedded in paraffin. Positivity was generally restricted to normal neuronal structures and neuronal tumors, including adrenal neuroblastoma, ganglioneuroblastoma, olfactory neuroblastoma, pheochromocytoma, carotid body paraganglioma, duodenal gangliocytic paraganglioma, and teratoma with neuroepithelial components. Three staining patterns of the normal or neoplastic neuronal structures were observed: two MAbs (CF and EB) stained predominantly the nerve fibers (axoplasm); one (AD) stained predominantly the cell bodies (perikaryon); and one (KB) stained both the axoplasm and the perikaryon. "Neuroendocrine" tumors such as pulmonary small cell carcinoma, pancreatic islet cell tumor, thyroid medullary carcinoma, and carcinoid tumors from various locations showed a variable staining pattern. Tumor cells undergoing mitotic division were usually positive regardless of type. Normal structures other than neuronal or "neuroendocrine," including normal glial cells, were negative. The authors also studied a range of glial cell tumors with MAbs CF and AD as well as with Dako polyclonal antiserum to NSE. The results showed that CF stained the axonal fibers in the normal white matter surrounding these tumors; it did not stain the tumor cells or the perikarya of neurons in the surrounding normal gray matter. AD stained the glioma cells as well as the perikarya and dendrites of neurons in the surrounding normal gray matter; it did not stain the axonal fibers in the surrounding normal white matter. By contrast, the polyclonal antiserum stained all of these structures. The high degree of staining specificity of the MAbs should prove them to be valuable in immunohistochemical diagnosis of tumors as well as in further understanding the role of NSE in neuronal differentiation.     

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.     

Isolation and partial characterization of a novel subset of human T lymphocytes defined by monoclonal antibodies

Delayed-type hypersensitivity (DTH) responses to alloantigens were found to correlate with both skin and tumour allograft rejection in 224 reconstituted ATXBM-CBA mice. Furthermore, DTH responses and allograft rejection were observed only in mice that had received Ly-1 cells. Depletion of Thy-1+ or Ly-1+ cells led to indefinite graft survival and the absence of DTH responses, whereas depletion of Ly-2+ cells led to rapid graft rejection and strong DTH responses. The same result was obtained with CBA mice responding to grafts of either C57BL/6 skin, the B16 melanoma, or the EL4 lymphoma; and for (CBA X A)F1 mice responding to H-2K region alloantigens of AQR skin grafts. Thus, DTH and allograft rejection are both mediated by a Ly-1 T cell and it is considered that these are two different manifestations of the same transplantation response.     

Production and partial characterization of monoclonal antibodies to Mycobacterium leprae.

Monoclonal antibodies to Mycobacterium leprae were produced by the fusion of BALB/c splenocytes and lymph node cells to BALB/c myeloma (NSI/1) cells. Eleven monoclonal antibodies were characterized as to their reactivity with M. leprae and 18 other mycobacterial species by enzyme-linked immunosorbent assay and immunofluorescence. Two monoclonal antibodies reacted only with M. leprae, and the other nine showed unique patterns of reactivity by enzyme-linked immunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000-dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M. gastri, and M. smegmatis. Potential uses for these antibodies in serological tests and immunochemical analyses are discussed.     

Generation and characterization of monoclonal antibodies to 28-, 35-, and 65-kilodalton proteins of Mycobacterium tuberculosis.

Three monoclonal antibodies (H60.15, H61.3, and H105.10) directed to protein antigens of Mycobacterium tuberculosis were obtained and characterized. H60.15 recognizes a protein with a molecular mass of 28 kilodaltons (kDa) with broad cross-reactivity on a panel of 12 species and strains of mycobacteria. H61.3 reacts with a 35-kDa protein present in M. tuberculosis, Mycobacterium bovis BCG, and M. africanum. On the basis of the antigen molecular masses and competition experiments with other monoclonal antibodies, H60.15 and H61.3 seem to be the first described monoclonal antibodies to these M. tuberculosis proteins. H105.10 binds to the cross-reactive 65-kDa protein present in mycobacteria. Epitope mapping of H105.10 was performed by using the M. leprae DNA sublibrary available in bacteriophage lambda gt11 for this antigen and revealed that its epitope resides in the region from amino acids 20 to 54. The 28-, 35-, and 65-kDa antigens isolated by immunoblotting and presented on nitrocellulose to pleural effusion T cells from tuberculosis patients induced a proliferative response, indicating the presence of T-cell epitopes. These observations indicate that two protein antigens should be added to the list of antigens detectable in M. tuberculosis by monoclonal antibodies. The common feature of such proteins, the elicitation of an immune response of limited or broad cross-reactivity for mycobacteria, encourages the search for their role in the pathogenesis of mycobacterioses.     

A 34- to 38-kilodalton Cryptococcus neoformans glycoprotein produced as an exoantigen bearing a glycosylated species-specific epitope.

Three monoclonal antibodies (MAbs), all of the immunoglobulin G1 subclass, were raised against Cryptococcus neoformans by using the technique of cyclophosphamide ablation of B-cell responses against shared epitopes of the cross-reactive fungus Trichosporon beigelii. MAb 3C2 was reactive against the encapsulated and nonencapsulated isolates of C. neoformans var. neoformans by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot), and in addition to a 34- to 38-kDa determinant, it recognized a series of lower-molecular-weight species. 3C2 also reacted strongly with culture supernatant preparations of C. neoformans var. neoformans by ELISA. 3C2 showed no recognition of either T. beigelii or C. neoformans var. gattii antigens. Enzymatic deglycosylation followed by reaction with 3C2 on Western blots revealed that sialic acid was an integral part of the determinant, together with N-acetylglucosaminyl-asparagine and alpha-mannose. Proteolytic digestion showed that the epitope was pepsin sensitive and that it also contained tryptophan and glycine and/or leucine as determinants of recognition by 3C2. The pI of the glycoprotein was 7.1. Affinity chromatography-purified antigen did not exhibit proteolytic activity on sodium dodecyl sulfate-polyacrylamide substrate gels. Indirect fluorescence antibody tests revealed that 3C2 labelling was confined to the cell membrane and cytoplasm of yeasts. The remaining MAbs, 7H4 and 5G5, recognized both capsulated and nonencapsulated strains of C. neoformans var. neoformans by both ELISA and Western Blot, identifying linear determinants with molecular masses of 36 and 30 kDa. They were unreactive against culture supernatant antigen (exoantigen) from either variant of C. neoformans.     

Characterization of an immunoreactive species-specific 19-kilodalton outer membrane protein from Helicobacter pylori by using a monoclonal antibody.

Immunoblotting experiments on hyperimmune rabbit serum and sera from patients with Helicobacter pylori gastritis showed a consistent antibody response to a 19-kDa outer membrane protein antigen. A monoclonal antibody, designated HP 40, which reacted by Western immunoblotting with this protein was produced. It was shared by all H. pylori strains tested (D 273, NCTC 11637, and 24 wild strains) but not by the thermophilic Campylobacter species, Campylobacter fetus, Helicobacter mustellae, or Helicobacter fennelliae. Immunogold staining suggested that the 19-kDa antigen was exposed on the outer surface of the bacteria. Its functional role and effectiveness as a serological diagnostic tool are under study.     

Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates.

Monoclonal antibodies (MAbs) were produced to four Chlamydia psittaci isolates: NJ1 and TT3 from turkeys, VS1 from a parakeet, and B577 from an ovine abortion. MAbs were tested for reactivity with each isolate by the indirect immunofluorescent antibody technique and for neutralization by an inclusion reduction neutralization technique in tissue culture. Two genus-specific and 14 serovar-specific MAbs were produced. Genus-specific MAbs reacted with all avian and mammalian isolates; however, each failed to neutralize its homologous chlamydial isolate. Turkey isolates NJ1 and TT3 were antigenically similar; serovar-specific MAbs produced to each reacted equally with both isolates yet showed little or no reaction with other serovars. Serovar-specific MAbs to the parakeet and abortion isolates were distinct; no cross-reactions were seen with other serovars. None of the serovar-specific MAbs reacted with an ovine arthritis isolate. Of the 14 serovar-specific MAbs, 13 partially neutralized homologous strains with or without the addition of complement. Because of the high specificity, the serovar-specific MAbs used with the immunofluorescence technique provided a rapid and precise method to identify three serovars of C. psittaci.     

Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages.

The yeast form of Paracoccidioides brasiliensis, the causative agent of a deep mycosis in humans, is known to be phagocytized by, and to multiply inside, macrophages. In this work we describe the involvement of gp43, a major antigenic protein of P. brasiliensis, in the initial steps of attachment of the fungus to macrophages. Anti-gp43 F(ab) polyclonal fragments were capable of inhibiting phagocytosis in a concentration-dependent manner. Sheep red blood cells sensitized with purified gp43 were more endocytized than SRBC alone, and this process was also inhibited by anti-gp43 F(ab) fragments. Inhibition tests indicated the involvement of fucose and mannose residues in the phagocytosis of the fungus and of SRBC-gp43 by macrophages. Taken together, these results suggest that gp43 may be involved in the adherence and uptake of the fungus by murine peritoneal macrophages, and that this binding may be dependent on monosaccharide residues that are part of the gp43 glycoprotein.     

Immunohistochemical characterization of fibrin(ogen)-related antigens in human tissues using monoclonal antibodies

A new approach to study the distribution of fibrin(ogen)-related antigens was investigated using three different monoclonal antibodies (MAbs) and the avidin-biotin complex immunoperoxidase technique. MAb I8C6 recognizes B beta 1-42 peptide and can react with either fibrinogen or fibrin I; MAb T2G1 recognizes B beta 15-42 peptide and detects fibrin II but does not cross-react with fibrinogen; MAb GC4 reacts with Fragments D/DD derived from plasmin degradation of fibrinogen or fibrin but not with intact fibrinogen. The method can be applied to frozen or Bouin's fixed paraffin-embedded tissues obtained at biopsy, surgery, and autopsy. The distribution of the three antigens observed with the three MAbs was compared with that obtained with a polyclonal antiserum to fibrinogen and with the more conventional histochemical stains used in pathology to demonstrate fibrin deposits in tissues (Lendrum and PTAH). The staining observed with the three monoclonals clearly detected three different populations of fibrin(ogen)-related antigen in the tissues examined. The staining with MAb T2G1 specifically detected fibrin II with greater sensitivity than did conventional stains. The results of this study suggest that this method allows the molecular form of fibrin(ogen)-related deposits in tissues to be determined and this information may help to elucidate the role of fibrin in various disease states, such as atherosclerosis and renal disease, and in tumor growth and metastasis.     

Immunohistochemical characterization of rat thymic non-lymphoid cells. II. Macrophages and granulocytes defined by monoclonal antibodies.

A panel of monoclonal antibodies (mAb) raised to antigens of rat thymic non-lymphoid cells (predominantly macrophages and granulocytes) was immunohistochemically characterized. Based on their staining patterns on cryostat thymic sections and double labellings using acid phosphatase activity, anti-cytokeratin mAb to exclude binding to epithelium or ED1 and ED2 mAb, specific for rat macrophages, antibodies were subdivided into four groups: (i) R-MC 39 mAb strongly reactive with macrophages in the cortex and cortico-medullary zone (CMZ) and weakly with some scattered macrophages in the medulla, blood vessels and thymocytes; (ii) R-MC 40, 41 and 42 mAb specific for cortical macrophages and most CMZ macrophages; (iii) R-MC 43 and 44 mAb predominantly recognizing CMZ and medullary macrophages; (iv) R-MC 45 mAb strongly labelling granulocytes and weakly a subset of macrophages throughout the thymus and isolated cells in the medulla. The obtained results show considerable heterogeneity within mobile thymic non-lymphoid cells and the presence of specific or common antigens in macrophages of particular topographic localization in the rat thymus.     

Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.