Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu,SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 °C (yeast form) and as cottonous colonies at 25 °C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population. This revised version was published online in August 2006 with corrections to the Cover Date.     

Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.     

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <. 001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.     

In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens

Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM). PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl. The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient. The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa. Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide. Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA. Further studies were designed to investigate the capacity of these fractions to induce cell proliferation. It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.     

The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-Galactosyl epitopes

Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.     

Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.     

Antigens of Cryptosporidium sporozoites recognized by immune sera of infected animals and humans

The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.     

Identification of antigens of Colombian Giardia duodenalis isolates recognized by total IgG and subclasses

Antigen profiles were described for Giardia duodenalis cysts and trophozoites that are recognized by IgG and its anti-G. dudodenalis subclasses (IgG1, IgG2, IgG3, IgG4). Antigens were identified by Western blot from G. duodenalis cyst and trophozoite isolates. Cysts and trophozoites were each subjected to protein separation by SDS-PAGE. The proteins were then transferred to nitrocellulose membranes by electroimmunoblot, and their antigenicity was determined by exposing them to sera from patients with confirmed diagnosis of G. duodenalis infection. The antigen-antibody reaction was revealed by specific alkaline phosphatase antibody conjugates against IgG, IgG2, IgG3, IgG4: bands were visualized by addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate and the stain nitro blue tetrazolium. The bands were read and analyzed by linear regression using Quantity One software. Thirty two antigens were simultaneously recognized by total IgG anti-G. duodenalis in the cyst and trophozoite stages. The antigens varied in molecular weight from 22 to 185 kDa. Nineteen antigens were identified by both IgG, and IgG3 anti-G duodenalis, with molecular weights ranging from 42 to 180 kDa. IgG2 and IgG4 did not identify any antigen in either stage. The antigens of molecular weights 27, 30, 31, 33, 45, 49, 57, 78, 89 and 170 kDa are shared with G. duodenalis isolates from other geographical regions of Colombia. The recognition of cyst and trophozoite antigens of Colombian G. duodenalis isolates by IgG, IgG1 and IgG3 anti-G. duodenalis suggested that they are involved in the induction of the host immune response.     

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self‐reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self‐antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2‐DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp‐2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic‐IVIg) and one with β‐propiolactone (propiolactone‐IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone‐IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self‐reactive IgG. Thus, protein spots recognized by IgG in propiolactone‐IVIg represent the core set of self‐antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as α‐enolase, RNA processing and cytoskeletal proteins such as lamin‐A/C.     

Metabolic labeling of Entamoeba histolytica antigens: characterization of a 28-kDa major intracellular antigen

The in vivo incorporation of radiolabeled amino acids into antigens of Entamoeba histolytica, HM-1:IMSS, is reported. Immunoprecipitation with sera from patients with invasive amebiasis revealed a 28-kDa antigen present in whole cell lysates of E. histolytica. This antigen was of cytoplasmic origin, as indicated by cell fractionation and Triton X-114 detergent-phase separation. Immunoprecipitation, using sera from patients with invasive amebiasis and symptomless cyst passers, revealed the 28-kDa antigen as the major antigen recognized by the sera tested. Immunoprecipitation analysis using radiolabeled-released proteins instead of whole cell lysates showed a number of bands, including the 28-kDa antigen. The data suggest that the 28-kDa antigen is of cytoplasmic origin or is released from the cytoplasmic compartment.     

Additive effect of rPb27 immunization and chemotherapy in experimental paracoccidioidomycosis

Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)(3), some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.     

Cross-reacting and specific antigenic components in cystic fluid from metacestodes of Echinococcus granulosus and Taenia solium

Sera from confirmed patients of 5 hydatidosis, 67 neurocysticercosis and 89 other parasitic diseases were tested for specific antibody (IgG) levels by ELISA to cystic fluid antigens from metacestodes of Echinococcus granulosus (HF) and Taenia solium (CF). All hydatidosis sera reacted positively to both HF and CF while neurocysticercosis sera did in 49.3% to HF and 85.1% to CF. The frequencies of cross-reactions were lower in other parasitic diseases to both antigens. By SDS-PAGE, protein bands of 64, 35, 22 and 7 kilodaltons (kDa) were found common in HF and CF. SDS-PAGE/immunoblot exhibited that hydatidosis sera reacted crossly to CF at 135, 110, 100, 86, 64, 45, 39, 35 and 24 kDa bands while neurocysticercosis sera did to HF at 135, 100, 86, 64, 52, 39, 35, 29 and 24 kDa bands. These results indicated that protein bands of 135, 100, 86, 64, 39, 35 and 24 kDa were major common components in HF and CF. Protein bands of 7 kDa in HF and 15, 10 and 7 kDa in CF did not react crossly and were specific components in respective antigens.     

Immunosuppressive effect of paracoccidioidomycosis sera on the proliferative response of normal mononuclear cells

In this paper we relate that sera from paracoccidioidomycosis patients inhibited the mitogen-induced proliferative responses of normal mononuclear cells. Treatment of these sera with 2.5% polyethyleneglycol (PEG), a method classically used to precipitate immune complexes, significantly reduced their inhibitory activity. Immunoblot analysis of the PEG precipitates identified a 34-kDa polypeptide, recognized by rabbit anti-P. brasiliensis IgG. Patient mononuclear cells showed partial restoration of their proliferative capacity after 24 h culture in medium alone, which suggests release of membrane-bound molecules in the culture medium. These findings indicate that circulating P. brasiliensis antigens, complexed or not with antibodies, may play a negative immunoregulatory effect in the mitogen-induced proliferative responses of paracoccidioidomycosis patients.Abbreviations CIC circulating immune complexes - DID double immunodiffusion test - IRMA two-site immunoradiometric assay - LT lymphocyte transformation assay - PBMC peripheral blood mononuclear cells - PCM paracoccidioidomycosis - PEG polyethyleneglycol - PHA phytohemagglutinin-P - SUPPEG supernatants from serum samples treated by PEG     

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.     

Patients with Chronic-form Paracoccidioidomycosis Present High Serum Levels of IgE Anti-paracoccidioides brasiliensis Gp70

Paracoccidioidomycosis (PCM) is a disease caused by the Paracoccidioides genus, which includes P. brasiliensis and the new phylogenetic species P. lutzii. Resistance to this infection has been correlated with a Th1 pattern of cellular immune response, while susceptibility is correlated to an intense humoral immune response with an increase in IgE levels. Serum levels of IgE and IgG anti-gp70 and anti-exoantigen in chronic PCM were analyzed by enzyme-linked immunosorbent assay. Results showed a higher gp70 concentration in somatic antigen (SA) than in cell-free antigen (CFA) preparation and significantly higher levels of IgE and IgG anti-gp70 in chronic PCM patients’ serum (n = 12) than in normal human serum (n = 12) (p < 0.05). Pearson’s correlation analysis showed a strong correlation between IgG and IgE anti-gp70 (r = 0.8424). Additionally, IgE purified from a pool of acute and chronic PCM patient’s serum was analyzed by immunoblotting. The patients with the acute form of the disease showed strong bands for gp43 and gp70 in SA but only for gp43 in CFA. In patients with the chronic form, solely the gp43 band was observed. In conclusion, we found that SA is a better source of gp70 than CFA is, and chronic PCM patients show high levels of IgE anti-gp70. This finding suggests that the Th2 immune response is potentially induced by gp70 in PCM disease, which calls for further study.     

Fasciola hepatica: an antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats

Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested for inducing protective immunity in rats. Two routes of vaccination were applied. The results showed that intraperitoneal vaccination induced significantly better protection (P <0.05) than intramuscular vaccination. Intraperitoneal vaccination with antigens from NEJs induced more effective protection: after challenge infection, rats that were so vaccinated had 92.6% (+/-2.5% SEM) fewer parasites in their liver and 57.3% (+/-13.3% SEM) fewer parasites penetrating the gut wall than control rats. Rats that were vaccinated with a low-molecular-weight fraction of antigen from NEJs were also highly protected against a challenge. F. hepatica antigens that are immunoreactive were identified on immunoblots, using sera collected from highly protected rats that had been vaccinated with NEJ antigens and also sera from cattle and rats that were experimentally infected with F. hepatica. The low-molecular-weight fraction of antigen from NEJs contained an immunodominant 32-kDa protein that was recognized by serum antibodies of vaccinated rats and immune cattle. This 32-kDa protein was not detected in partially purified antigens from adult flukes. We conclude that antigens of NEJs of F. hepatica, when injected intraperitoneally in rats, are highly protective. In particular, the 32-kDa protein contained in these antigens may be highly valuable for the development of an effective vaccine against F. hepatica.     

Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.