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    Comparison of the amino acid sequence of the Leishmania (Leishmania) chagasi cysteine proteinase 1 gene (rLdccys1) with those from other Leishmania species. CPB from L. (L.) infantum, CPB18 from L. (L.) mexicana, and Lpcys2 from L. (L.) pifanoi. Alignment of the amino acid sequence was performed using the MegAlign-DNAstar (Clustal W) and Gene Doc programs. Dots correspond to identical amino acids. The pre-pro-region is represented in lower case letters and the mature protein in upper case letters. Conserved cysteine proteinase residues are indicated: papain substrate binding (dark gray), catalytic domains (black), glycosylation site (*), and C-terminal cysteine residues (light gray).

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    Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting analysis of the recombinant protein (rLdccys1) resulting from the Ldccys1 gene expression. A, Extracts from Escherichia coli BL21(DE3) transformed with either the pHis vector alone (lane 2) or pHis/Ldccys1 gene (lane 3) and the recombinant protein purified on a nickel column (lane 4) were subjected to electrophoresis on a 12% sodium dodecyl sulfate–polyacrylamide gel and stained with Coomassie blue. B, The same samples were transferred to a nitrocellulose membrane and incubated with monoclonal antibody 2E5D3. Lane 2, pHis alone; lane 3, pHis/Ldccys1 gene. Low molecular mass markers are represented in lanes 1 and indicated in kilodaltons (kDa).

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    Enzyme-linked immunosorbent assay and Western blotting of sera from humans with visceral leishmaniasis (VL) against recombinant cysteine proteinase. A, Microtiter plates were coated with either 200 ng/well of rLdccys1 or 100 ng/well of Leishmania (Leishmania) chagasi amastigote and promastigote extracts. Anti-human peroxidase-conjugated antibody at a 1:1,000 dilution was used. Results are given as the mean ± SD optical density (O.D.) of 10 human VL sera assayed individually at the indicated dilutions. B, Western blotting analysis was performed using 25 μg/lane of promastigote and amastigote extracts (lanes 1 and 2, respectively) and 5 μg/lane of rLdccys1 (lane 3) and five serum samples diluted to 1:100. Low molecular mass markers are indicated in kilodaltons (kDa) on the left.

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    Reactivity of sera from patients with visceral leishmaniasis, other infectious diseases, and healthy controls with rLdccys1 (200 ng/well) and Leishmania (Leishmania) chagasi amastigote (AMA) and promastigote (PRO) extracts (100 ng/well). An enzyme-linked immunofsorbent assay was performed with sera from all patients diluted to 1:500. The horizontal lines represent cut-off values for each antigen, which were calculated by adding 2 SD values to the mean optical density (O.D.) of 74 human normal sera. NHS = normal human sera; VL = visceral leishmaniasis; TB = tuberculosis; CL = cutaneous leishmaniasis; Chagas = Chagas disease.

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A RECOMBINANT CYSTEINE PROTEINASE FROM LEISHMANIA (LEISHMANIA) CHAGASI SUITABLE FOR SERODIAGNOSIS OF AMERICAN VISCERAL LEISHMANIASIS

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  • 1 Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil; Associação de Ensino Superior e Tecnológico do Piauí, Teresina, Piauí, Brazil

A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.

INTRODUCTION

Leishmaniasis comprises a group of diseases caused by protozoan parasites of the Leishmania genus that includes cutaneous, mucocutaneous and visceral leishmaniasis.1 Visceral leishmaniasis (VL) is caused mainly by Leishmania (Leishmania) donovani in Indian and east Africa, Leishmania (L.) infantum in the Mediterranean area, and Leishmania (L.) chagasi in South America. The most severe form of the disease affects 500,000 people worldwide.2 In Brazil, rural to urban migration contributes to the spreading of VL in towns and suburbs of large cities.3,4 The disease symptoms include irregular fever, splenomegaly, hepatomegaly, anemia, weight loss, and pancytopenia, and may lead to death if untreated. Development of clinical disease may be dependent on other factors including malnutrition, use of immunosuppressive drugs, and, especially, co-infection with human immunodeficiency virus.5–7

Routine diagnosis of VL has been based on the visualization of the parasite in bone marrow smears or splenic aspirates; however, this method is limited by a low sensitivity.8 Serologic diagnosis of VL is based on different methods of antibody detection that include the direct agglutination test,9 the indirect immunofluorescence test,10 immunoblot analysis,11–14 and the enzyme-linked immunosorbent assay (ELISA).15–18 However, use of whole parasite extracts in these serologic tests is limited because of assay reproducibility and specificity mainly due to the cross-reactivity with other diseases such as Chagas disease, cutaneous leishmaniasis, and tuberculosis.18–20 To obtain a specific diagnosis for VL, the following purified and recombinant Leishmania antigens have been used in serologic tests: SLA, gp63, rK39, rGBP, H2A, H2B, rLACK, rP20, rPSA-2, and A2.21–29 Comparative evaluation of ELISAs using these antigens showed that several of them are suitable in the diagnosis of Mediterranean VL.30

Leishmania cysteine proteinases have been used as targets for vaccines,31–33 chemotherapy,34,35 and serodiagnosis of cutaneous leishmaniasis and VL.36,37 Previous studies from our laboratory demonstrated the implication of a 30 kDa cysteine proteinase from L. (L.) chagasi (p30) in partially protective cellular immune responses against homologous infection in BALB/c mice.32 In the present study, we have expressed the gene encoding the p30 antigen from L. (L.) chagasi, Ldccys1, in Escherichia coli and tested the recombinant protein (rLdccys1) for use in serodiagnosis of American VL. In L. (L.) chagasi, the Ldccys1 gene is organized as a multicopy gene tandemly arranged and expressed in promastigote and amastigote forms.38 The amino acid sequence shows high identity, 78%, 79%, and 76%, to cysteine proteinases from Leishmania (L.) mexicana cpb18, Leishmania (L.) pifanoi Lpcys2, and Leishmania (L.) major cathepsin L-like protease, respectively, and also shows all conserved characteristic residues of cysteine proteinases. Sera from patients living in areas of Brazil endemic for VL, healthy controls, and from patients with other diseases were used to determine the sensitivity and specificity of the recombinant Ldccys1 antigen in comparison with crude L. (L.) chagasi lysates in ELISAs.

MATERIALS AND METHODS

Parasites.

Eight-week-old female golden hamsters were obtained from breeding stocks maintained at the Universidade of Campinas (São Paulo, Brazil). All animal procedures were reviewed and approved by the Ethical Committee for Animal Care at the Universidade Federal de São Paulo Escola Paulista de Medicina. The L. (L.) chagasi strain used (MHOM/BR/1972/LD) was characterized and kindly provided by Dr. J. J. Shaw (Instituto Evandro Chagas, Belém, Pará, Brazil) and maintained as amastigotes by inoculation of hamsters by the intraperitioneal route. Two months after infection, the hamsters were killed and the spleens were homogenized, treated with 0.2% saponin for 10 minutes, and centrifuged at 1,400 × g for 5 minutes for isolation of amastigotes. The resulting pellet was resuspended in phosphate-buffered saline (PBS), centrifuged at 250 × g for 5 min, the supernatant was centrifuged at 1,400 × g for 5 minutes, and the pellet was resuspended in PBS. The suspension was agitated for 3 hours at room temperature and centrifuged at 1,400 × g for 5 minutes. The final pellet containing purified amastigotes was frozen at −20°C until use.39

Isolation of genomic DNA and polymerase chain reaction (PCR) amplification of the L. (L.) chagasi cysteine proteinase gene 1 (Ldccys1).

Leishmania (L.) chagasi genomic DNA was extracted from 1 × 109 amastigotes as previously described.40 Amplification of the L. (L.) chagasi Ldccys1 gene was performed using primers corresponding to its open reading frame (ORF), previously published in GeneBank.41 The 5′ end primer (5′-TGC GGG ATC CCC ATG GCG ACG TCG AGG-3′) and the 3′ end primer (5′-CGA TGA ATT CCC CTA CGT GTA CTG GCA-3′) containing Bam HI and Eco RI restriction site sequences, respectively, were used for directional cloning. The PCR amplification was carried out using 100 ng of L. (L.) chagasi genomic DNA and 100 pmol of each primer in a mixture containing 0.2 mM of each dNTP, 10 mM Tris-HCl, pH 9.0, 1 mM MgCl2, 50 mM KCl, and 1 unit of Taq DNA polimerase in a final volume of 50 μL. Amplification conditions were 30 cycles at 94°C for 1 minute, 40°C for 2 minutes, and 72°C for 2 minutes. The PCR products were gel purified (Geneclean II kit; Bio-Rad Laboratories, Hercules, CA), digested with Bam HI and Eco RI, and cloned in the Bam HI and Eco RI sites of plasmid pUC18. The DNA sequencing was carried out according to the dideoxy-chain method in an ABI377 automatic sequencer (Applied bio-systmes, Foster City, CA).

Expression and purification of the recombinant cysteine proteinase (rLdccys1).

The PCR product corresponding to the ORF of the Ldccys1 gene was subcloned into the Bam HI and Eco RI restriction sites of the pHis-parallel 3 expression vector in frame with an amino-terminal six histidine tag.42 The recombinant plasmids were used to transform E. coli BL21(DE3) and protein expression was carried out by inoculating 500 mL of Luria Bertani medium containing 100 μg/mL of ampicilin with a 25 mL overnight bacterial culture. The suspension was incubated on a rotatory shaker at 37°C until log phase (absorbance at 600 nm = 0.6) was reached. Protein expression was then induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside for an additional 3 hours at 37°C. After growth, the recombinant bacteria were centrifuged at 4,000 × g for 10 minutes and the recombinant antigen was purified from the insoluble inclusion bodies by affinity chromatography using a Ni-NTA Superflow agarose matrix (Qiagen, Valencia, CA) according to the method of Skeiky and others.43 Purified protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoesis (SDS-PAGE) and Western blotting using a previously described monoclonal antibody (2E5D3) directed against a 30 kDa cysteine proteinase of L. (L.) amazonensis that cross-reacts with a 30 kDa antigen of L. (L.) chagasi amastigotes.31,32

Human sera.

Sera used in this study were classed as normal, active cases of VL, and other diseases. Human VL serum samples were collected from 131 individuals living in areas of Brazil endemic for VL. All patients had a VL diagnosis confirmed by the identification of amastigotes in bone marrow tissue, an indirect immunofluorescence reaction, a positive ELISA result, and showed clinical symptoms of VL: fever, splenomegaly, anemia, coughing, and wasting. Normal human sera were collected from 74 individuals living in a non-endemic area. Sera from individuals with other diseases included confirmed cases of cutaneous leishmaniasis (n = 26), Chagas disease (n = 35), and tuberculosis (n = 12).

Western blotting analysis.

Extracts from L. (L.) chagasi amastigotes and promastigotes and the recombinant protein were subjected to electrophoresis on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes as described by Towbin and others.44 Membranes were blocked with 5% powdered skim milk in PBS and incubated for 1 hour with monoclonal antibody 2E5D3 and VL human sera diluted 1:100. The strips were then washed in 0.05% Tween 20 in PBS and incubated with peroxidase-conjugated anti-mouse and anti-human secondary antibodies, and developed with diaminobenzidine and H2O2.

Ezyme-linked immunosorbent assay.

Microtiter plates (high-binding Costar plates; Corning, Inc., Corning, NY) were coated with the recombinant protein (200 ng/well) or L. (L.) chagasi extracts from amastigotes and promastigotes (100 ng/well) in coating buffer (0.05 M Na2CO3/NaHCO3, pH 9.6). The plates were incubated overnight at 4°C and then blocked with 5% powdered skim milk in PBS for 1 hour. Human sera diluted 1:500 were added and incubated for 1 hour at room temperature. After three washes with 0.05% Tween 20 in PBS, anti-human IgG horseradish peroxidase conjugate (Bio-Rad Laboratories) (diluted 1:1,000) was added and incubated for 1 hour at 37°C. The plates were washed three times in 0.05% Tween 20 in PBS and the reaction was developed with 0.5 mg/mL of o-phenylenediamine in 0.05 M sodium citrate, pH 4.5, containing 0.03% H2O2. The reaction was stopped by adding 4 N H2SO4, and the absorbance was measured at 492 nm in a Multiskan Plate Reader (Labsystems Oy, Helsinki, Finland). The cut-off values for each antigen were calculated by adding 2 SD values to the mean absorbance of 74 normal sera.

RESULTS

Isolation of the cysteine proteinase gene 1 (Ldccys1) from L. (L.) chagasi.

Using genomic DNA from L. (L.) chagasi and a pair of primers corresponding to the ORF of the L. (L.) chagasi Ldccys1 gene,41 we amplified a 1.3 kb fragment by the PCR. The nucleotide sequence analysis of the 1.3 kb fragment showed 99.8% identity with the published sequence of the Ldccys1 gene, with an ORF encoding a protein that contained all the characteristics of a cysteine proteinase as previously shown by Omara-Opyene and Gedamu41 and observed by the deduced amino acid sequence of the 1.3 kb fragment shown in Figure 1. It contains the conserved cysteine and histidine residues at positions 150 and 287, respectively, which are present in the catalytic domain of cysteine proteinases. Glycine, which is involved in substrate binding in papain, is also present at position 148. In addition, another amino acid residue important in catalysis, asparagine, is present at position 307. The Ldccys1 protein is composed of pre-region and pro-region followed by a mature protease core and a C-terminal extension. The pre-region contains the hydrophobic amino acid represented by residues 1-37. The pro-region is cleaved between amino acid residues 124 and 125, generating an alanine as in the case of other cysteine proteinases. In the C-terminal region, Ldccys1 contains 10 conserved cysteine residues present in other cysteine proteinases.45

Expression and purification of the recombinant cysteine proteinase.

The 1.3 kb fragment, which corresponds to the ORF of the Ldccys1 gene, was cloned into the pHis parallel 3 expression vector in frame with a six-histidine tag. Expression of the Ldccys1 gene in E. coli resulted in a recombinant 47 kDa protein that was solubilized in 8 M urea and extracted on a nickel column, and its purity was demonstrated by SDS-PAGE. Figure 2A shows the lysates from E. coli BL21 transformed with pHis parallel vector alone (lane 2) or carrying the Ldccys1 gene (lane 3) and purified rLdccys1 (lane 4). Western blotting analysis of bacterial extracts showed the reactivity of the recombinant protein with monoclonal antibody 2E5D3 and confirmed the expression of rLdccys1 (Figure 2B).

Reactivity of sera from patients with VL with recombinant cysteine proteinase.

To evaluate the antigenicity of L. (L.) chagasi rLdccys1, 10 individual sera from patients with VL were analyzed by an ELISA using the purified rLdccys1 and extracts from L. (L.) chagasi amastigotes and promastigotes as antigens. A titration curve was performed and the optimal concentration was found to be 200 ng/well for rLdccys1 and 100 ng/well for L. (L.) chagasi amastigote and promastigote extracts. Figure 3A shows that sera from patients with VL reacted strongly with rLdccys1, showing titers comparable to those obtained with promastigote lysates up to a dilution of 1:1,600, indicating that patients with VL show a strong antibody response against L. (L.) chagasi cysteine proteinase. These findings are corroborated by results shown in Figure 3B that represent sera from patients with active VL (A to E) that were also tested for their reactivity to antigens from L. (L.) chagasi promastigotes and amastigotes and rLdccys1 by Western blotting. There are several antigens in both L. (L.) chagasi lysates that react strongly with sera from cases with VL. These antigens have molecular masses of 70, 56, and 17 kDa and are common in the two forms of L. (L.) chagasi, as well as 90 kDa and 14 kDa antigens, which are present only in promastigotes. All sera were also reactive with rLdccys1, as well as with a 30 kDa antigen from amastigote and promastigote lysates that corresponds to the cysteine proteinase encoded by the L. (L.) chagasi Ldccys1 gene (Figure 3B).

Determination of sensitivity and specificity of L. (L.) chagasi cysteine proteinase by ELISA.

The sensitivity of L. (L.) chagasi rLdccys1 in ELISAs for sera from patients with VL was determined to compare reactivity with the recombinant antigen and amastigote and promastigote L. (L.) chagasi extracts. Of 131 sera from patients with active VL, 80% reacted with rLdccys1, while 98% and 99% were reactive with promastigote and amastigote extracts, respectively (Figure 4).

The specificity of the rLdccys1 was evaluated using sera from healthy controls and from patients with other diseases including cutaneous leishmaniasis, tuberculosis, and Chagas disease (Figure 4). The rLdccys1 antigen was very specific (96%), showing no cross-reactivity with sera from patients with Chagas disease, and very low rates of positive reactions with sera from patients with cutaneous leishmaniasis (3%, n = 26) and tuberculosis (8%, n = 12), and only four false-positive results in sera from 74 healthy controls. In contrast, amastigote and promastigote extracts showed high rates of cross-reactivity with sera from patients with cutaneous leishmaniasis (65% and 80%, respectively, n = 26), tuberculosis (83% for both extracts, n = 12), and Chagas disease (14% and 34%, respectively, n = 35). Table 1 shows the sensitivity, specificity, positive predictive value, and negative predictive value of the three antigens used in the ELISAs. The data demonstrate that the rLdccys1 antigen was significantly more specific than L. (L.) chagasi extracts for diagnosis of VL by ELISA.

DISCUSSION

The 30 kDa cysteine proteinase of L. (L.) chagasi was first implicated in cellular immune responses mediated by Th1 cells partially protective against homologous infection in BALB/c mice.32 The present work shows the antigenicity of this cysteine proteinase for humoral responses in clinically and parasitologically confirmed cases of human VL by use of a recombinant protein obtained after expression of the previously cloned and characterized Ldccys1 gene.41 The recombinant Ldccys1 antigen was obtained by expression of the Ldccys1 gene in the pHis vector, resulting in a 47 kDa protein whose predicted amino acid sequence analysis showed that it shares 99% identity with the Ldccys1 cysteine proteinase from L. (L.) chagasi amastigotes (Figure 1). Reactivity of recombinant E. coli expressing the Ldccys1 gene with a monoclonal antibody directed to the 30 kDa cysteine proteinase of L. (L.) chagasi showed that the recombinant protein corresponds to the 30 kDa cysteine proteinase from L. (L.) chagasi (Figure 2B).

Previous screening using 10 individual sera from patients with VL and the recombinant protein in an ELISA showed that the reactivity of rLdccys1 is comparable with that exhibited by promastigote extract to a dilution of 1:1,600 (Figure 3A). This demonstrates the feasibility of using this antigen for antibody detection in sera from patients with VL. Immunoblotting of L. (L.) chagasi promastigote and amastigote extracts against sera from patients with VL identified several reactive antigens, some of them common to both parasite forms, and showed that the 30 kDa cysteine proteinase is recognized by sera from patients with VL (Figure 3B). The antigenicity of Leishmania cysteine proteinases in humoral responses has been reported. Cysteine proteinases from L. (L.) major (CPA and CPB) were shown to be recognized by sera from patients infected with L. (L.) major, and CPA and CPB from L. (L.) infantum were used as targets for serodiagnosis of active VL cases in humans and dogs in Iran.36,37 Comparison of the predict amino acid sequence between L. (L.) chagasi Ldccys1 and CPB from L. (L.) infantum showed an identity of 99% (Figure 1). This result is consistent with previous data showing the high identity among cysteine proteinases expressed in Leishmania.45,46 The level of sensitivity with rLdccys1 was comparable with that obtained with rCPB (76%), strengthening the identity of these cysteine proteinases, and supporting the similarity of L. (L.) chagasi and L. (L.) infantum, which has been demonstrated by genotypic relationships within the L. (L.) donovani complex.47,48

Antibodies to rLdccys1 were detected in 80% of the samples from humans with active VL. This value is comparable to those obtained by use of the recombinant L. (L.) donovani A2 antigen by Carvalho and others29 (77%) in sera from Brazilian patients with symptomatic VL and by Ghedin and others28 in sera from Sudanese (80%) or Indian patients (60%), and is lower than the level of sensitivity reported with other Leishmania recombinant antigens.30 Conversely, the specificity of rLdccys1 is high and comparable to that observed with recombinant proteins analyzed,25,49 and is consistent when one considers infectious diseases that occur in same VL-endemic areas and whose cross-reactivity with Leishmania is well documented. Moreover, the spectrum of absorbance values obtained with rLdccys1 is comparable to that obtained with promastigote and amastigote extracts (70%, 84%, and 66% of VL sera showed an optical density greater than 1 with rLdccys1, promastigote, and amastigote lysates, respectively), allowing easy interpretation of ELISA results.

Our findings are consistent with data in the literature that have shown the importance of using recombinant antigens in serodiagnosis of VL by ELISA, and indicate that recombinant Ldccys1 from L. (L.) chagasi represents an additional tool suitable for the diagnosis of American VL.

Table 1

Diagnostic performance of rLdccys1 and crude antigens of Leishmania (Leishmania) chagasi in enzyme-linked immunosorbent assays

Percentage (no. of positive sera/total no. of sera)
AntigenSensibilitySpecificityPositive predictive valueNegative predictive value
* Total number of visceral leishmaniasis sera.
†Total number of normal and other diseases sera.
Promastigote98 (129/131*)69 (46/147†)7498
Amastigote99 (130/131)76 (35/147)7999
rLdccys180 (105/131)96 (6/147)9584
Figure 1.
Figure 1.

Comparison of the amino acid sequence of the Leishmania (Leishmania) chagasi cysteine proteinase 1 gene (rLdccys1) with those from other Leishmania species. CPB from L. (L.) infantum, CPB18 from L. (L.) mexicana, and Lpcys2 from L. (L.) pifanoi. Alignment of the amino acid sequence was performed using the MegAlign-DNAstar (Clustal W) and Gene Doc programs. Dots correspond to identical amino acids. The pre-pro-region is represented in lower case letters and the mature protein in upper case letters. Conserved cysteine proteinase residues are indicated: papain substrate binding (dark gray), catalytic domains (black), glycosylation site (*), and C-terminal cysteine residues (light gray).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.126

Figure 2.
Figure 2.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting analysis of the recombinant protein (rLdccys1) resulting from the Ldccys1 gene expression. A, Extracts from Escherichia coli BL21(DE3) transformed with either the pHis vector alone (lane 2) or pHis/Ldccys1 gene (lane 3) and the recombinant protein purified on a nickel column (lane 4) were subjected to electrophoresis on a 12% sodium dodecyl sulfate–polyacrylamide gel and stained with Coomassie blue. B, The same samples were transferred to a nitrocellulose membrane and incubated with monoclonal antibody 2E5D3. Lane 2, pHis alone; lane 3, pHis/Ldccys1 gene. Low molecular mass markers are represented in lanes 1 and indicated in kilodaltons (kDa).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.126

Figure 3.
Figure 3.

Enzyme-linked immunosorbent assay and Western blotting of sera from humans with visceral leishmaniasis (VL) against recombinant cysteine proteinase. A, Microtiter plates were coated with either 200 ng/well of rLdccys1 or 100 ng/well of Leishmania (Leishmania) chagasi amastigote and promastigote extracts. Anti-human peroxidase-conjugated antibody at a 1:1,000 dilution was used. Results are given as the mean ± SD optical density (O.D.) of 10 human VL sera assayed individually at the indicated dilutions. B, Western blotting analysis was performed using 25 μg/lane of promastigote and amastigote extracts (lanes 1 and 2, respectively) and 5 μg/lane of rLdccys1 (lane 3) and five serum samples diluted to 1:100. Low molecular mass markers are indicated in kilodaltons (kDa) on the left.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.126

Figure 4.
Figure 4.

Reactivity of sera from patients with visceral leishmaniasis, other infectious diseases, and healthy controls with rLdccys1 (200 ng/well) and Leishmania (Leishmania) chagasi amastigote (AMA) and promastigote (PRO) extracts (100 ng/well). An enzyme-linked immunofsorbent assay was performed with sera from all patients diluted to 1:500. The horizontal lines represent cut-off values for each antigen, which were calculated by adding 2 SD values to the mean optical density (O.D.) of 74 human normal sera. NHS = normal human sera; VL = visceral leishmaniasis; TB = tuberculosis; CL = cutaneous leishmaniasis; Chagas = Chagas disease.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.126

Authors’ addresses: Suzana de Souza Dias, Simone Katz, Márcia Regina Machado dos Santos, and Clara Lúcia Barbiéri, Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 862, 60 Andar, 04023-062, São Paulo, SP, Brazil, Telephone: 55-11-5576-4532, Fax: 55-11-5571-1095, E-mails: suzi.dias@bol.com.br, simone.epm@uol.com.br, mrmachados@ecb.epm.br, and barbiericl@ecb.epm.br. Paulo Henrique da Costa Pinheiro, Associação de Ensino Superior e Tecnológico do Piauí, Rua Vitorino Ortiges Fernandes, 6123, 64057-100, Teresina, Piauí, Brazil, Telephone: 55-86-231-0700, Fax: 55-86- 231-0284, E-mail: ph.pinhe@bol.com.br.

Acknowledgments: We are grateful to Drs. Maurício Martins Rodrigues, Miriam Dorta, and Reinaldo Salomão for providing the human sera. We also thank Dr. Lindsay Pirrit for a critical reading of the manuscript.

Financial support: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and the Fundação de Amparo à Pesquisa do Estado de Sao Paulo of Brazil.

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Author Notes

Reprint requests: Clara Lúcia Barbiéri, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 60 Andar, 04023-062, São Paulo, SP, Brazil.
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