Cysticercosis Immunodiagnosis Using 18- and 14-Kilodalton Proteins from Taenia crassiceps Cysticercus Antigens Obtained by Immunoaffinity Ch
http://www.100md.com
微生物临床杂志 2005年第7期
Laboratorio de Imunologia, Faculdade de Ciências Farmacêuticas, Universidade de So Pulo, So Paulo
Laboratorio de Imunopatologia do Instituto Butantan, So Paulo
Departamento de Neurologia, Faculdade de Medicina de Ribeiro Preto, Universidade de So Paulo, Ribeiro Preto
Faculdade de Medicina, Universidade de So Paulo, So Paulo
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de So Paulo, Pirassununga
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
ABSTRACT
Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18- and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.
INTRODUCTION
Neurocysticercosis (NC) is caused by Taenia solium cysticerci in the central nervous system. Serological tests are helpful for the precise diagnosis because they confirm or supplement clinical and laboratorial diagnosis based on brain image investigation (12).
Although several serological methods have been evaluated to date, these tests still present problems. False-negative results can be obtained in cerebrospinal fluid (CSF) and serum samples from proven NC patients, and false-positive results have been reported for patients with other pathologies, particularly other parasitic diseases (11), and even for healthy individuals (1, 2). The detection of serum antibodies is impaired by cross-reactivity with other parasites, mainly when crude antigens are used. These data point out a need for the use of purified preparations to circumvent these problems.
Glycoprotein fractions obtained from T. solium cysticerci antigen by lentil-lectin (Lens culinaris) affinity chromatography were considered efficient for the serological diagnosis of NC (8, 17, 23). However, its use has been limited due to the assay technology, high cost, and complexity and length of the antigen purification process. Fractions with molecular masses below 15 kDa purified by affinity chromatography with anti-T. solium monoclonal antibody (MAb) specifically detected anti-T. solium antibodies in samples from NC patients (4, 12). The limited source of T. solium cysticerci hampers the large-scale production of specific antigens by these purification methods (24).
More recently, the use of recombinant proteins or even synthetic peptides from T. solium has been reported, and investigations are under way (5, 9, 10). Probably due to the complexity of the immune response in NC patients, a mixture of several specific and well-characterized proteins will give the desired levels of sensitivity and specificity.
On the other hand, the method for obtaining antigenic extracts from Taenia crassiceps cysticerci and their cross-reactivity with T. solium cysticerci antigens (13, 15, 27, 28) made them an interesting alternative antigen source for diagnosis (2, 21, 22) and immunological investigation of cysticercosis (3, 7, 18).
Vesicular fluid of T. crassiceps has been efficiently used in the diagnosis of cysticercosis, and the 18- and 14-kDa fractions from T. crassiceps have been considered specific for the immunodiagnosis of NC using an immunoblotting assay (1). High-molecular-weight peptides have been associated with cross-reactivity when human (1) and swine (21) serum samples were assayed.
Purified proteins from T. crassiceps antigens and their use in a simple test, such as the enzyme-linked immunosorbent assay (ELISA) format, may contribute to the improvement of the specificity of immunological tests applied for clinical diagnostic and surveillance studies of human and pig cysticercosis infection.
In this study, we report a simple method for the purification of native specific proteins of T. crassiceps cysticerci antigens, using two anti-T. crassiceps MAbs selected from a panel of MAbs cross-reacting with T. solium and T. crassiceps antigens in an ELISA to detect antibodies in CSF and serum samples from NC patients.
MATERIALS AND METHODS
Samples. Serum and CSF samples were obtained from patients attending the Faculty of Medicine Hospital at the University of So Paulo, campi So Paulo and Ribeiro Preto, Brazil. Twenty-three CSF and 20 serum samples from patients with NC were used. These patients had NC diagnosis confirmed by imaging exam (computed tomography and/or magnetic resonance imaging) and clinical and immunological data. Additionally, 9 CSF samples from patients with clinical findings and positive immunological tests for NC were also tested (Table 1).
The CSF control group (CG) consisted of 112 CSF samples obtained from patients with other neurological disorders (OND). Forty-two of these CSF samples did not show laboratory alteration and had the diagnosis of meningitis excluded (noninflammatory CSF), while 70 were inflammatory CSF, including viral or bacterial meningitis (19 samples), neurosyphilis (6 samples), cytomegalovirus meningoencephalitis (4 samples), neurotoxoplasmosis (1 sample), human T-cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis (1 sample), and human immunodeficiency virus (HIV)-associated meningoencephalitis (39 samples) (Table 2).
The CG consisted of 194 serum samples from apparently healthy individuals and 46 samples from patients with other parasitic diseases, including 13 intestinal parasites (Giardia lamblia [2 samples], Entamoeba histolytica [3 samples], Ascaris lumbricoides [2 samples], and Strongyloides stercoralis [6 samples]) and 33 systemic parasites (hydatidosis, Echinococcus granulosus [30 samples], and schistosomiasis, Schistosoma mansoni [3 samples]) (Table 2).
This study was approved by the Ethics Committee for the Analysis of Research Projects of the FCF/USP (approval 188/2003) and complied with Resolution 196/96 of the National Health Council of the Brazilian Ministry of Health.
Experimental animals. Animal manipulations were approved by the Ethics Committee for Experimental Animals of the FCF/USP (project 13/2003), adopted by the Brazilian Committee for Experimental Animals.
Parasites and antigens. T. crassiceps and T. solium cysts were obtained as described by Espíndola et al. (7). Four different antigen preparations were used: two from T. crassiceps cysticerci and two from T. solium cysticerci. Phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.) was added to each preparation, at a final concentration of 0.4 mM, to inhibit protease activity. Excretory/secretory T. crassiceps (ES-T. crassiceps), vesicular fluid of T. crassiceps (VF-T. crassiceps), or total T. solium (T-T. solium) antigens were produced according to the method of Espíndola et al. (7). Vesicular fluid of T. solium (VF-T. solium) was prepared from intact cysticerci of T. solium. After at least 10 washes with phosphate-buffered saline (PBS, 0.01 M; 0.0075 M Na2HPO4, 0.025 M NaH2PO4, 0.15 M NaCl, pH 7.2), vesicular fluid was aspirated from the cysts with a syringe coupled with a 13- by 0.4-mm needle. The fluid was centrifuged at 15,000 x g for 60 min at 4°C. The supernatant was sonicated (at 20 kHz and 1 mA, for four 30-s periods in an ice bath) and then centrifuged at 15,000 x g for 60 min at 4°C. The supernatant was collected corresponding to the VF-T. solium antigen.
Monoclonal antibody production. BALB/c mice were immunized with each antigenic preparation (ES-T. crassiceps, VF-T. crassiceps, VF-T. solium, and T-T. solium), and MAbs were obtained as described by Espíndola et al. (7). The MAb isotypes were determined using a commercial kit (mouse monoclonal antibody isotyping reagents; Sigma).
Antigen purification by immunoaffinity chromatography. MAbs were coupled to CNBr-Sepharose 4B according to the manufacturer's instructions (Amershan Pharmacia Biotech, Piscataway, N.J.), using 10 mg of anti-VF-T. crassiceps antigen MAb or anti-ES-T. crassiceps antigen MAb per 3 g of CNBr-Sepharose 4B. A total of 14 mg of protein from VF-T. crassiceps antigen was applied to the column. Elution of unbound protein was monitored by UV monitor (Bio-Rad Laboratories, Inc., Hercules, CA). After column washes with borate/saline buffer (0.1 M H3BO3, 0.1 M Na2B4O7, and 1.0 M NaCl, pH 8.5, diluted 1:20 in 0.15 M NaCl), desorption buffer (0.2 M glycine and 0.15 M NaCl, pH 2.8) was applied to elute the bound protein. Fractions were immediately neutralized with 1.0 M Tris (Tris hydroxymethylaminoethane) solution, concentrated, dialyzed against borate/saline buffer (pH 8.5), using a YM-10 membrane (Millipore Corp.), and then stored at –20°C.
SDS-PAGE, IB, and ELISA. The nonpurified or MAb immunoaffinity chromatography-purified VF-T. crassiceps antigens were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by following the method described by Laemmli (14). The antigens were solubilized with sample buffer (0.01 M Tris-HCl, pH 6.8, containing 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol) at 100°C for 5 min and separated electrophoretically on a 15% polyacrylamide gel. For immunoblotting (IB) analysis, the separated antigens were electrophoretically transferred to a 0.22-μm-pore-size membrane of polyvinylidene difluoride (Millipore Corp., Bedford, Mass.). The membranes were blocked by treatment with 5% skim milk (Molico skim milk; Nestle, Araatuba, SP, Brazil) in PBS for 2 h, washed in PBS containing 0.05% Tween 20 (Merck, Schudart, Munich, Germany), and then incubated for 18 h at 4°C with MAbs diluted 1:1,000 or sera or CSF diluted 1:50 and 1:10, respectively, in 1% skim milk in PBS. After further washes, strips were incubated for 1 h with goat anti-human immunoglobulin G (IgG)-biotin/avidin peroxidase (Sigma) or goat anti-mouse IgG-alkaline phosphatase (Bio-Rad Laboratories, Inc.) conjugates. After additional washes, the antigen-antibody complexes were developed by incubation with an appropriate substrate: 4-chloro-1-naphthol (Sigma) predissolved in methanol (20% of the volume) and then diluted to 0.05% with Tris-buffered saline (0.01 M Tris, 0.15 M NaCl, pH 7.4) containing 0.06% H2O2 (for the peroxidase conjugate) or 0.01% 5-bromo-4-chloro-3-indolylphosphate predissolved in N,N-dimethyl formamide (Sigma) and 0.02% nitroblue tetrazolium (Sigma) predissolved in 70% N,N-dimethyl formamide and then diluted in 0.01 M NaHCO3 and 0.001 M MgCl2 (for the phosphatase conjugate). The conjugate-substrate control was performed by incubating the membrane with 1% skim milk and, after further washes, incubating the membrane with anti-mouse IgG-alkaline phosphatase conjugate and substrate.
ELISA was carried out using the immunoaffinity-purified fraction eluted from the MAb anti-VF-T. crassiceps column as the antigen. Each well of 96-well ELISA polystyrene high-binding plates (Costar Corning, Inc., Cambridge, Mass.) was coated with 100 μl of antigen (0.5 μg/ml) in 0.5 M carbonate-bicarbonate buffer (pH 9.6) for 18 h in a humidified chamber at 4°C. The wells were blocked for 1 h with 5% milk in PBS containing 0.05% Tween 20 and then incubated for 1 h with serum or CSF samples diluted 1:50 and 1:10, respectively. Peroxidase-conjugated goat anti-human IgG was added, and plates were incubated for 1 h. After each incubation step, the plates were washed using an automatic washer, with four cycles of saline (0.15 M NaCl) containing 0.05% Tween 20. ortho-Phenylenediamine (1 mg/ml) and H2O2 (1 μl/ml) diluted in 0.2 M citrate buffer (pH 5.0) were added (in the dark) as the chromogenic substrate, and plates were incubated for 20 min. The reactions were stopped by adding 100 μl of 2 M H2SO4. Color intensity was quantified using an ELISA plate reader (Diagnostics Pasteur, Strasburg-Schiltigheim, France) at 492 nm. All incubations were carried out at 37°C.
Statistical analysis. The cutoff values were determined using the receiver operating characteristic curve (Win Episcope 2.0). Comparisons in the median values among independent groups were performed using the nonparametric Kruskal-Wallis analysis of variance and Dunn's test. Analyses were made using Sigma Stat (Jandel Scientific Software). Significance was defined at the 5% level.
RESULTS
A total of eight MAbs (1 anti-ES-T. crassiceps, 1 anti-VF-T. crassiceps, 2 anti-VF-T. solium, and 4 anti-T-T. solium) obtained from four fusions cross-reacted with both T. solium and T. crassiceps antigens in an ELISA. In IB, these MAbs reacted with the 14-kDa protein from T-T. solium antigen and the 18- and 14-kDa proteins from VF-T. crassiceps antigen. All MAbs belong to the IgG isotype, one was IgG2b (anti-ES-T. crassiceps), and the others were IgG1 (Table 3).
From a total of 14.0 mg from VF-T. crassiceps antigen, 5.4 mg (38.5%) and 6.0 mg (43.0%) of purified fractions were obtained when anti-VF-T. crassiceps and anti-ES-T. crassiceps MAb columns, respectively, were used. These purified fractions presented relative molecular masses of 67, 18, and 14 kDa by SDS-PAGE (Fig. 1).
All MAbs (anti-ES-T. crassiceps, anti-VF-T. crassiceps, anti-VF-T. solium, and anti-T-T. solium) and serum or CSF samples from patients with NC recognized the 18- and 14-kDa proteins of VF-T. crassiceps purified antigen (Fig. 2). Although the 30-kDa protein was not observed by SDS-PAGE (Fig. 1), it was recognized by some (Fig. 2; see also Fig. 4). No reactivity was observed with the 67-kDa protein for any sample or MAb.
ELISA analysis of 23 CSF and 20 serum samples from patients with confirmed NC, using the T. crassiceps purified fraction as the antigen (ELISA-18/14), showed a sensitivity of 100%. All nine CSF samples with clinical findings and positive immunological tests were also positive by this assay (Fig. 3). The medians of absorbance to CSF and serum samples from patients with NC were 1.056 and 1.415, respectively.
ELISA-18/14 analysis of CSF samples from the 42 noninflammatory CSF controls showed a specificity of 100% (median of absorbance, 0.002). When 70 inflammatory CSF samples were included in the control group, the specificity was found to be 99.1% (confidence interval, 97.3% to 100%), and the median absorbance increased to 0.033 (Fig. 3). A single inflammatory CSF sample positive in the ELISA was from a patient with HIV-associated meningoencephalitis and showed strong reactivity in IB-18/14 (Fig. 4).
The specificity for the serum samples from 194 healthy individuals was 100%. When the 46 samples from patients with other parasitic diseases were included, the specificity was 98.7% (confidence interval, 97.3 to 100%). The median absorbances obtained for serum samples from healthy individuals, from patients with systemic parasitic diseases, and from patients with intestinal parasitic diseases were 0.130, 0.251, and 0.174, respectively. Three samples from patients with hydatidosis (ELISA-18/14 positive) were also positive in the ELISA with T. solium cysticerci antigens, and two of them were also positive by IB-18/14. Some CSF and serum samples with negative ELISA results were analyzed by IB-18/14 and found to be negative (Fig. 4).
DISCUSSION
In the present study, anti-T. crassiceps (anti-ES and anti-VF) MAbs selected by ELISA showed cross-reactivity with the T. solium antigen, and all anti-T. solium MAbs also presented cross-reactivity with the T. crassiceps antigen. This cross-reactivity confirms the presence of common antigenic determinants shared by the cysticerci, as reported before (2, 7, 13, 19, 27, 28). The reactivity of anti-T. crassiceps MAbs as well as of some polyclonal antibodies (CSF and serum samples from NC patients) toward the T. crassiceps antigen was abolished by treatment of the antigen with sodium periodate (data not shown), indicating that the chosen MAbs were specific for common immunodominant epitopes involved in the human immune response to cysticercus.
The reactivity of anti-T. crassiceps MAbs with T. solium and T. crassiceps antigenic fractions with relative molecular masses below 30 kDa and the specificity of these peptides in detecting antibodies in CSF and serum samples from patients with NC directed us to the purification of such peptides in the present study. Purified fractions of VF-T. crassiceps using MAb immunoaffinity chromatography corresponded to the native 18- and 14-kDa proteins. Immunoblot analyses showed that all anti-T. crassiceps and anti-T. solium MAbs, as well as CSF and serum samples from patients with NC, identified both purified proteins. The importance of the 18- and 14-kDa proteins from T. solium was also demonstrated by others (8, 26). Greene et al. (8) showed that the purified fractions from lentil lectin-bound glycoprotein antigen contain multiple subunits that have potential as diagnostic antigens, particularly the 14-kDa subunit. The authors also suggest that, since the subunits are glycoproteins, differences between them could be due to the amino acid chain lengths and/or posttranslational modifications.
The 30-kDa protein of the antigen purified by immunoaffinity was only identified by IB, probably due to the higher sensitivity of the immunological methods compared with staining techniques used in SDS-PAGE, including silver staining (data not shown). A protein with a similar molecular mass was observed in the excretory/secretory antigens after in vitro culture of T. crassiceps cysticerci (7).
The 67-kDa protein identified by SDS-PAGE (Fig. 1) was not recognized by MAbs or antibodies from CSF and serum samples from patients with NC. Although no reactivity was observed with the goat anti-mouse IgG-alkaline phosphatase conjugate (data not shown), the 67-kDa protein seems to be an artifact that appeared during the acidic desorption process and could be detectable only by SDS-PAGE, as reported by Kim et al. (12), using an anti-T. solium MAb.
The large amount and excellent fraction yield (38.5% and 43.0%) of 18- and 14-kDa purified peptides obtained from VF-T. crassiceps were reproducible in two batches using each of the MAbs (data not shown).
Using 18- and 14-kDa T. crassiceps purified proteins, we developed an immunodiagnostic method based on the ELISA system. The sensitivity of the test was 100% when 20 serum samples and 23 CSF samples from the NC group were assayed. For two patients with positive serum results, the paired CSF samples were negative for all antigens (VF-T. crassiceps, T. solium, and the 18- and 14-kDa proteins) by ELISA and IB. These two CSF samples were obtained after albendazole treatment of the patients, possibly during the calcification process. The calcification phase has been associated with a lower antibody concentration, especially in CSF, regardless of the antigen used (2, 13, 16, 29). Otherwise, five NC patients with ELISA-positive results for CSF samples presented negative results when paired serum samples were tested by ELISA with all antigens. However, very faint reactivity with the 30-, 18-, and 14-kDa proteins was observed by IB (data not shown). Three of the five negative serum samples were also negative by IB using VF-T. crassiceps and T-T. solium antigens, and the other two showed a very faint reactivity to the 14-kDa protein in the IB T. crassiceps and IB T. solium antigens (data not shown). The use of a larger quantity of purified antigen as well as another solid phase (e.g., dot blot) or the application of a lower cutoff would improve the sensitivity of ELISA-18/14 for these serum samples but may result in a lower specificity.
The ELISA-18/14 used on the control group showed 100% specificity for the noninflammatory CSF samples and 99.1% specificity when the inflammatory CSF samples were included. A significant difference was observed between absorbance results for CSF samples from the NC patients and CSF samples from the control group (P < 0.0001). In the control group, the single CSF sample that gave a positive result was from a patient with HIV-associated meningoencephalitis. This CSF sample was also strongly reactive for VF-T. crassiceps by IB and ELISA. We were unable to confirm the diagnosis of cysticercosis in this case because complementary clinical and neuroimaging data were not available. Detecting antibodies in CSF always seems to be specific unless immunoglobulins cross the blood-brain barrier during meningitis episodes, as reported with HIV infection (25).
The specificity for 194 serum samples in the control group (healthy individuals) was 100%, excluding serum samples from patients with hydatidosis. Three (10%) of the 30 serum samples from patients with hydatidosis were ELISA-18/14 positive (Fig. 3), and two of them were confirmed by IB-18/14 (Fig. 4). The specificity of the test including these samples was 98.7%, with a significant difference between the results for serum samples from patients with NC and the control group (P < 0.0001). Ishida et al. (11) reported 36 (77%) ELISA VF-T. crassiceps-positive results among 50 serum samples from patients with hydatidosis, but reactivity was confirmed in only 5 (10.9%) samples by IB using glycoproteins (18 to 14 kDa) from VF-T. crassiceps purified by affinity chromatography concanavalin A-Sepharose (20). Other authors have demonstrated the presence of common antigenic components of the cestodes Taenia and Echinococcus (6, 11). The 18- and 14-kDa proteins obtained in the present work were found to be specific for cysticercosis, and we are planning further studies to select those specific peptides from VF-T. crassiceps which are common to hydatid fluid from E. granulosus. It is possible that the conformation of the proteins should be highly important for either the immunogenicity or the antigenicity, and it is expected that more than one epitope is recognized by each individual, particularly in human cases, where immune responses are highly heterogeneous.
VF-T. crassiceps ELISA has shown a low specificity for serum samples, which indicates the need to perform confirmatory IB testing (1, 2). Bragazza et al. (1) described a 20% rate of false-positive results in 1,863 serum samples when a population from an area of cysticercosis endemicity was tested. By testing these samples by IB, the authors demonstrated that proteins of >30 kDa from VF-T. crassiceps were responsible for the unspecific reactions or cross-reactions with other parasitic infectious diseases. The cross-reactivities affecting the specificity index should be better evaluated in further clinical epidemiological studies using serum samples from different regions, including the most frequent heterologous parasitosis and other infections in each respective area.
In the present study, the use of 18- and 14-kDa T. crassiceps purified proteins in ELISA demonstrated high specificity and sensitivity for the diagnosis of human cysticercosis. Future analysis should validate its usefulness for screening seropositive individuals in immunoepidemiological studies with human and swine serum samples.
ACKNOWLEDGMENTS
This work was supported by FAPESP (grants 00/00141-3 and 02-012061-0 to A.J.V.), by CNPq (proc. 300937/2003-2 to O.M.T.), by a FAPESP fellowship to N.M.E. (03/10747-4), and by a CNPq fellowship to A.H.I. (107974/2003-7) and J.M.P.
We are indebted to Lucia Martins Teixeira and Walter Martin Oelemann from UFRJ, RJ, for comments on and review of the manuscript; to Fatima Tiecher, Instituto de Pesquisas Biologicas (Porto Alegre, RS), for providing serum samples from hydatidosis; and to Maria Carmen Arroyo Sanchez from Instituto de Medicina Tropical, USP, SP, Brazil, for statistical analysis.
REFERENCES
Bragazza, L. M., A. J. Vaz, A. D. C. Passos, O. M. Takayanagui, P. M. Nakamura, N. M. Espíndola, A. X. Pardini, and E. C. Bueno. 2002. Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens. Rev. Inst. Med. Trop. So Paulo 44:7-12.
Bueno, E. C., A. J. Vaz, L. R. Machado, J. A. Livramento, and S. R. Mielle. 2000. Specific Taenia crassiceps and Taenia solium antigenic peptides for neurocysticercosis immunodiagnosis using serum samples. J. Clin. Microbiol. 38:146-151.
Bueno, E. C., A. J. Vaz, L. R. Machado, J. A. Livramento, S. L. M. ávila, and A. W. Ferreira. 2001. Antigen-specific suppression of cultured lymphocytes from patients with neurocysticercosis. Clin. Exp. Immunol. 126:304-310.
Cho, S., S. Kim, S. Kang, and Y. Kong. 1988. Biochemical properties of a purified protein in cystic fluid of Taenia solium metacestodes. Korean J. Parasitol. 26:87-94.
Chung, J. Y., Y. Y. Bahk, S. Huh, S. Y. Kang, Y. Kong, and S. Y. Cho. 1999. A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis. J. Infect. Dis. 180:1307-1315.
Coltorti, E., and G. Cammarieri. 1993. Inmunodiagnostico de hidatidosis: evaluacion de antígenos de líquido hidatídico y de líquido vesicular de cisticerco de Taenia crassiceps. Rev. Inst. Med. Trop. So Paulo 35:155-162.
Espíndola, N. M., A. J. Vaz, A. X. Pardini, and I. Fernandes. 2002. Excretory/secretory antigens (ES) from in-vitro cultures of Taenia crassiceps cysticerci, and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis. Ann. Trop. Med. Parasitol. 96:361-368.
Greene, R. M., P. P. Wilkins, and V. C. W. Tsang. 1999. Diagnostic glycoproteins of Taenia solium cysts share homologous 14- and 18-kDa subunits. Mol. Biochem. Parasitol. 99:257-261.
Greene, R. M., K. Hancock, P. P. Wilkins, and V. C. W. Tsang. 2000. Taenia solium: molecular cloning and serologic evaluation of 14- and 18-kDa related, diagnostic antigens. J. Parasitol. 86:1001-1007.
Hancock, K., S. Pattabhi, R. M. Greene, M. L. Yushak, F. Williams, A. Khan, J. W. Priest, M. Z. Levine, and V. C. W. Tsang. 2004. Characterization and cloning of GP50, a Taenia solium antigen diagnostic for cysticercosis. Mol. Biochem. Parasitol. 133:115-124.
Ishida, M. M. I., G. Rubinsky-Elefant, A. W. Ferreira, S. Hoshino-Shimisu, and A. J. Vaz. 2003. Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals. Acta Trop. 89:73-84.
Kim, S., S. Kang, and S. Cho. 1986. Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization. Korean J. Parasitol. 24:145-158.
Kunz, J., B. Kalinna, V. Watschke, and E. Geyer. 1989. Taenia crassiceps metacestode vesicular fluid antigens shared with the Taenia solium larval stage and reactive with serum antibodies from patients with neurocysticercosis. Zentbl. Bakteriol. 271:510-520.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Larralde, C., J. Sotelo, R. M. Montoya, G. Palencia, A. Padilla, T. Govezensky, M. L. Diaz, and E. Sciutto. 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium. Arch. Pathol. Lab. Med. 114:926-928.
Michault, A., B. Riviere, P. Fressy, J. P. Laporte, G. Bertil, and C. Mignard. 1990. Apport de l'enzime-linked immunoelectrotransfer blot assay au diagnostic de la neurocysticercose humanaine. Pathol. Biol. 38:119-125.
Obregon-Henao, A., D. L. Gil, D. I. Gomez, F. Sanzon, J. M. Teale, and B. I. Restrepo. 2001. The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kD. Mol. Biochem. Parasitol. 144:209-215.
Pardini, A. X., A. J. Vaz, L. R. Machado, and J. A. Livramento. 2001. Cysticercus antigens in cerebrospinal fluid samples from patients with neurocysticercosis. J. Clin. Microbiol. 39:3368-3372.
Pardini, A. X., R. H. Peralta, A. J. Vaz, L. R. Machado, and J. M. Peralta. 2002. Use of Taenia crassiceps cysticercus antigen preparations for detection of antibodies in cerebrospinal fluid samples from patients with neurocysticercosis (T. solium). Clin. Diagn. Lab. Immunol. 9:190-193.
Peralta, R. H., A. J. Vaz, A. X. Pardini, H. W. Macedo, L. R. Machado, S. G. De Simone, and J. M. Peralta. 2002. Evaluation of an antigen from Taenia crassiceps cysticercus for the serodiagnosis of neurocysticercosis. Acta Trop. 83:159-168.
Pinto, P. S. A., A. J. Vaz, P. M. L. Germano, and P. M. Nakamura. 2000. Performance of the ELISA test for swine cysticercosis using antigens of T. solium and T. crassiceps cysticerci. Vet. Parasitol. 88:127-130.
Pinto, P. S. A., A. J. Vaz, P. M. Nakamura, and P. M. L. Germano. 2001. Immunoblot analysis using antigen from T. crassiceps cysticerci in the diagnosis of swine cysticercosis. Bol. Chil. Parasitol. 56:36-42.
Restrepo, B. I., A. Obregon-Henao, M. Mesa, D. L. Gil, B. L. Ortiz, J. S. Mejía, G. E. Villota, F. Sanzon, and J. M. Teale. 2000. Characterization of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int. J. Parasitol. 30:689-696.
Sako, Y., M. Nakao, T. Ikejima, X. Z. Piao, K. Nakaya, and A. Ito. 2000. Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes. J. Clin. Microbiol. 38:4439-4444.
Singer, E. J., K. Syndulko, B. Fahy-Chandon, P. Schimid, A. Conrad, and W. W. Tourtellotte. 1994. Intrathecal IgG synthesis and albumin leakage are increased in subjects with HIV-1 neurologic disease. J. Acquir. Immune Defic. Syndr. 7:265-271.
Tsang, V. C. W., J. A. Brand, and A. E. Boyer. 1989. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J. Infect. Dis. 159:50-59.
Vaz, A. J., P. M. Nakamura, C. C. Barreto, A. W. Ferreira, and J. A. Livramento. 1997. Immunodiagnosis of human neurocysticercosis: use of heterologous antigenic particles (Cysticercus longicollis) in indirect immunofluorescence test. Serodiagn. Immunother. Infect. Dis. 8:157-161.
Vaz, A. J., C. M. Nunes, R. M. F. Piazza, J. A. Livramento, M. V. Silva, P. M. Nakamura, and A. W. Ferreira. 1997. Immunoblot with cerebrospinal fluid from patients with neurocysticercosis using antigen from cysticerci of Taenia solium and Taenia crassiceps. Am. J. Trop. Med. Hyg. 57:354-357.
Wilson, M. R., R. T. Bryan, J. A. Fried, D. A. Ware, P. M. Schantz, J. B. Pilcher, and V. C. W. Tsang. 1991. Clinical evolution of the cysticercosis enzyme-linked immunoelectrotransfer blot in patients with neurocysticercosis. J. Infect. Dis. 164:1007-1009.(Noeli Maria Espíndola, Al)
Laboratorio de Imunopatologia do Instituto Butantan, So Paulo
Departamento de Neurologia, Faculdade de Medicina de Ribeiro Preto, Universidade de So Paulo, Ribeiro Preto
Faculdade de Medicina, Universidade de So Paulo, So Paulo
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de So Paulo, Pirassununga
Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
ABSTRACT
Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18- and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.
INTRODUCTION
Neurocysticercosis (NC) is caused by Taenia solium cysticerci in the central nervous system. Serological tests are helpful for the precise diagnosis because they confirm or supplement clinical and laboratorial diagnosis based on brain image investigation (12).
Although several serological methods have been evaluated to date, these tests still present problems. False-negative results can be obtained in cerebrospinal fluid (CSF) and serum samples from proven NC patients, and false-positive results have been reported for patients with other pathologies, particularly other parasitic diseases (11), and even for healthy individuals (1, 2). The detection of serum antibodies is impaired by cross-reactivity with other parasites, mainly when crude antigens are used. These data point out a need for the use of purified preparations to circumvent these problems.
Glycoprotein fractions obtained from T. solium cysticerci antigen by lentil-lectin (Lens culinaris) affinity chromatography were considered efficient for the serological diagnosis of NC (8, 17, 23). However, its use has been limited due to the assay technology, high cost, and complexity and length of the antigen purification process. Fractions with molecular masses below 15 kDa purified by affinity chromatography with anti-T. solium monoclonal antibody (MAb) specifically detected anti-T. solium antibodies in samples from NC patients (4, 12). The limited source of T. solium cysticerci hampers the large-scale production of specific antigens by these purification methods (24).
More recently, the use of recombinant proteins or even synthetic peptides from T. solium has been reported, and investigations are under way (5, 9, 10). Probably due to the complexity of the immune response in NC patients, a mixture of several specific and well-characterized proteins will give the desired levels of sensitivity and specificity.
On the other hand, the method for obtaining antigenic extracts from Taenia crassiceps cysticerci and their cross-reactivity with T. solium cysticerci antigens (13, 15, 27, 28) made them an interesting alternative antigen source for diagnosis (2, 21, 22) and immunological investigation of cysticercosis (3, 7, 18).
Vesicular fluid of T. crassiceps has been efficiently used in the diagnosis of cysticercosis, and the 18- and 14-kDa fractions from T. crassiceps have been considered specific for the immunodiagnosis of NC using an immunoblotting assay (1). High-molecular-weight peptides have been associated with cross-reactivity when human (1) and swine (21) serum samples were assayed.
Purified proteins from T. crassiceps antigens and their use in a simple test, such as the enzyme-linked immunosorbent assay (ELISA) format, may contribute to the improvement of the specificity of immunological tests applied for clinical diagnostic and surveillance studies of human and pig cysticercosis infection.
In this study, we report a simple method for the purification of native specific proteins of T. crassiceps cysticerci antigens, using two anti-T. crassiceps MAbs selected from a panel of MAbs cross-reacting with T. solium and T. crassiceps antigens in an ELISA to detect antibodies in CSF and serum samples from NC patients.
MATERIALS AND METHODS
Samples. Serum and CSF samples were obtained from patients attending the Faculty of Medicine Hospital at the University of So Paulo, campi So Paulo and Ribeiro Preto, Brazil. Twenty-three CSF and 20 serum samples from patients with NC were used. These patients had NC diagnosis confirmed by imaging exam (computed tomography and/or magnetic resonance imaging) and clinical and immunological data. Additionally, 9 CSF samples from patients with clinical findings and positive immunological tests for NC were also tested (Table 1).
The CSF control group (CG) consisted of 112 CSF samples obtained from patients with other neurological disorders (OND). Forty-two of these CSF samples did not show laboratory alteration and had the diagnosis of meningitis excluded (noninflammatory CSF), while 70 were inflammatory CSF, including viral or bacterial meningitis (19 samples), neurosyphilis (6 samples), cytomegalovirus meningoencephalitis (4 samples), neurotoxoplasmosis (1 sample), human T-cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis (1 sample), and human immunodeficiency virus (HIV)-associated meningoencephalitis (39 samples) (Table 2).
The CG consisted of 194 serum samples from apparently healthy individuals and 46 samples from patients with other parasitic diseases, including 13 intestinal parasites (Giardia lamblia [2 samples], Entamoeba histolytica [3 samples], Ascaris lumbricoides [2 samples], and Strongyloides stercoralis [6 samples]) and 33 systemic parasites (hydatidosis, Echinococcus granulosus [30 samples], and schistosomiasis, Schistosoma mansoni [3 samples]) (Table 2).
This study was approved by the Ethics Committee for the Analysis of Research Projects of the FCF/USP (approval 188/2003) and complied with Resolution 196/96 of the National Health Council of the Brazilian Ministry of Health.
Experimental animals. Animal manipulations were approved by the Ethics Committee for Experimental Animals of the FCF/USP (project 13/2003), adopted by the Brazilian Committee for Experimental Animals.
Parasites and antigens. T. crassiceps and T. solium cysts were obtained as described by Espíndola et al. (7). Four different antigen preparations were used: two from T. crassiceps cysticerci and two from T. solium cysticerci. Phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo.) was added to each preparation, at a final concentration of 0.4 mM, to inhibit protease activity. Excretory/secretory T. crassiceps (ES-T. crassiceps), vesicular fluid of T. crassiceps (VF-T. crassiceps), or total T. solium (T-T. solium) antigens were produced according to the method of Espíndola et al. (7). Vesicular fluid of T. solium (VF-T. solium) was prepared from intact cysticerci of T. solium. After at least 10 washes with phosphate-buffered saline (PBS, 0.01 M; 0.0075 M Na2HPO4, 0.025 M NaH2PO4, 0.15 M NaCl, pH 7.2), vesicular fluid was aspirated from the cysts with a syringe coupled with a 13- by 0.4-mm needle. The fluid was centrifuged at 15,000 x g for 60 min at 4°C. The supernatant was sonicated (at 20 kHz and 1 mA, for four 30-s periods in an ice bath) and then centrifuged at 15,000 x g for 60 min at 4°C. The supernatant was collected corresponding to the VF-T. solium antigen.
Monoclonal antibody production. BALB/c mice were immunized with each antigenic preparation (ES-T. crassiceps, VF-T. crassiceps, VF-T. solium, and T-T. solium), and MAbs were obtained as described by Espíndola et al. (7). The MAb isotypes were determined using a commercial kit (mouse monoclonal antibody isotyping reagents; Sigma).
Antigen purification by immunoaffinity chromatography. MAbs were coupled to CNBr-Sepharose 4B according to the manufacturer's instructions (Amershan Pharmacia Biotech, Piscataway, N.J.), using 10 mg of anti-VF-T. crassiceps antigen MAb or anti-ES-T. crassiceps antigen MAb per 3 g of CNBr-Sepharose 4B. A total of 14 mg of protein from VF-T. crassiceps antigen was applied to the column. Elution of unbound protein was monitored by UV monitor (Bio-Rad Laboratories, Inc., Hercules, CA). After column washes with borate/saline buffer (0.1 M H3BO3, 0.1 M Na2B4O7, and 1.0 M NaCl, pH 8.5, diluted 1:20 in 0.15 M NaCl), desorption buffer (0.2 M glycine and 0.15 M NaCl, pH 2.8) was applied to elute the bound protein. Fractions were immediately neutralized with 1.0 M Tris (Tris hydroxymethylaminoethane) solution, concentrated, dialyzed against borate/saline buffer (pH 8.5), using a YM-10 membrane (Millipore Corp.), and then stored at –20°C.
SDS-PAGE, IB, and ELISA. The nonpurified or MAb immunoaffinity chromatography-purified VF-T. crassiceps antigens were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by following the method described by Laemmli (14). The antigens were solubilized with sample buffer (0.01 M Tris-HCl, pH 6.8, containing 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol) at 100°C for 5 min and separated electrophoretically on a 15% polyacrylamide gel. For immunoblotting (IB) analysis, the separated antigens were electrophoretically transferred to a 0.22-μm-pore-size membrane of polyvinylidene difluoride (Millipore Corp., Bedford, Mass.). The membranes were blocked by treatment with 5% skim milk (Molico skim milk; Nestle, Araatuba, SP, Brazil) in PBS for 2 h, washed in PBS containing 0.05% Tween 20 (Merck, Schudart, Munich, Germany), and then incubated for 18 h at 4°C with MAbs diluted 1:1,000 or sera or CSF diluted 1:50 and 1:10, respectively, in 1% skim milk in PBS. After further washes, strips were incubated for 1 h with goat anti-human immunoglobulin G (IgG)-biotin/avidin peroxidase (Sigma) or goat anti-mouse IgG-alkaline phosphatase (Bio-Rad Laboratories, Inc.) conjugates. After additional washes, the antigen-antibody complexes were developed by incubation with an appropriate substrate: 4-chloro-1-naphthol (Sigma) predissolved in methanol (20% of the volume) and then diluted to 0.05% with Tris-buffered saline (0.01 M Tris, 0.15 M NaCl, pH 7.4) containing 0.06% H2O2 (for the peroxidase conjugate) or 0.01% 5-bromo-4-chloro-3-indolylphosphate predissolved in N,N-dimethyl formamide (Sigma) and 0.02% nitroblue tetrazolium (Sigma) predissolved in 70% N,N-dimethyl formamide and then diluted in 0.01 M NaHCO3 and 0.001 M MgCl2 (for the phosphatase conjugate). The conjugate-substrate control was performed by incubating the membrane with 1% skim milk and, after further washes, incubating the membrane with anti-mouse IgG-alkaline phosphatase conjugate and substrate.
ELISA was carried out using the immunoaffinity-purified fraction eluted from the MAb anti-VF-T. crassiceps column as the antigen. Each well of 96-well ELISA polystyrene high-binding plates (Costar Corning, Inc., Cambridge, Mass.) was coated with 100 μl of antigen (0.5 μg/ml) in 0.5 M carbonate-bicarbonate buffer (pH 9.6) for 18 h in a humidified chamber at 4°C. The wells were blocked for 1 h with 5% milk in PBS containing 0.05% Tween 20 and then incubated for 1 h with serum or CSF samples diluted 1:50 and 1:10, respectively. Peroxidase-conjugated goat anti-human IgG was added, and plates were incubated for 1 h. After each incubation step, the plates were washed using an automatic washer, with four cycles of saline (0.15 M NaCl) containing 0.05% Tween 20. ortho-Phenylenediamine (1 mg/ml) and H2O2 (1 μl/ml) diluted in 0.2 M citrate buffer (pH 5.0) were added (in the dark) as the chromogenic substrate, and plates were incubated for 20 min. The reactions were stopped by adding 100 μl of 2 M H2SO4. Color intensity was quantified using an ELISA plate reader (Diagnostics Pasteur, Strasburg-Schiltigheim, France) at 492 nm. All incubations were carried out at 37°C.
Statistical analysis. The cutoff values were determined using the receiver operating characteristic curve (Win Episcope 2.0). Comparisons in the median values among independent groups were performed using the nonparametric Kruskal-Wallis analysis of variance and Dunn's test. Analyses were made using Sigma Stat (Jandel Scientific Software). Significance was defined at the 5% level.
RESULTS
A total of eight MAbs (1 anti-ES-T. crassiceps, 1 anti-VF-T. crassiceps, 2 anti-VF-T. solium, and 4 anti-T-T. solium) obtained from four fusions cross-reacted with both T. solium and T. crassiceps antigens in an ELISA. In IB, these MAbs reacted with the 14-kDa protein from T-T. solium antigen and the 18- and 14-kDa proteins from VF-T. crassiceps antigen. All MAbs belong to the IgG isotype, one was IgG2b (anti-ES-T. crassiceps), and the others were IgG1 (Table 3).
From a total of 14.0 mg from VF-T. crassiceps antigen, 5.4 mg (38.5%) and 6.0 mg (43.0%) of purified fractions were obtained when anti-VF-T. crassiceps and anti-ES-T. crassiceps MAb columns, respectively, were used. These purified fractions presented relative molecular masses of 67, 18, and 14 kDa by SDS-PAGE (Fig. 1).
All MAbs (anti-ES-T. crassiceps, anti-VF-T. crassiceps, anti-VF-T. solium, and anti-T-T. solium) and serum or CSF samples from patients with NC recognized the 18- and 14-kDa proteins of VF-T. crassiceps purified antigen (Fig. 2). Although the 30-kDa protein was not observed by SDS-PAGE (Fig. 1), it was recognized by some (Fig. 2; see also Fig. 4). No reactivity was observed with the 67-kDa protein for any sample or MAb.
ELISA analysis of 23 CSF and 20 serum samples from patients with confirmed NC, using the T. crassiceps purified fraction as the antigen (ELISA-18/14), showed a sensitivity of 100%. All nine CSF samples with clinical findings and positive immunological tests were also positive by this assay (Fig. 3). The medians of absorbance to CSF and serum samples from patients with NC were 1.056 and 1.415, respectively.
ELISA-18/14 analysis of CSF samples from the 42 noninflammatory CSF controls showed a specificity of 100% (median of absorbance, 0.002). When 70 inflammatory CSF samples were included in the control group, the specificity was found to be 99.1% (confidence interval, 97.3% to 100%), and the median absorbance increased to 0.033 (Fig. 3). A single inflammatory CSF sample positive in the ELISA was from a patient with HIV-associated meningoencephalitis and showed strong reactivity in IB-18/14 (Fig. 4).
The specificity for the serum samples from 194 healthy individuals was 100%. When the 46 samples from patients with other parasitic diseases were included, the specificity was 98.7% (confidence interval, 97.3 to 100%). The median absorbances obtained for serum samples from healthy individuals, from patients with systemic parasitic diseases, and from patients with intestinal parasitic diseases were 0.130, 0.251, and 0.174, respectively. Three samples from patients with hydatidosis (ELISA-18/14 positive) were also positive in the ELISA with T. solium cysticerci antigens, and two of them were also positive by IB-18/14. Some CSF and serum samples with negative ELISA results were analyzed by IB-18/14 and found to be negative (Fig. 4).
DISCUSSION
In the present study, anti-T. crassiceps (anti-ES and anti-VF) MAbs selected by ELISA showed cross-reactivity with the T. solium antigen, and all anti-T. solium MAbs also presented cross-reactivity with the T. crassiceps antigen. This cross-reactivity confirms the presence of common antigenic determinants shared by the cysticerci, as reported before (2, 7, 13, 19, 27, 28). The reactivity of anti-T. crassiceps MAbs as well as of some polyclonal antibodies (CSF and serum samples from NC patients) toward the T. crassiceps antigen was abolished by treatment of the antigen with sodium periodate (data not shown), indicating that the chosen MAbs were specific for common immunodominant epitopes involved in the human immune response to cysticercus.
The reactivity of anti-T. crassiceps MAbs with T. solium and T. crassiceps antigenic fractions with relative molecular masses below 30 kDa and the specificity of these peptides in detecting antibodies in CSF and serum samples from patients with NC directed us to the purification of such peptides in the present study. Purified fractions of VF-T. crassiceps using MAb immunoaffinity chromatography corresponded to the native 18- and 14-kDa proteins. Immunoblot analyses showed that all anti-T. crassiceps and anti-T. solium MAbs, as well as CSF and serum samples from patients with NC, identified both purified proteins. The importance of the 18- and 14-kDa proteins from T. solium was also demonstrated by others (8, 26). Greene et al. (8) showed that the purified fractions from lentil lectin-bound glycoprotein antigen contain multiple subunits that have potential as diagnostic antigens, particularly the 14-kDa subunit. The authors also suggest that, since the subunits are glycoproteins, differences between them could be due to the amino acid chain lengths and/or posttranslational modifications.
The 30-kDa protein of the antigen purified by immunoaffinity was only identified by IB, probably due to the higher sensitivity of the immunological methods compared with staining techniques used in SDS-PAGE, including silver staining (data not shown). A protein with a similar molecular mass was observed in the excretory/secretory antigens after in vitro culture of T. crassiceps cysticerci (7).
The 67-kDa protein identified by SDS-PAGE (Fig. 1) was not recognized by MAbs or antibodies from CSF and serum samples from patients with NC. Although no reactivity was observed with the goat anti-mouse IgG-alkaline phosphatase conjugate (data not shown), the 67-kDa protein seems to be an artifact that appeared during the acidic desorption process and could be detectable only by SDS-PAGE, as reported by Kim et al. (12), using an anti-T. solium MAb.
The large amount and excellent fraction yield (38.5% and 43.0%) of 18- and 14-kDa purified peptides obtained from VF-T. crassiceps were reproducible in two batches using each of the MAbs (data not shown).
Using 18- and 14-kDa T. crassiceps purified proteins, we developed an immunodiagnostic method based on the ELISA system. The sensitivity of the test was 100% when 20 serum samples and 23 CSF samples from the NC group were assayed. For two patients with positive serum results, the paired CSF samples were negative for all antigens (VF-T. crassiceps, T. solium, and the 18- and 14-kDa proteins) by ELISA and IB. These two CSF samples were obtained after albendazole treatment of the patients, possibly during the calcification process. The calcification phase has been associated with a lower antibody concentration, especially in CSF, regardless of the antigen used (2, 13, 16, 29). Otherwise, five NC patients with ELISA-positive results for CSF samples presented negative results when paired serum samples were tested by ELISA with all antigens. However, very faint reactivity with the 30-, 18-, and 14-kDa proteins was observed by IB (data not shown). Three of the five negative serum samples were also negative by IB using VF-T. crassiceps and T-T. solium antigens, and the other two showed a very faint reactivity to the 14-kDa protein in the IB T. crassiceps and IB T. solium antigens (data not shown). The use of a larger quantity of purified antigen as well as another solid phase (e.g., dot blot) or the application of a lower cutoff would improve the sensitivity of ELISA-18/14 for these serum samples but may result in a lower specificity.
The ELISA-18/14 used on the control group showed 100% specificity for the noninflammatory CSF samples and 99.1% specificity when the inflammatory CSF samples were included. A significant difference was observed between absorbance results for CSF samples from the NC patients and CSF samples from the control group (P < 0.0001). In the control group, the single CSF sample that gave a positive result was from a patient with HIV-associated meningoencephalitis. This CSF sample was also strongly reactive for VF-T. crassiceps by IB and ELISA. We were unable to confirm the diagnosis of cysticercosis in this case because complementary clinical and neuroimaging data were not available. Detecting antibodies in CSF always seems to be specific unless immunoglobulins cross the blood-brain barrier during meningitis episodes, as reported with HIV infection (25).
The specificity for 194 serum samples in the control group (healthy individuals) was 100%, excluding serum samples from patients with hydatidosis. Three (10%) of the 30 serum samples from patients with hydatidosis were ELISA-18/14 positive (Fig. 3), and two of them were confirmed by IB-18/14 (Fig. 4). The specificity of the test including these samples was 98.7%, with a significant difference between the results for serum samples from patients with NC and the control group (P < 0.0001). Ishida et al. (11) reported 36 (77%) ELISA VF-T. crassiceps-positive results among 50 serum samples from patients with hydatidosis, but reactivity was confirmed in only 5 (10.9%) samples by IB using glycoproteins (18 to 14 kDa) from VF-T. crassiceps purified by affinity chromatography concanavalin A-Sepharose (20). Other authors have demonstrated the presence of common antigenic components of the cestodes Taenia and Echinococcus (6, 11). The 18- and 14-kDa proteins obtained in the present work were found to be specific for cysticercosis, and we are planning further studies to select those specific peptides from VF-T. crassiceps which are common to hydatid fluid from E. granulosus. It is possible that the conformation of the proteins should be highly important for either the immunogenicity or the antigenicity, and it is expected that more than one epitope is recognized by each individual, particularly in human cases, where immune responses are highly heterogeneous.
VF-T. crassiceps ELISA has shown a low specificity for serum samples, which indicates the need to perform confirmatory IB testing (1, 2). Bragazza et al. (1) described a 20% rate of false-positive results in 1,863 serum samples when a population from an area of cysticercosis endemicity was tested. By testing these samples by IB, the authors demonstrated that proteins of >30 kDa from VF-T. crassiceps were responsible for the unspecific reactions or cross-reactions with other parasitic infectious diseases. The cross-reactivities affecting the specificity index should be better evaluated in further clinical epidemiological studies using serum samples from different regions, including the most frequent heterologous parasitosis and other infections in each respective area.
In the present study, the use of 18- and 14-kDa T. crassiceps purified proteins in ELISA demonstrated high specificity and sensitivity for the diagnosis of human cysticercosis. Future analysis should validate its usefulness for screening seropositive individuals in immunoepidemiological studies with human and swine serum samples.
ACKNOWLEDGMENTS
This work was supported by FAPESP (grants 00/00141-3 and 02-012061-0 to A.J.V.), by CNPq (proc. 300937/2003-2 to O.M.T.), by a FAPESP fellowship to N.M.E. (03/10747-4), and by a CNPq fellowship to A.H.I. (107974/2003-7) and J.M.P.
We are indebted to Lucia Martins Teixeira and Walter Martin Oelemann from UFRJ, RJ, for comments on and review of the manuscript; to Fatima Tiecher, Instituto de Pesquisas Biologicas (Porto Alegre, RS), for providing serum samples from hydatidosis; and to Maria Carmen Arroyo Sanchez from Instituto de Medicina Tropical, USP, SP, Brazil, for statistical analysis.
REFERENCES
Bragazza, L. M., A. J. Vaz, A. D. C. Passos, O. M. Takayanagui, P. M. Nakamura, N. M. Espíndola, A. X. Pardini, and E. C. Bueno. 2002. Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens. Rev. Inst. Med. Trop. So Paulo 44:7-12.
Bueno, E. C., A. J. Vaz, L. R. Machado, J. A. Livramento, and S. R. Mielle. 2000. Specific Taenia crassiceps and Taenia solium antigenic peptides for neurocysticercosis immunodiagnosis using serum samples. J. Clin. Microbiol. 38:146-151.
Bueno, E. C., A. J. Vaz, L. R. Machado, J. A. Livramento, S. L. M. ávila, and A. W. Ferreira. 2001. Antigen-specific suppression of cultured lymphocytes from patients with neurocysticercosis. Clin. Exp. Immunol. 126:304-310.
Cho, S., S. Kim, S. Kang, and Y. Kong. 1988. Biochemical properties of a purified protein in cystic fluid of Taenia solium metacestodes. Korean J. Parasitol. 26:87-94.
Chung, J. Y., Y. Y. Bahk, S. Huh, S. Y. Kang, Y. Kong, and S. Y. Cho. 1999. A recombinant 10-kDa protein of Taenia solium metacestodes specific to active neurocysticercosis. J. Infect. Dis. 180:1307-1315.
Coltorti, E., and G. Cammarieri. 1993. Inmunodiagnostico de hidatidosis: evaluacion de antígenos de líquido hidatídico y de líquido vesicular de cisticerco de Taenia crassiceps. Rev. Inst. Med. Trop. So Paulo 35:155-162.
Espíndola, N. M., A. J. Vaz, A. X. Pardini, and I. Fernandes. 2002. Excretory/secretory antigens (ES) from in-vitro cultures of Taenia crassiceps cysticerci, and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis. Ann. Trop. Med. Parasitol. 96:361-368.
Greene, R. M., P. P. Wilkins, and V. C. W. Tsang. 1999. Diagnostic glycoproteins of Taenia solium cysts share homologous 14- and 18-kDa subunits. Mol. Biochem. Parasitol. 99:257-261.
Greene, R. M., K. Hancock, P. P. Wilkins, and V. C. W. Tsang. 2000. Taenia solium: molecular cloning and serologic evaluation of 14- and 18-kDa related, diagnostic antigens. J. Parasitol. 86:1001-1007.
Hancock, K., S. Pattabhi, R. M. Greene, M. L. Yushak, F. Williams, A. Khan, J. W. Priest, M. Z. Levine, and V. C. W. Tsang. 2004. Characterization and cloning of GP50, a Taenia solium antigen diagnostic for cysticercosis. Mol. Biochem. Parasitol. 133:115-124.
Ishida, M. M. I., G. Rubinsky-Elefant, A. W. Ferreira, S. Hoshino-Shimisu, and A. J. Vaz. 2003. Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals. Acta Trop. 89:73-84.
Kim, S., S. Kang, and S. Cho. 1986. Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization. Korean J. Parasitol. 24:145-158.
Kunz, J., B. Kalinna, V. Watschke, and E. Geyer. 1989. Taenia crassiceps metacestode vesicular fluid antigens shared with the Taenia solium larval stage and reactive with serum antibodies from patients with neurocysticercosis. Zentbl. Bakteriol. 271:510-520.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Larralde, C., J. Sotelo, R. M. Montoya, G. Palencia, A. Padilla, T. Govezensky, M. L. Diaz, and E. Sciutto. 1990. Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium. Arch. Pathol. Lab. Med. 114:926-928.
Michault, A., B. Riviere, P. Fressy, J. P. Laporte, G. Bertil, and C. Mignard. 1990. Apport de l'enzime-linked immunoelectrotransfer blot assay au diagnostic de la neurocysticercose humanaine. Pathol. Biol. 38:119-125.
Obregon-Henao, A., D. L. Gil, D. I. Gomez, F. Sanzon, J. M. Teale, and B. I. Restrepo. 2001. The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kD. Mol. Biochem. Parasitol. 144:209-215.
Pardini, A. X., A. J. Vaz, L. R. Machado, and J. A. Livramento. 2001. Cysticercus antigens in cerebrospinal fluid samples from patients with neurocysticercosis. J. Clin. Microbiol. 39:3368-3372.
Pardini, A. X., R. H. Peralta, A. J. Vaz, L. R. Machado, and J. M. Peralta. 2002. Use of Taenia crassiceps cysticercus antigen preparations for detection of antibodies in cerebrospinal fluid samples from patients with neurocysticercosis (T. solium). Clin. Diagn. Lab. Immunol. 9:190-193.
Peralta, R. H., A. J. Vaz, A. X. Pardini, H. W. Macedo, L. R. Machado, S. G. De Simone, and J. M. Peralta. 2002. Evaluation of an antigen from Taenia crassiceps cysticercus for the serodiagnosis of neurocysticercosis. Acta Trop. 83:159-168.
Pinto, P. S. A., A. J. Vaz, P. M. L. Germano, and P. M. Nakamura. 2000. Performance of the ELISA test for swine cysticercosis using antigens of T. solium and T. crassiceps cysticerci. Vet. Parasitol. 88:127-130.
Pinto, P. S. A., A. J. Vaz, P. M. Nakamura, and P. M. L. Germano. 2001. Immunoblot analysis using antigen from T. crassiceps cysticerci in the diagnosis of swine cysticercosis. Bol. Chil. Parasitol. 56:36-42.
Restrepo, B. I., A. Obregon-Henao, M. Mesa, D. L. Gil, B. L. Ortiz, J. S. Mejía, G. E. Villota, F. Sanzon, and J. M. Teale. 2000. Characterization of the carbohydrate components of Taenia solium metacestode glycoprotein antigens. Int. J. Parasitol. 30:689-696.
Sako, Y., M. Nakao, T. Ikejima, X. Z. Piao, K. Nakaya, and A. Ito. 2000. Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes. J. Clin. Microbiol. 38:4439-4444.
Singer, E. J., K. Syndulko, B. Fahy-Chandon, P. Schimid, A. Conrad, and W. W. Tourtellotte. 1994. Intrathecal IgG synthesis and albumin leakage are increased in subjects with HIV-1 neurologic disease. J. Acquir. Immune Defic. Syndr. 7:265-271.
Tsang, V. C. W., J. A. Brand, and A. E. Boyer. 1989. An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J. Infect. Dis. 159:50-59.
Vaz, A. J., P. M. Nakamura, C. C. Barreto, A. W. Ferreira, and J. A. Livramento. 1997. Immunodiagnosis of human neurocysticercosis: use of heterologous antigenic particles (Cysticercus longicollis) in indirect immunofluorescence test. Serodiagn. Immunother. Infect. Dis. 8:157-161.
Vaz, A. J., C. M. Nunes, R. M. F. Piazza, J. A. Livramento, M. V. Silva, P. M. Nakamura, and A. W. Ferreira. 1997. Immunoblot with cerebrospinal fluid from patients with neurocysticercosis using antigen from cysticerci of Taenia solium and Taenia crassiceps. Am. J. Trop. Med. Hyg. 57:354-357.
Wilson, M. R., R. T. Bryan, J. A. Fried, D. A. Ware, P. M. Schantz, J. B. Pilcher, and V. C. W. Tsang. 1991. Clinical evolution of the cysticercosis enzyme-linked immunoelectrotransfer blot in patients with neurocysticercosis. J. Infect. Dis. 164:1007-1009.(Noeli Maria Espíndola, Al)