Antigen extracts obtained from the vesicular fluid of Taenia crassiceps cysticerci and from fractions purified by affinity chromatography with the lectin concanavalin A and the glycoprotein antigen separated by electrophoresis were used for the detection of Taenia solium anticysticercus antibodies. The sensitivity and specificity obtained for all antigens were 100% in enzyme-linked immunosorbent assay with good reproducibility. Using immunoblotting of the three antigens, low-molecular-mass peptides (18 and 14 kDa) were characterized only in cerebrospinal fluid samples from patients with neurocysticercosis. The results confirm that antigen fractions purified from T. crassiceps cisticerci are important sources of specific peptides and proved to be efficient in detecting anti-T. solium antibodies.Neurocysticercosis (NC), caused by the larval form of Taenia solium, is considered to be an endemic disease in underprivileged regions such as Latin America, Asia, Africa, China, and Indonesia (2,16,23,26), and it represents an important public health problem in these regions.The diagnosis of NC is based on clinical and laboratory criteria and on imaging examinations such as axial computed tomography and magnetic resonance imaging, which are efficient methods for the visualization of cysticerci and of an inflammatory response but are very expensive and inaccessible to most of the affected population (4).Although the immunological tests used for the diagnosis of cysticercosis have been considerably refined, the selection of an adequate antigen is required to improve diagnostic efficiency, and this continues to be an area of interest.Earlier studies have demonstrated the importance of the use of purified extracts obtained mainly from the glycoprotein fractions of T. solium for the detection of antibodies against cysticercus antigens in immunological assays (6,7,10,11,22). The difficulty of obtaining parasites from naturally infected pigs for the isolation of T. solium antigen continues to be a problem. Therefore, our group has been studying the use of antigens obtained from cysticerci of the Taenia crassiceps ORF strain (1, 25). The objective of the present study was to determine the efficiency of antigen fractions purified from T. crassiceps cysticerci in the detection of specific antibodies in cerebrospinal fluid (CSF) samples from patients with NC.Parasites and antigens. Vesicular fluid antigen was obtained from the larval form of T. crassiceps (VF-Tcra) ORF strain (8) as described previously (25). The purified fractions of concanavalin A (ConA-Tcra) were obtained by affinity chromatography with lectin. Total antigen was subjected to gel filtration on a PD-10 Sephadex G-25M column, purified by affinity chromatography on a ConA-Sepharose 4B column (Pharmacia Fine Chemicals) equilibrated with Tris-HCl buffer (pH 7.4) (0.5 M NaCl, 0.05 M Tris), and eluted with 0.2 M ␣-methylmannopyranoside solution. The protein peak was detected by spectrophotometry at 280 nm and concentrated by ultrafiltration through a YM-10 membrane (Amic...
Antigens were obtained from cysticerci of the ORF strain of Taenia crassiceps, by culture of cysts in protein-free hybridoma medium (PFHM). Budding of new vesicles was observed after 24-48 h. Excretory/secretory (ES) antigens (peptides of <20 kDa) were recovered in the medium after culture for 48 h. SDS-PAGE analysis of vesicular-fluid (VF) antigens (obtained by rupturing T. crassiceps cysticerci in PFHM) and the ES antigens indicated partial homology between the two preparations. ES peptides of 18- and 14-kDa were recognized by polyclonal antibodies produced in rabbits immunized either with the VF antigens or with a total-antigen preparation of T. solium cysticerci. Antibodies present in samples of serum or cerebrospinal fluid (CSF) from patients with neurocysticercosis also reacted with ES peptides. An anti-ES monoclonal antibody detected antigens in the CSF from 10 patients with neurocysticercosis, showing the antigenic homology of the ES antigens with those of T. solium cysticerci in human infections.
Antigens were detected in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis (NC) by enzyme-linked immunosorbent assay (ELISA) using polyclonal sera of rabbit anti-Taenia solium cysticerci (anti-Tso) and anti-Taenia crassiceps cysticerci vesicular fluid (anti-Tcra or anti-Tcra <30 kDa). A group of NC patients (n ؍ 174) were studied (NC), including 40 patients in different phases of the disease. ELISAs carried out with the anti-Tso, anti-Tcra, and anti-Tcra <30 kDa showed sensitivities of 81.2, 90, and 95.8% and specificities of 82, 98, and 100%, respectively. The 14-and 18-kDa low-molecular-weight peptides were only detected in CSF samples from patients with NC by immunoblotting with anti-Tso and anti-Tcra sera.
Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.
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