[Show abstract][Hide abstract] ABSTRACT: To study diagnostic epitopes within the Taenia solium 8 kDa antigen family, six overlapping synthetic peptides from an 8 kDa family member (Ts8B2) were synthesized and evaluated by ELISA and MABA with sera from patients with neurocysticercosis (NCC), from infected pigs and from rabbits immunized with recombinant Ts8B2 protein. The pre-immune rabbit sera and the Ts8B2 recombinant protein served as negative and positive controls, respectively. A similar analysis was done with the already described antigenic peptides from another member of the 8 kDa family, highly similar to Ts8B2, the CyDA antigen. Surprisingly, neither the Ts8B2 peptides nor the CyDA peptides were recognized by infected human and porcine sera. However, the entire Ts8B2 recombinant, as well as amino and carboxy-terminal halves were recognized by the positive serum samples. The observed lack of recognition of linear Ts8B2 peptides suggests that the principal serological response to the Ts8B2 family is focused on conformational epitopes in contrast to the previously observed antigenicity of the CyDA peptides. This differential antigenicity of 8 kDa family peptides could be related with parasite antigenic variability. The fact that rabbits experimentally immunized with Ts8B2 did make anti-peptide antibodies to peptides Ts8B2-6 and CyDA-6, located in the carboxy-terminal region demonstrated that the Ts8B2 peptides are not intrinsically non-immunogenic.
[Show abstract][Hide abstract] ABSTRACT: Ts8B2 is a gene which encodes for a member of the Taenia solium metacestode 8kDa antigen family. Since the Ts8B2-GST recombinant protein compares very favourably with other diagnostic antigens, and in order to study the antigenic nature and structure of this molecule, the Ts8B2 was expressed in prokaryotic and eukaryotic systems. The diagnostic potential of the recombinant Ts8B2 proteins was evaluated by enzyme-linked immunosorbent assays (ELISA) using a collection of serum and cerebrospinal fluid (CSF) samples from patients with clinically defined neurocysticercosis (NCC), and also sera from T. solium infected pigs. Despite the predicted glycosylation of the Ts8B2-Bac recombinant protein, there was very little difference in assay sensitivity/specificity when the Ts8B2 reagent was expressed in either prokaryotic or eukaryotic systems, suggesting that peptidic Ts8B2 epitopes are immunodominant in porcine cysticercosis and human neurocysticercosis. Conveniently, production of recombinant Ts8B2 in Escherichia coli is economical and facile, making it a feasible and practical choice as a diagnostic reagent for use in endemic areas. The Ts8B2 ELISA is particularly useful for the diagnosis of active as opposed to inactive cases of NCC and conduct of the assay is also facilitated by the fact that assay sensitivity is significantly greater when serum as opposed to CSF samples are employed.
Molecular and Biochemical Parasitology 09/2009; 168(2):168-71. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the objective of providing inexpensive and reproducible assays for the detection of antibodies indicating exposure to Taenia saginata and Taenia solium, we have evaluated the diagnostic utility of the T. saginata oncosphere adhesion protein (HP6-Tsag), expressed in baculovirus (HP6-Bac) and bacteria (HP6-GST [glutathione S-transferase]), employing enzyme-linked immunosorbent assays (ELISAs) and sera from T. saginata infected cattle, T. solium infected pigs and serum and cerebrospinal fluid (CSF) samples from clinically defined T. solium neurocysticercosis (NCC) patients. The two recombinant proteins were antigenic in all three systems, with the signal to background ratio of the HP6-Bac ELISA slightly higher than that for the HP6-GST ELISA. Assay performance in cattle was similar to previously described peptide-based ELISA assays, although NCC sample sensitivity/specificity was marginally better. The sensitivity of the HP6-Bac and HP6-GST ELISAs was close for active human NCC (77.4 and 80.6% for serum and 76.9 and 73.1% for CSF samples, respectively). In inactive human NCC, however, the sensitivity of the HP6-Bac ELISA was almost twice that of the HP6-GST ELISA. Because peptides are relatively expensive and recombinant proteins are simple and economical to produce, the latter may provide useful reagents for antibody detection in countries with endemic cysticercosis/NCC.
Parasitology Research 09/2007; 101(3):517-25. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibody screening of a lambdaZAP-XR Taenia solium metacestode cDNA library yielded a clone (Ts8B1), with an insert of 345 bp, and an open reading frame of 258 bp, that coded for a protein with 85 amino acid residues. Alignment of the predicted amino acid sequence with sequences from SWISSPROT revealed an 88% identity with TcA5.5, a 10 kDa immunodiagnostic antigen of T. crassiceps, 75% identity with CyDA a T. solium metacestode antigen, 40-50% identity with several variants of the 8 kDa subunit of antigen B of Echinococcus spp. and with members of the T. solium metacestode 8 kDa antigen family. Two other Ts8B1 related molecules, Ts8B2 and Ts8B3, were identified in the metacestode cDNA library by PCR, coding for 85 and 66 amino acid polypeptides, respectively. Both Ts8B1 and Ts8B2 were characterized as E/S antigens through their subcellular localisation in the secretory membrane system when expressed in NRK cells. The three cDNA inserts were expressed, purified and probed in enzyme-linked immunosorbent assays (ELISA) with sera and cerebro-spinal fluid from patients with confirmed neurocysticercosis, and with sera from pigs infected with T. solium. The most promising antigen, Ts8B2, performed with a sensitivity of 96.8% and specificity of 93.1% in the detection of active NCC when using serum samples in the assay and performed similarly in the porcine system. The implications of these findings are discussed.
Molecular and Biochemical Parasitology 04/2007; 152(1):90-100. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study evaluates five synthetic peptides derived from four, potentially protective, Taenia saginata oncosphere molecules for the serodiagnosis of T. solium cysticercosis/neurocysticercosis in three distinct Venezuelan endemic regions. The peptides, all of which have been described previously, are designated HP6-3, Ts45W-1, Ts45W-5, Ts45S-10 and TEG-1. In clinically verified and seropositive hospital cases, combining the results of three of the individual peptide-based ELISAs (HP6-3, Ts45W-1 and Ts45W-5) afforded the best balance between sensitivity (85%) and specificity (83.5%), a significant improvement on the 63.6% specificity obtained with the routinely employed T. solium cyst-fluid-based ELISA. Similarly, in the seropositive Venezuelan endemic zone samples, 89.09% of Amerindians, 77.27% of symptomatic rural subjects and 67.83% of non-symptomatic rural subjects were also classed as seropositive by the combined peptide-based ELISAs. The profile of antibody recognition to individual peptides varied between the different groups of samples examined. The relevance of the above findings for the serology and prognosis of T. solium cysticercosis/neurocysticercosis in hospital- and field-based situations is discussed.
Transactions of the Royal Society of Tropical Medicine and Hygiene 09/2005; 99(8):568-76. · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding for a predicted small heat shock protein (sHSP), Tsol-sfISP35.6, has been isolated by antibody screening of a Taenia solium c-DNA library. The clone was a full-length sequence (1172 bp) with an open reading frame of 945 bp and encoded for a 314 amino acid protein with deduced molecular mass of 35.6 kDa, isoelectric point of 5.6 arid the characteristic HSP20/alpha-crystallin domain duplicated. It was highly conserved, with a high sequence similarity with other platyhelminth sHSPs. Western blot analysis, using serum from neurocysticercosis patients (NCC), indicated that the purified Tsol-sHSP35.6 expression product was immunogenic, while in indirect ELISA, using the purified Tsol-sHSP35.6 expression product as antigen and serum samples from pigs and humans, 80% of T. solium infected pigs and 84% of patients with active, or 71% of patients with inactive NCC were sero-positive. The possible relevance of Tsol-sHSP35.6 in the diagnosis and pathogenesis of NCC is discussed.
[Show abstract][Hide abstract] ABSTRACT: A lambdaZAP-express cDNA library of Taenia saginata metacestodes was constructed. Antibody screening yielded a clone with an insert of 3,408 bp, an open reading frame of 2,589 bp, a deduced sequence of 863 amino acid and a molecular mass of 98.89 kDa. Alignments of the predicted amino acid sequence showed identity with paramyosins from several species: 98.8% with Taenia solium, 96.3% with Echinococcus.granulosus and about 70% with Schistosoma spp. The insert was expressed and purified. A collagen binding assay was performed which showed that T. saginata GST-paramyosin retained this property in a dose-dependent manner. Problems were encountered due to high backgrounds in serological assays in the homologous T. saginata system. However, the recombinant paramyosin was recognized by antibodies present in 31.6% of sera from T. solium seropositive cysticercosis patients and 100% of the sera from acute cysticercosis patients. The immunodominant epitope was the carboxyl-terminal fragment of the molecule.
Parasitology Research 10/2003; 91(1):60-7. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examined the seroprevalence and serum antibody isotype profile for Taenia solium cysticercosis in an Amerindian community in the Amazonas state of Venezuela. An antigen-trapping enzyme-linked immunosorbent assay (Ag-ELISA) was used to detect viable cysticercosis. Indirect ELISA (Ab-ELISA) and enzyme-linked immunoelectrotransfer blot (EITB) was performed by using antigens prepared from T. solium metacestodes to detect anti-parasite antibodies. The Ag-ELISA and Ab-ELISAs revealed 64.7% and 79.0% seropositivity, respectively, in the Amerindian population. Immunoglobulin (Ig) M was the predominant antibody class, suggesting recent infection. In comparison sera from, clinically defined, hospital neurocysticercosis cases revealed only 27% seropositivity by Ag-ELISA, compared with 86-92% seropositivity by Ab-ELISA, and IgG4 was the predominant antibody subclass detected. The EITB antigen recognition patterns of the hospitalized patients were very similar to that of the Amerindians, confirming exposure to the parasite. These results, combined with the predominance of IgM antibody responses and the marked detection of secreted products of viable parasites, strongly suggest that recent exposure to T. solium had occurred in the Amerindian population.
The American journal of tropical medicine and hygiene 03/2002; 66(2):170-4. · 2.53 Impact Factor