Abstract
BackgroundThe identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.MethodsTwenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay.ResultsThe peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%.ConclusionsThe epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.
Highlights
The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines
Chagas disease is caused by the protozoan Trypanosoma cruzi, which belongs to the Trypanosamatidae family [1]
Linear epitope mapping Linear epitopes often contain between two and six amino acids that are critical for recognition by an antibody [41,42]
Summary
The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. Epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. During the chronic phase of the disease, diagnosis of an infection relies on serological assays since there is a major decline in the number of parasites circulating in patients’ blood [3,4]. The most common techniques used are ELISA, indirect hemagglutination (IH), indirect immunofluorescence (IIF), western blot and immunochromatography [4,5]. While these methods are usually simple to perform and have a low cost, they can demonstrate low sensitivity and/or specificity, or even cross-reactions with other pathogens, especially Leishmania spp. Crossreactivity is a major concern when whole or semi-purified extracts of T. cruzi epimastigotes are used as antigens in serological tests [5]
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