Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

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.

Molecular Characterization of Tc964, A Novel Antigenic Protein from Trypanosoma cruzi

“…These results could be explained by the fact that the T. cruzi specific epitopes are located in the non-conserved regions of the proteins, contrary to what happens with the Leishmania epitopes which could map to the conserved ones. Altogether, these findings prove, as other studies reported, that antigenic peptides can be a useful tool for the diagnosis of CCD [59][60][61].…”

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

“…All serological techniques are used primarily during the latent and chronic stages of infection, when it is difficult to identify the parasites. Cross-reactivity of Chagas disease tests with circulating antibodies related to other infections and diseases, such as leishmaniasis, malaria, toxoplasmosis, hepatitis, leprosy, syphilis and collagen vascular diseases, is common ( Carvalho et al 1993 , Bottino et al 2013 , Guevara et al 2013 ). This cross-reactivity inspired us to conduct a study of five different serologic tests to determine the statistical concordance among these tests and identify the true seroprevalence rate, as has been previously described by the WHO ( Foti et al 2009 ).…”

Epidemiological Aspects of Chagas Disease - a Review

“…The CRA (cytoplasmic repetitive antigen) and FRA (flagellar repetitive antigen) proteins from T. cruzi have been used in studies on the diagnosis of ChD; an ELISA-based diagnostic test that used either of CRA antigens achieved 100 % sensibility and specificity, while FRA antigen showed a lower sensitivity of 91.5 % with a specificity of 60 % [54]. The IgM IFA is useful for detection of IgM-specific T. cruzi antibodies; a positive IgM IFA result is indicative of acute infection [55], and hence an IgM assay could be an important diagnostic tool.…”