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

BMC Infectious Diseases

Salvatore G De-Simone¹  David William Provance²  José R Coura³  José B Pereira³  Luciano P Gomes⁴  Carolina G Bottino⁴ 

[1] Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil;Centro de Desenvolvimento Tecnológico em Saúde (CDTS)/Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças Negligenciadas (INCT-IDN), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil;Laboratório de Doenças Parasitárias, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil;Laboratório de Bioquímica de Proteínas e Peptídeos, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil

关键词: Peptide-ELISA;    Spot-synthesis;    Epitopes;    Flagellar repetitive antigen;    Cytoplasmic repetitive antigen;    Trypanosoma cruzi;    Chagas disease;   

Others  :  1145347
DOI  :  10.1186/1471-2334-13-568

 received in 2013-05-03, accepted in 2013-11-08,  发布年份 2013

【 摘 要 】

Background

The 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.

Methods

Twenty-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.

Results

The 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%.

Conclusions

The 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.

【 授权许可】

   
2013 Bottino et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

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