Dados do Trabalho

Título

Linear vs. Conformational determinants: ELISA reactivity of L. infantum recombinant proteins and linear epitope mapping through microarray analysis

Introdução

In Brazil serological diagnosis is the main diagnostic method used for the detection of Leishmania infantum infection. The diagnostic protocol recommended is screening using rapid test (DPP) in dogs and humans, and as confirmatory test ELISA (EIE-LVC) for dogs and immunofluorescence for humans. Despite the low cost and the ease of performance, the diagnostic tests recommended present accuracy issues and cross-reactivity with other species of the Trypanosomatidae family.

Objetivo (s)

To improve the accuracy of available diagnostic tests, four recombinant proteins of L. infantum (rLci1, rLci2, rLci5 and rLci12) were identified, which, when used in the ELISA and DPP, presented sensitivity and specificity superior to the tests recommended by the Brazilian Ministry of Health (Accuracy: 84% - 92%). However, some level of cross-reactivity with Trypanosoma spp. was found.

Material e Métodos

To improve specificity, bioinformatics (BLAST-p, Clustal Omega) and microarray studies were carried out, to identify the main conserved epitopes among the species L. infantum, T. cruzi, Ehrlichia canis and Babesia canis as well as Leishmania braziliensis, and select those specific to Leishmania for use in a diagnostic test. To assess the linear reactivity of each protein an indirect IgG ELISA were conducted with proteins in its original conformation and denaturated at 80ºC for 20 minutes.

Resultados e Conclusão

After in silico evaluation similarity matrices were constructed. High similarity (>80%) with T. cruzi was found only for rLcis 1 and 12. While rLcis 5 and 2 had only similarity to proteins within the Leishmania species. Only rLci 5 showed a significant difference in sensitivity after denaturation process. In addition, the linear epitopes with greater reactivity for anti-L. infantum antibodies (IgG) were evaluated by a microarray assay, and subsequent visualization and quantification of the intensity of the reactivity by fluorescence detection.Sixty-four candidate sequences for dogs and two hundred and fifty candidate sequences for humans were selected for posterior validation analysis. Specific candidate epitopes for an accurate diagnosis were identified from all four proteins and are very promising targets to develop new VL tests.

Palavras-chave

LEISHMANIA; RECOMBINANT PROTEINS; EPITOPE MAPPING

Agradecimentos

Funded by Fiocruz (PROEP and INOVA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) PROEX, Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPQ and Fundação de Apoio a Fiocruz - FIOTEC.