In silico-predicted B-cell epitopes for bovine brucellosis serodiagnosis: Preliminary analytical evaluation of synthetic peptide- and multi-epitope protein-based indirect ELISAs

Serological methods are valuable diagnostic tools for bovine brucellosis, a zoonotic infectious disease with a worldwide distribution. Currently employed diagnostic methods utilize crude bacterial extract or regions of LPS, a molecule that can exhibit cross-reactions with other infectious agents. The goal of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISAi) using synthetic peptides or a multi-epitope protein based on in silico-predicted B cell epitopes. Peptides were synthesized on a cellulose membrane using spot synthesis, and immunoblots were performed to evaluate reactivity by densitometry against bovine sera of interest. Peptides reacting with serum from positive cattle and non-reactive to the serum from negative controls were selected, synthesized in a soluble form, and used as antigens for the development of the ELISAi. Two peptides (P1 and P2) were selected and, after standardization of the ELISAi, positive (25) and negative (175) samples were tested, resulting in a sensitivity of 84% (21/25) and specificity of 83.43% (146/175). The sequence of the two peptides in replicates with spacers were inserted into a multi-epitope protein, which was also used as antigen in an ELISAi resulting in a sensitivity of 72% (18/25) and a specificity of 61.71% (108/175). This study provided a preliminary analytical assessment of in silico-predicted epitopes for developing novel serologic diagnostic tests.