B-cell epitope specificity of carboxy terminus of mycobacterium paratuberculosis ModD

Epitope mapping of ModD of Mycobacterium paratuberculosis was performed using overlapping peptides. In total, 80 overlapping peptides, covering the entire mature ModD, were commercially synthesized. Each peptide spanned 14amino acids with an offset of 4amino acids, i.e., with an overlap of 10amino acids. Synthetic peptide antigenicity was evaluated by enzyme-linked immunosorbent assay (ELISA) using rabbit antisera to culture filtrate (CF) of M. avium or M. paratuberculosis or recombinant ModD (rModD). The peptides o f ModD reacting most strongly (ELISA OD1.0) were clustered near the N- and C-terminal ends. The peptides around the C-terminal end only showed the greatest specificity for M. paratuberculosis, yielding high ELISA OD values with rabbit anti-M. paratuberculosis CF serum and low ELISA OD values with rabbit anti-M. avium CF serum. Sera from naturally M. paratuberculosis-infected cattle, however, bound poorly to the short, 14-amino-acid peptides. Thus, two longer peptides covering amino acids 100 to 125 and 328 to 353 were synthesized based on their broad reactivity to rabbit serum against M. paratuberculosis CF. The peptide covering amino acids 328 to 353 showed the highest level of specific bovine antibody binding.