New technologies such as immunomagnetic separation have immediate applicability in Johne's disease research, but specific antibodies available in unlimited quantities have not been produced. Furthermore, the development of monoclonal antibodies to M. avium subsp. paratuberculosis (MAP) was identified as an unmet need at the 7th ICP in Bilbao, Spain. To fill this gap in Johne's disease research, monoclonal antibodies (mAbs) against MAP were produced from BALB/c mice immunized with sonicated MAP bacilli or recombinant fusion proteins. A total of ten stable hybridomas producing monoclonal antibodies to MAP proteins ranging from 12-40 kDa were identified in immunoblot assays. Six mAbs were classified as broadly cross reactive and four showed limited cross reactivity when analyzed against a panel of whole cell protein lysates comprising seven different mycobacterial species. The mAbs were characterized for their isotype, binding specificity, nature of binding epitope, reactivity in immunoassays, etc. The identity of the epitopes that bind selected monoclonal antibodies was determined by screening a MAP-phage expression library. This approach revealed that 9G10 detects MAP1643 (isocitrate lyase), 11G4 detects MAP3840 (70-kDa HSP) and 8G2 detects MAP2121c (a membrane protein), three proteins present in high relative abundance in MAP bacilli. The epitopes for 11G4 and 8G2 were mapped to the N-terminal half of each protein whereas 9G10 binds to the C-terminal half of MAP1643. Among the panel of mAbs generated in this study, 9G10 and 14D4 appear to label MAP best by immuno-electron microscopy. Aptamers, or nucleic acids that bind specific protein sequences, were also generated against the hypothetical protein encoded by MAP0105c and tested for specificity to MAP. Finally, a phage produced single-chain antibody (ptb9) was identified that binds to the MAP-specific protein encoded by MAP0858. These detection reagents will be beneficial in many Johne's disease research applications.