Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp

2 Specific antibodies, available in unlimited quantities, have not been produced against 4 Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium 6 subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of ten hybridomas were obtained 8 producing monoclonal antibodies to proteins ranging from 25-85 kDa. All mAbs showed some degree of cross reactivity when analyzed against a panel of whole cell protein 10 lysates comprising seven different mycobacterial species. The mAbs were characterized by several methods, which included isotype analysis, specificity, epitope, reactivity in 12 immunoblot assays and by electron microscopy. The identity of the antigens that bind two selected monoclonal antibodies was determined by screening a M. avium subsp. 14 paratuberculosis-lambda phage expression library. This approach revealed that 9G10 detects MAP1643 (isocitrate lyase) and 11G4 detects MAP3840 (70-kDa heat shock 16 protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for 11G4 were mapped to the N-terminal half of 18 MAP3840 whereas 9G10 binds to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind specific protein sequences, were also generated against the hypothetical 20 protein encoded by MAP0105c and tested for binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be 22 beneficial in many JD research applications.