Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/8/2007
Publication Date: 5/1/2007
Citation: Bannantine, J.P., Radosevich, T.J., Stabel, J.R., Sreevatsan, S., Kapur, V., Paustian, M. 2007. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunology. 14(5):518-526. Interpretive Summary: This study reports on the development and characterization of highly specific antibodies, termed monoclonal antibodies, that detect a variety of antigenic proteins present in Mycobacterium paratuberculosis. This work is important because there are currently no antibodies available to any known M. paratuberculosis proteins and new detection reagents are sorely needed for studying this pathogenic bacterium. Nine monoclonal antibodies that detect the same protein were used in a variety of assays and showed good utility in each one. The antibodies also showed some reactivity with a similar protein present in other mycobacterial species. Finally, the identity and location of antibody binding within selected proteins was determined.
Technical Abstract: Specific antibodies, available in unlimited quantities, have not been produced against 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 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 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 lysates comprising seven different mycobacterial species. The mAbs were characterized by several methods, which included isotype analysis, binding specificity, nature of binding epitope, reactivity in 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. paratuberculosis-phage expression library. This approach revealed that 9G10 detects MAP1643 (isocitrate lyase) and 11G4 detects MAP3840 (70-kDa heat shock 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 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 protein encoded by MAP0105c and tested for binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.