Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2007
Publication Date: 3/1/2007
Citation: Bannantine, J.P., Radosevich, T.J., Stabel, J.R., Berger, S., Griffin, J.F., Paustian, M. 2007. Production and Characterization of Monoclonal Antibodies Against a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunology. 14(3):312-317. Interpretive Summary: This study reports on the development and characterization of highly specific antibodies, termed monoclonal antibodies, that detect a surface protein present in Mycobacterium paratuberculosis. This work is important because there are currently no antibodies available to any known M. paratuberculosis surface proteins and new detection reagents are sorely needed for studying this pathogenic bacterium. Two 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 location of antibody binding within the protein was determined.
Technical Abstract: The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c has been shown to play a role in invasion of epithelial cells and is an important membrane antigen recognized by cattle with Johne’s disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies (mAbs), termed 8G2 and 13E1, which bind both the native and recombinant MMP. These antibodies were examined for cross-reactivity to M. avium subsp. avium, M. abscessus, M. avium subsp. silvaticum, M. intracellulare, M. kansasii, M. scrofulaceum, M. phlei and M. bovis by immunoblot analysis. 13E1 showed the least cross-reactivity, detecting MMP in mycobacteria within the MAC complex, but not in M. abscessus, M. kansasii, M. scrofulaceum, M. phlei or M. bovis. 8G2 showed a similar reactivity pattern with the MAC complex; however, this antibody also recognized a smaller size protein present in M. abscessus and M. kansasii. The 8G2 epitope was mapped to a 77 amino acid region on the N-terminal half of MMP whereas 13E1 detected only the full length MMP protein. Reactivity with cytosol-enriched and membrane-enriched fractions produced for five M. avium subsp. paratuberculosis isolates provided additional evidence that MAP2121c is surface or membrane localized. Both antibodies labeled M. avium subsp. paratuberculosis by immunofluorescence and immunoelectron microscopy. These are the first published mAbs against a known M. avium subsp. paratuberculosis protein and will provide an unlimited source of useful reagents for studies with this significant veterinary pathogen.