Development and Characterization of Monoclonal Antibodies and Aptamers against Major Antigens ofMycobacterium aviumsubsp.paratuberculosis

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 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were 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, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.The genus Mycobacterium comprises a diverse group of animal and human pathogens as well as saprophytes, many of which are ubiquitous in the environment. Mycobacterium avium subsp. paratuberculosis is a member of the Mycobacterium avium complex (MAC) and an animal pathogen that is highlighted by the large financial burden that it places on the dairy industry due to Johne's disease (JD). Figures extrapolated from the 1996 NAHMS dairy survey suggest that the cost of this disease was over $200 million per year (25). The growing recognition of M. avium subsp. paratuberculosis infection in wildlife species is of considerable concern, since it may affect our ability to control or eradicate JD from domesticated animals (10, 11).Despite the research difficulties and economic consequences of JD, very few reports have described specific, antigen-based detection reagents for M. avium subsp. paratuberculosis. With the exception of a single study published 10 years ago (24), the scientific literature is silent on the subject of M. avium subsp. paratuberculosis monoclonal antibodies (MAbs) and their use in JD research. Very recently, single-chain antibodies were selected by cloning heavy and light chains from sheep with JD (6). This effort has resulted in two very promising recombinant antibodies; however, the M. avium subsp. paratuberculosis proteins that these antibodies react with remain unknown. The over...

Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis

“…Although we were not able to fine-map these epitopes, this finding shows that Hsp70 contains multiple targets for antibody interactions. Immunization of mice with whole-cell extracts of MAP also led to the generation of monoclonal antibodies specific for Hsp70 (MAP3840), indicating that this protein is immunogenic and abundantly present in MAP [25].…”

Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

“…paratuberculosis was reported by Metzger-Boddien et al (17). Other groups have been attempting to produce monoclonal antibodies for application in IMS (3,4). However, as an alternative to either polyclonal or monoclonal antibodies for the capture of M. avium subsp.…”

Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array

“…Among the strongest detected protein by the rabbit and mouse was DnaK (MAP3840). Two mAb have already been developed to this protein following immunization with a whole cell lysate of M. paratuberculosis [49], further supporting that this is a strong immunogen in mice. This antigen was also detected in cattle sera but not as strongly.…”