Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2006
Publication Date: 9/13/2006
Citation: Berger, S., Hinz, D., Bannantine, J.P., Griffin, J.F. 2006. Isolation of High-Affinity Single-Chain Antibodies against Mycobacterium avium subsp. paratuberculosis Surface Proteins from Sheep with Johne's Disease. Clinical and Vaccine Immunology.13(9):1022-1029. Interpretive Summary: In this study we basically took 3 sheep that clearly had signs of Johne’s disease and cloned all of the antibodies these sheep produced. This was done by taking the spleen, blood and bone marrow at necropsy and extracting the RNA from these tissues. We then made DNA copies of these RNAs and cloned them. This represented all of the antibodies produced in these diseased sheep. The next step was to identify which of these antibodies would bind tightly to Mycobacterium avium subspecies paratuberculosis (MAP), the bacteria that causes Johne’s disease. Through a screening process, two such antibodies were identified. Their strong binding to MAP has enabled them to be used as reagents to separate the bacteria from complex environments, such as milk or feces. The true novelty is obtaining these antibodies from a diseased host, making the antibodies biologically relevant and not just synthetically created. These results are discussed in terms of the potential use of single chain phage display monoclonal antibodies as novel diagnostic reagents.
Technical Abstract: Johne’s disease, due to infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools - necessary to understand disease processes and to identify sub-clinical infection - are much sought after. Here, we describe the production of single chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne’s disease by cloning heavy-chain and lambda light-chain variable regions, and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34 kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against M. avium subsp. avium but non-reactive against M. bovis or M. phlei cells, and were shown capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10**6-fold when employed in magnetic bead separation of mixed Mycobacterium spp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10**3 M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single chain phage display monoclonal antibodies as novel diagnostic reagents.