Abstract
Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne’s disease research and diagnostics. Johne’s disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.
Highlights
Monoclonal antibodies are important detection reagents for diagnostics and therapeutic purposes
This suggests potential new therapeutic strategies for the systemic treatment with monoclonal antibodies against gut-colonizing bacteria, of which Mycobacterium avium subspecies paratuberculosis (Map) is a member
Plaques reacting with Monoclonal antibodies (mAbs) were subcloned by infection of E. coli XLOLR strain (Stratagene) with the phage lysate obtained from the positive plaque and the addition of ExAssist helper phage (Stratagene)
Summary
Monoclonal antibodies (mAbs) are important detection reagents for diagnostics and therapeutic purposes. To close a research gap and facilitate detection of Map, which causes a chronic intestinal disease in cattle, sheep, and goats, we previously generated a set of stable hybridomas against Map whole cell extracts or membrane enriched extracts [4,5] In those studies, only five antigens were successfully identified when screening a phage lambda expression library with the antibodies. The antibody was obtained by immunizing mice with the human gastric cell line MGC803 [10] It was later discovered after a failed cDNA expression library screen that the antigen to PD4 was a membrane protein of Mycoplasma hyorhinis that could bind directly to tumor cells [11]. This catalog of monoclonal antibodies should prove useful for Map research
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
