Membrane and cytoplasmic proteins of Mycobacterium avium subspecies paratuberculosis that bind to novel monoclonal antibodies

Authors: Bannantine, John; Stabel, Judith; Lippolis, John; Reinhardt, Timothy

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2018
Publication Date: 12/11/2018
Citation: Bannantine, J.P., Stabel, J.R., Lippolis, J.D., Reinhardt, T.A. 2018. Membrane and cytoplasmic proteins of Mycobacterium avium subspecies paratuberculosis that bind to novel monoclonal antibodies. Microorganisms. 6(4):127. https://doi.org/10.3390/microorganisms6040127.
DOI: https://doi.org/10.3390/microorganisms6040127

Interpretive Summary: This study examines antibodies to the pathogen that causes Johne's disease, termed Mycobacterium avium subspecies paratuberculosis (Map). A catalog of 36 antibodies bind to unknown proteins on this bacterium. The goal of this study was to identify which Map proteins the antibodies bind to. Through several different biochemical and molecular approaches, the identities were discovered. With this information, we can now use the antibodies to quickly assess the quality of different proteomic preparations from this bacterium when grown in different environments or conditions. This work will be of interest primarily to research investigators working in the field.

Technical Abstract: Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne’s disease research and diagnostics. 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 by protein array. When both 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 their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes.