Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies

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