Production and characterization of monoclonal antibodies against a major membrane protein of Mycobacterium avium subsp. paratuberculosis

Abstract

The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c is an important membrane antigen recognized in cattle with Johne's disease. In this study, purified recombinant MMP was used to produce two stable monoclonal antibodies, termed 8G2 and 13E1, which were characterized by immunoblotting, epitope mapping, and immunofluorescence microscopy.

FIG. 1.

Immunoblot analysis of mAbs 8G2 and 13E1 shows reactivity with the E. coli-expressed recombinant protein as well as the native M. avium subsp. paratuberculosis 35-kDa protein. Shown are three identical immunoblots that were exposed to the antibodies indicated beneath each filter. Note that anti-MBP (α-MBP) is a mAb to the MBP affinity tag that does not detect any M. avium subsp. paratuberculosis proteins whereas 8G2 and 13E1 detect only the E. coli-expressed MBP-MAP2121c gene product fusion protein and the MAP2121c gene product in M. avium subsp. paratuberculosis. Lanes: 1, MBP-LacZ; 2, MBP-MAP2121c gene product fusion protein; 3, M. avium subsp. paratuberculosis whole-cell lysate. Kilodalton size markers are shown in the left margin and indicated by corresponding notches between the blots.

FIG. 2.

MMP mAbs are not subspecies specific for M. avium subsp. paratuberculosis. Two identical immunoblots were probed with 13E1 (A) and 8G2 (B). In all lanes, 0.6 μg of a whole-cell lysate was loaded. Lanes: 1, protein standards; 2, M. avium subsp. silvaticum; 3, M. scrofulaceum; 4, M. abscessus; 5, M. avium subsp. paratuberculosis K-10; 6, M. avium subsp. avium (TMC702); 7, M. bovis (strain 95-1315); 8, M. phlei; 9, M. bovis BCG; 10, M. avium subsp. paratuberculosis ATCC 19698; 11, M. avium subsp. avium (TMC715); 12, M. avium subsp. paratuberculosis (isolate Linda); 13, M. intracellulare; 14, M. kansasii. Sizes in kilodaltons of protein standards are indicated in the left margin.

FIG. 3.

Localization of mAb epitopes to regions of the MAP2121c gene product. Four constructs were produced and expressed in E. coli. (A) The full-length protein and truncated versions of the protein are shown schematically. Protein sizes and relative positions within the full-length protein are depicted to scale based on the start and stop codons contained within the expression clone. Each of four quadrants of the protein can be distinguished based on mAb reactivity patterns. (B) For determination of the approximate locations of mAb epitopes in the primary sequence of the MAP2121c gene product, purified recombinant peptides representing the full-length (lane 2), N-terminal half (lane 3), central region (lane 4), and C-terminal half (lane 5) of the protein were immunoblotted and probed with selected mAbs as indicated beneath each blot. Anti-MBP binds the affinity tag, and therefore the blot probed with anti-MBP shows all the proteins present and indicates their relative amounts and positions within each blot. The MBP-LacZ control protein is present in lane 6. No reactivity with the protein size standards (lane 1) was observed. The positions of the standards are indicated in the left margin. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

FIG. 4.

MMP was present in the membrane-enriched fractions of five M. avium subsp. paratuberculosis isolates. The resulting immunoblot was exposed to mAb 8G2. This antibody labeled a 35-kDa protein in the membrane-enriched fractions of the five M. avium subsp. paratuberculosis isolates. Sizes in kilodaltons of protein standards are indicated in the left margin.

FIG. 5.

Immunofluorescent-antibody staining of 48-h M. avium subsp. paratuberculosis-infected BoMac macrophages with Alexa Fluor 488-labeled 8G2 and 13E1. Each four-panel image shows cells visualized by using DIC, DAPI, Alexa Fluor 488, and DAPI merged with Alexa Fluor 488. Both 8G2 and 13E1 Alexa Fluor 488-labeled antibodies stained intracellular M. avium subsp. paratuberculosis. Arrows in the DIC and Alexa Fluor 488 panels indicate the locations of the mycobacteria. Note that the DAPI nuclear staining is in close proximity to the Alexa Fluor 488-labeled mycobacteria. The bar indicates 5 μm for all panels.