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The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anionexchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

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Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of a-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Tritondissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.

Enzyme-linked immunosorbent assay (ELISA) protocols for the detection of antibodies to Mycobacterium paratuberculosis have employed a variety of antigens. These include crude protoplasmic antigens from M. paratuberculosis strains 18, 3,4