Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep

Abstract

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.

Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin

“…Identification and characterization of antigen D has greatly facilitated the development of a procedure to obtain relatively large amounts of this component (30). Studies are in progress to evaluate the purified D component as an antigen in various serological assays for paratuberculosis beyond its current diagnostic use in agar gel immunodiffusion.…”

“…Purified antigen D was also chromatographically prepared in larger amounts as described previously (30). Briefly, the ammonium sulfate-precipitated sonicated preparation was chromatographed on Sephacryl S-200 and the high-molecular-mass antigen D was collected in the exclusion peak.…”

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

“…However, the relevance of M. leprae bacterioferritin as an antigen for purposes of diagnosis has not been explored by these investigators. In the context of Johne's disease, M. paratuberculosis bacterioferritin is known to be an immunodominant B cell antigen, and is the key component of a diagnostic test [13]. MLP has been used in this study to evaluate its potential application in the laboratory diagnosis of leprosy.…”

Purification of Mycobacterium bovis BCG Tokyo antigens by chromatofocusing, lectin-affinity chromatography, and hydrophobic interaction chromatography

“…The effort to purify these for future study is justified from previous investigations of their activities. For example, the BCG85 complex antigens were shown to elicit skin test (7,16,28,40) and humoral (13,14,18,36,42) responses. These antigens appear to play an important role in pathogenesis through an interaction with fibronectins (1,16), including an inhibition of the skin test response by binding T-cell fibronectins (16).…”

“…Purification of antigens from both M. bovis and M. tuberculosis has been achieved through a variety of separation techniques, as reviewed by Daniel and Janicki (6). More recent studies include isoelectric focusing (9), chromatofocusing (15), anion-exchange chromatography on DEAE columns (7,15,17), gel filtration (7,15), hydrophobic interaction or reversephase chromatography (7,31,32,36,41), lectin-affinity chromatography on concanavalin A (ConA)-Sepharose (12,15), and immunosorbent affinity chromatography with anti-MPB64 monoclonal antibodies (2). In contrast to anion-exchange chromatography on DEAE Sepharose, chromatofocusing demonstrated a better resolution of the major M. bovis antigens (15).…”