title: 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 anion-exchange (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.
authors.affiliation: Agriculture Canada, Animal Diseases Research Institute, Nepean, Ottawa, Ontario, Canada.
authors.forename: E A
authors.initials: EA
authors.lastname: Sugden
authors.forename: B W
authors.initials: BW
authors.lastname: Brooks
authors.forename: N M
authors.initials: NM
authors.lastname: Young
authors.forename: D C
authors.initials: DC
authors.lastname: Watson
authors.forename: K H
authors.initials: KH
authors.lastname: Nielsen
authors.forename: A H
authors.initials: AH
authors.lastname: Corner
authors.forename: C
authors.initials: C
authors.lastname: Turcotte
authors.forename: A
authors.initials: A
authors.lastname: Michaelides
authors.forename: R B
authors.initials: RB
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006360:Heat-Shock Proteins; D007123:Immunoelectrophoresis, Two-Dimensional; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008199:Lymphadenitis / Q000175:diagnosis; D008969:Molecular Sequence Data; D010283:Paratuberculosis / Q000175:diagnosis* / Q000276:immunology; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; C065036:antigen 85, Mycobacterium bovis; C072166:antigen A, Mycobacterium paratuberculosis; C072165:antigen D, Mycobacterium paratuberculosis; C058749:groES protein, Mycobacterium tuberculosis; C050016:lipoarabinomannan