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.
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.
Sugden
E A
EA
Agriculture Canada, Animal Diseases Research Institute, Nepean, Ottawa, Ontario, Canada.
Brooks
B W
BW
Young
N M
NM
Watson
D C
DC
Nielsen
K H
KH
Corner
A H
AH
Turcotte
C
C
Michaelides
A
A
Stewart
R B
RB
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
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