Purification and characterization of the 30,000 dalton native antigen of Mycobacterium tuberculosis and characterization of six monoclonal antibodies reactive with a major epitope of this antigen.
The 30,000 dalton native antigen of Mycobacterium tuberculosis is a major constituent of this organism and is secreted into culture medium. We purified this antigen by ammonium sulfate precipitation, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography to yield a single 29 to 30 kd component. The first 20 N-terminal amino acid sequence was determined and found to be identical to that reported for M. bovis alpha-antigen. Immunoelectrophoresis studies demonstrated the purified 30,000 dalton antigen to be immunologically identical with antigen 6 and antigen 85B. The 30,000 dalton native antigen was a potent skin test antigen in sensitized guinea pigs. Six immunoglobulin G1 murine monoclonal antibodies against the 30,000 dalton antigen were generated. By enzyme-linked immunosorbent assay and western immunoblotting, all six monoclonal antibodies reacted with the 30,000 dalton antigen, perhaps with the same epitope. When used with culture filtrates of other mycobacteria, the monoclonal antibodies demonstrated reactivity with M. gordonae and M. kansasii and to a lesser extent with M. avium and M. scrofulaceum.
Salata
R A
RA
Department of Medicine, Case Western Reserve University, University Hospitals of Cleveland, OH 44106.
Sanson
A J
AJ
Malhotra
I J
IJ
Wiker
H G
HG
Harboe
M
M
Phillips
N B
NB
Daniel
T M
TM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D000939:Epitopes; D006168:Guinea Pigs; D007123:Immunoelectrophoresis, Two-Dimensional; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012882:Skin Tests
D000596:Amino Acids; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes