[Production, characterization and purification of monoclonal antibodies against antigens of Mycobacterium tuberculosis H37Rv strain].
OBJECTIVE
Using monoclonal antibody (McAb) technique to analyze polypeptide antigens of Mycobacterium strains and try to find out the best method for purification of McAb.


METHODS
McAbs against H37Rv strain were produced by routine procedure. McAb-producing hybridomas were detected by ELISA and immunoblots technique. 24 strains of mycobacterium were grown on Loewenstein-Jensen medium for 1 to 3 weeks at 37 degrees C. H37Rv strain were grown on Loewenstein-Jensen medium or Sauton liquid medium. Ammonium sulfate, caprylic acid precipitation and ion exchange chromatography methods were used to purify McAbs against antigens of H37Rv strain.


RESULTS
14 McAb-produing hybridomas were obtained. C2 McAb reacted with all 24 strains of mycobacterium and 7C12 only reacted with H37Rv, H37Ra, M. bovis, BCG strains, C2 and 7C12 reacted with secreted proteins of H37Rv strain. 40,000/38,000 antigen of H37Rv strain was expressed differently in Loewenstein-Jensen medium and Sauton liquid medium. Relative activity of all McAbs was between 10(-4)-10(-7). The results of purified McAb showed that ammonium sulfate precipitation method could have the highest rate of recovery. Ion exchange chromatography method could have the highest purity. Caprylic acid method can remove albumin effectively and the purification of McAbs in ascific fluid was simple and easy to perform.


CONCLUSIONS
Polypeptide antigens of H37Rv against by C2, 7C12 McAb were the secreted proteins. For purification of McAb, the results suggested that ion exchange chromatography method can be applied for analysis and caprylic acid method for a very large volume of ascitic fluid.
Lu
X
X
Laboratory of Microbiology, Dept. of Shannxi Provincial Geriatric Research Center, Shaanxi Provincial Hospital, Xi'an 710068, China.
Zhang
X
X
Zhang
Y
Y
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D009169:Mycobacterium tuberculosis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial