Use of the recombinant 38-kDa antigen of Mycobacterium tuberculosis as an immunogen for specific antisera production.
The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.
D'Souza
C D
CD
Radiation Medicine Center, Bhabha Atomic Research Center, Bombay, India.
Kadival
G V
GV
Samuel
A M
AM
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007114:Immunization / Q000379:methods; D051379:Mice; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D011994:Recombinant Proteins