Immunological evaluation of a 12-kilodalton protein of Mycobacterium tuberculosis by enzyme-linked immunosorbent assay

Objective: To purify and study the seroreactivity of native and recombinant 12-kilodalton protein of Mycobacterium tuberculosis H37Rv.Design: M. tuberculosis H37Rv cells and Escherichia coli XL-1 containing the plasmid PRL4 encoding the M. tuberculosis heat shock protein GroES homolog were used as sources for the purification of native and recombinant 12 kD of M. tuberculosis respectively. The seroreactivity of the 12 kDs was studied by ELISA using sera from 35 leprosy and 25 active pulmonary tuberculosis (TB) patients, and from 10 normal healthy controls.Results: The 12 kD protein was purified from H37Rv extract (s12 kD) and from recombinant E. coli (r12 kD) by ultrafiltration and MonoQ fast pressure liquid chromatography (FPLC). Analysis of s12 kD and r12 kD by SDS-PAGE revealed a single protein band in both cases with an approximate molecular weight of 12 000 which was recognized by monoclonal antibody SA-12 in immunoblotting. Both the proteins exhibited a pI of ~4.6 by isoelectric focusing. Both the 12 kD proteins exhibited 96% positivity with TB sera as compared to normal control sera (P < 0.01). Only one serum sample from the 35 leprosy sera tested exhibited binding to both the s12 kD and r12 kD proteins. Delayed type hypersensitivity reaction to the 12 kD proteins was elicited in guinea pigs that had been immunized with H37Rv sonicate.Conclusion: The 12 kD protein could be easily purified and could serve as a valuable serodiagnostic tool in the screening of TB cases from a large population in an endemic area.