Isolation, purification and immunological characterization of novel low molecular weight protein antigen CFP 6 from culture filtrate of M-tuberculosis

A novel immunogenic antigen, CFP 6 was purified from culture filtrate of M. tuberculosis by a preparatory 2-D electrophoresis method. The protein focu sed at pI of 4.0 during isoelectric focusing. Molecular weight of the purif ied protein by ES MS was found to be 11.61 kD. N-terminal amino acid sequen ce of CFP 6 could be aligned to the deduced amino acid sequence from ORF Rv 3004 and was found to be a novel protein with 112 aa residues. Single N-ter minal sequence showed that the purified protein was essentially free from c ontaminants and the amino acid analysis of the antigen was in good agreemen t with the DNA sequence deduced amino acid composition. Purified CFP 6 was studied for its ability to induce proliferative responses of peripheral blo od lymphocytes from five categories of human subjects, These were: untreate d, active pulmonary tuberculosis patients; patients after 2-3 months of che motherapy; vaccinated professional contacts; vaccinated/nonvaccinated healt hy controls. CFP 6 elicited high proliferative responses in health; contact s and patients recovering from the disease. This protein also induced the r elease of a significantly high amount of IFN-gamma in cell culture supernat ant of healthy contacts as compared to other categories of subjects. This p rotein was further evaluated and compared with PPD and total CS for its DTH inducing ability in guinea pigs immunised with BCG or M. tuberculosis H(37 )Rv. CFP 6 elicited a powerful immune response in vitro and in vivo animal model, hence seems to be an immunologically important protein. (C) 2000 Els evier Science Ltd. AII rights reserved.