Development of immunoassays specific for the diagnosis of tuberculosis requires antigens unique to Mycobacterium tuberculosis. In a search for such antigens we tested six proteins encoded by RD1, a region present in M. tuberculosis and virulent M. bovis genomes but missing from the DNA of all substrains of M. bovis Bacillus Calmette-Guerin (BCG). The six proteins (Rv3871, Rv3872, Rv3873, MTSA-10, ESAT-6 and Rv3878) were purified to near-homogeneity from recombinant Escherichia coli. When tested for the ability to elicit antibody responses and delayed type hypersensitivity in tuberculous guinea pigs, only two of six antigens, ESAT-6 and MTSA-10, elicited strong skin reactions, while vigorous antibody responses were observed to all six proteins. When antibody responses to RD1 antigens were evaluated in sera from patients having pulmonary tuberculosis and from control subjects (patients having mycobacterioses other than tuberculosis, and healthy persons), a sizeable proportion (25%) of tuberculosis patients but none of the control subjects, had antibodies against MTSA-10 and/or ESAT-6. We conclude that MTSA-10 and ESAT-6 are promising candidates for immunodiagnostic assays specific for tuberculosis.

Antibodies against Mycobacterium tuberculosis antigens were detected by enzyme-linked immunosorbent assay in cerebrospinal fluid (CSF) samples obtained from 442 patients with tuberculous meningitis (TBM) and 102 control patients. Antibodies were found in the CSF of 87% of patients with clinical (culture-negative) TBM, 72% of patients with culture-positive TBM, and 65% of patients with autopsy-proven TBM. That anti-M. tuberculosis antibodies were detected in the CSF of patients with clinically diagnosed cases more frequently than in patients with culture-positive cases suggests that the detection of antibodies in CSF tends to decrease as bacillary load increases. Of the patients with clinical TBM who were coinfected with human immunodeficiency virus (HIV), 70% exhibited anti-M. tuberculosis antibody in CSF, which suggests that antibody responses in this group were substantially weaker than those in HIV-negative patients with clinical TBM. Some groups showed a stronger response to certain antigens, which suggests that antigen recognition patterns may be specific for the stage of disease.

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.Proteins secreted into the extracellular environment by Mycobacterium tuberculosis are usually targets of immune responses in the infected host. Thus, the filtrate of M. tuberculosis cultures has constituted an important source of antigens that induce protective immunity and immune responses having diagnostic value (reviewed in references 1, 6, and 20). In the 1980s and early 1990s S. Nagai and his collaborators purified several proteins from culture filtrates of M. tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG). These proteins were termed MPT (for M. tuberculosis) or MPB (for M. bovis BCG) followed by a number indicating the relative mobility during nondenaturing polyacrylamide gel electrophoresis (13). Of the MPT and MPB proteins known to be secreted (18), many elicit immune responses that are specific for the M. tuberculosis complex and therefore of diagnostic value (for example, MPT64 [2], MPB70 [7], and MPT63 [12]). Others confer protective immunity (MPT44, MPT59, and MPT45, i.e., the members of the Ag85 complex [8,17]). While all other genes encoding MPT and MPB proteins were identified and sequenced in the pregenome era, the mpt53 gene was identified only recently (19) from the analysis of the NH 2 -terminal sequence of the purified protein (13) and from the genome sequence of M. tuberculosis (mpt53 is Rv2878c) (5). MPT53 is a 15-kDa protein (13) that induces antibody responses in tuberculous cattle (19). In the present work, we characterized the specificity of MPT53 for the M. tuberculosis complex and the involvement of MPT53 in immune responses to tuberculosis (TB) in guinea pigs and in humans.The distribution of mpt53 among tuberculous and nontuberculous mycobacteria was determined by Southern transfer and hybridization. The gene was present in the DNA of members of the M. tuberculosis complex (Fig. 1, lanes 1, 4, and 8, and data not shown). Analysis of 50 clinical isolates of M. tuberculosis identified one isolate bearing a chromosomal deletion encompassing mpt53 (data not shown), indicating a relative gene instability. We next investigated the distribution of mpt53 among nontuberculous mycobacteria. Analysis of the partial M. avium genome sequence (http://www.tigr.org/tdb/) by BLAST homology programs indicated that M. avium contains a homolog of mpt53 (Ϸ80% identity with nucleotide and amino acid sequences corresponding to the extracellular MPT53 protein of M. tuberculosis) (data not sho...

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