Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.Tuberculosis (TB) is a chronic infectious disease caused by an intracellular pathogen, Mycobacterium tuberculosis, and is a major worldwide health problem. According to the World Health Organization (WHO), one-third of the world's population is latently infected with the bacterium and more than 10 million new cases of TB and 2 million deaths occur across the globe annually (11). Controlling the incidence of TB depends on rapid, sensitive diagnostic tests, efficient drug treatment, and the development of an improved vaccine. Currently, attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only available vaccine against TB, but it has proven to be problematic because of inconsistent protection and false-positive results during diagnosis due to cross-reactivity (4, 12). The purified protein derivative of M. tuberculosis is the most commonly used antigen for the diagnosis of TB, but its use is also problematic because of poor specificity due to cross-reactivity with antigens from other Mycobacterium species (36). Thus, an individual's BCG vaccination status and the prevalence of environmental mycobacteria are important factors to consider in the diagnosis of TB.Since accurate and reliable diagnostic methods for M. tuberculosis infection are urgently required for the global control of this pathogen, the diagnostic potential of M. tuberculosis-specific antigens has been explored. In an attempt to identify sensitive and specific serodiagnostic antigens or novel immunogenic antigens of M. tuberculosis...

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