Identification of a Novel 27‐kDa Protein from Mycobacterium tuberculosis Culture Fluid by a Monoclonal Antibody Specific for the Mycobacterium tuberculosis Complex

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Abstract
Mycobacterium tuberculosis antigens inducing species-specific immune responses are likely to be particularly important for serodiagnosis or for skin testing of tuberculosis. In the present study, we describe the characterization of two novel monoclonal antibodies (MoAbs) A3h4 (IgG2a) and B5g1 (IgM) that are directed to M. tuberculosis 27-kDa and 25-kDa proteins respectively. Specificity analysis by immunoblotting using 20 different species of mycobacterial sonicates revealed that MoAb A3h4 was specific for M. tuberculosis complex alone while MoAb B5g1 showed a limited cross-reactivity. Direct comparison with previously characterized MoAbs revealed that these MoAbs A3h4 and B5g1 defined new antigenic determinants of M. tuberculosis. By using M. tuberculosis complex-specific MoAb A3h4 we have identified a distinct 27-kDa protein in the M. tuberculosis H37Rv culture fluid. Since this MoAb did not bind to the previously characterized MPT44, MPT59, MPT45, MPT51 and MPT64 proteins as well as the 23-kDa superoxide dismutase (SOD) protein of M. tuberculosis, we conclude that MoAb A3h4 recognizes a novel protein in the M. tuberculosis H37Rv culture fluid. Studies of the subcellular distribution of these MoAb-reactive proteins indicate that the MoAb A3h4-reactive 27-kDa protein is present not only in the culture fluid but also in the cytosol and the cell wall of M. tuberculosis. By contrast, B5g1-reactive protein is mainly a cytosolic protein. When these MoAbs were tested in a previously established ELISA with intact mycobacteria derived from early cultures, only MoAb A3h4 showed the positive reactivity to mycobacteria belonging to the M. tuberculosis complex. In addition, during the present comparative studies of MoAbs we have also found that the previously described MoAb F116-5, which is known to recognize the mycobacterial 23-kDa SOD protein [17], cross-reacted with the MPT44, MPT59, MPT45 and MPT51 secreted proteins but not with MPT64 and MPB70. These findings indicate that the family of four secreted proteins of M. tuberculosis share a common epitope with M. tuberculosis SOD protei

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