The data presented show that we have identified several antigens that augment the 38-kDa antigen in the serodiagnosis of TB. This was achieved by screening genomic expression libraries with pulmonary and extrapulmonary TB sera, 38-kDa antibody-negative sera, and a rabbit antibody to TB-soluble proteins. In particular, four antigens (Mtb8, Mtb11, Mtb48, and DPEP) were identified and subsequently expressed as recombinant proteins. These antigens were extensively tested with a large panel of sera. A fifth antigen, Mtb81, was subsequently identified by two-dimensional gel electrophoresis in combination with mass spectrometry and also expressed as a recombinant. This antigen was found to be particularly effective in the serodiagnosis of HIV-TB coinfections. The reactivity of each of these antigens with smear-negative and smear-positive pulmonary TB sera and extrapulmonary sera is presented.
Mtb8, Mtb11, and Mtb48 were each shown to react by ELISA with subpopulations of sera from TB patients, consistent with other observations that antibody responses in TB patients are heterogeneous (29
). In addition, all three were shown to provide an incremental increase in sensitivity for TB detection when used in conjunction with the 38-kDa antigen (Table ), indicating the need for appropriate multiantigen cocktails to increase sensitivity (30
). Further increases in clinical sensitivity were achievable with the addition of the proline-rich antigen DPEP.
To facilitate assay development and cost when using multiple antigens, we have combined several antigens into multiepitope polyproteins. Once such polyprotein including three of these antigens (Mtb8, Mtb11, and Mtb48) in combination with the 38-kDa antigen (TbF6) was constructed and expressed in E. coli. This multiepitope polyprotein was evaluated by ELISA alone and in combination with the proline-rich protein DPEP to determine if the sensitivity was increased by the combination of these antigens. The ELISA data (Table ) showed that while TbF6 provided the sensitivity expected based on its four component antigens, the 38-kDa antigen, Mtb11, Mtb8, and Mtb48, a further increase in sensitivity could be observed with the addition of DPEP into the ELISA well with TbF6. Similar sensitivity was achievable with TbF10 (the 38-kDa antigen and Mtb11 and Mtb8) in a cocktail together with Mtb48 and DPEP (Table ). The functionality of the individual antigens in the polyproteins TbF6 and TbF10 was also demonstrated in the membrane-based MAPIA assay, indicating their potential use in developing rapid lateral-flow formats (Fig. ). In addition, the specificity of the TbF6 and TbF10 antigens was tested by MAPIA with 12 sera from mycobacteria other than TB and shown to be negative (data not shown).
Further increases in sensitivity are predicted if Mtb81 was used in cocktails with the polyproteins. The theoretical inclusion of Mtb81 in the antigen composition (which was tested by ELISA alone) predicted improved sensitivity with the HIV-TB population from 46.9% (30 of 64 samples) with TbF6 and DPEP to 79.7% (51 of 64 samples) with Mtb81 and 84.4% (54 of 64 samples) for TbF6-DPEP-Mtb81. The above studies have served to identify antigens that, when used together, predict a high degree of clinical sensitivity in the detection of active TB. In addition, these antigens provide the basis for cocktails that maintain a high degree of specificity in TB diagnosis.
The six antigens described here (the 38-kDa antigen, Mtb8, Mtb11, Mtb48, DPEP, and Mtb81), several of which are incorporated into the polyproteins TbF6 or TbF10, provide the basis for the development of sensitive and specific tests for evaluating TB infections, particularly as an adjunctive test to the AFB smear typically used for TB diagnosis. By combining multiple antigens into polyproteins, this provides a potentially cost-effective way of developing tests for use in developing countries where cost per test is important. Efforts are now under way to provide the optimal combinations of antigens and/or polyproteins for the development of commercially viable rapid lateral-flow tests and ELISA for TB serodiagnosis that can be used alone or in combination with smear testing. In this context, the combination of TbF6 and DPEP seems more amenable to ELISA development and that of TbF10 with DPEP and Mtb48 to rapid lateral-flow test development. Incorporation of Mtb81 into these formats to increase sensitivity in HIV-TB population will also be necessary and could require further polyprotein engineering. In this context, we are epitope mapping Mtb81 to identify a smaller more antigenic fragment that would be more amenable to incorporation into a polyprotein. The incremental increases in sensitivity demonstrated with the described antigens, along with incorporation into polyproteins, should facilitate the development of ELISA and rapid tests with improved characteristics over current technology. Algorithms to incorporate such tests into TB diagnosis will also need to be evaluated. Rapid formats, providing they are sensitive and specific enough, would have field utility and, since they detect many AFB-negative but TB-positive individuals, would complement AFB smear analyses to increase clinical sensitivity.