Monoclonal antibodies against a Mycobacterium tuberculosis Ag85B-Hsp16.3 fusion protein

The secreted Mycobacterium tuberculosis (MTB) proteins, Ag85B and Hsp16.3, have been the focus of intensive research in recent years. These proteins have high sensitivity in bacterium-negative tuberculosis (TB) patients, and are valuable for the rapid diagnosis of bacterium-negative TB. Fusion proteins including multiple antigens such as Ag85B and Hsp16.3 provide improved sensitivity and specificity for serological diagnosis of active TB compared with a single antigen. Many studies have shown that the production of MAbs recognizing a specific repertoire of M. tuberculosis antigens and the tests based on monoclonal antibodies have been found to be valuable in positive detection of TB, particularly for smear-positive pulmonary TB. A number of MAbs are currently used for serodiagnosis of TB. Therefore, an Ag85B-Hsp16.3 fusion protein was expressed and purified using an E. coli system in this study. Three Ag85B-Hsp16.3 fusion protein-specific MAbs were generated by routine murine hybridoma techniques. The titer, specificity, and relative affinity of all three MAbs were determined by ELISA and the serological responses were analyzed. The levels of antigens in a proportion of TB patients were shown to be significantly higher than those in healthy controls. The sensitivity and specificity of the currently available detection systems is likely to be improved by the employment of a combination of these MAbs with others that are already in use.