Purification of MPB70 and production of specific monoclonal antibodies

MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was not detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans.