Identification by Two-Dimensional Gel Electrophoresis

We have previously identified proteins in fractions of culture filtrate ofMycobacterium tuberculosis with the capacity to induce cytokine production in monocytes, by using a technique we defined as "monocyte Western blotting" (immunoblotting). In this series of experiments, we have extended this technique to two-dimensional gel electrophoresis and have identified a novel 58-kDa protein ofM. tuberculosis which induces production of tumor necrosis factor by human monocytes. Nitrocellulose particles bearing this protein were used to develop murine monoclonal antibodies by the technique of intrasplenic immunization. The protein was purified by preparative isoelectric focusing and gel electrophoresis and subjected to N-terminal amino acid sequence analysis. As tumor necrosis factor is a mediator of both pulmonary necrosis and macrophage activation for intracellular killing, this 58-kDa protein may play an important role both in the immunopathogenesis of tuberculosis and in mycobacterial immunity.