Preparation of monoclonal antibodies against DNA-cytosine-5-methyltransferase from human placenta.

Mouse monoclonal antibodies were prepared against DNA-cytosine-5-methyltransferase (EC 2.1.1.37) from human placenta by conventional hybridoma technology. Spleen cells from BALB/c mice immunized with highly purified enzyme were fused to X-63 Ag8.653 mouse myeloma cells. After the hybrid selection in HAT medium individual clones were screened for production of antibodies directed against the enzyme by use of solid-phase ELISA or RIA in which highly purified DNA methyltransferase was either immobilized on microtiter plates or 125I-labeled enzyme was used as a tracer. Positive clones were subcloned, re-screened in the same system and the presence of antibodies directed against DNA methyltransferase was definitively proved in a test system in which the enzyme activity was removed from the solution in immune complexes precipitated by anti mouse immunoglobulin antibodies. From more than 3800 constructed clones 8 were selected which produced antibodies against DNA methyltransferase from human placenta. These antibodies may serve as a useful tool for analysis of DNA methyltransferase structure, intracellular localization and molecular heterogeneity of this enzyme.