Immunologic and biochemical analysis of TNT-1 and TNT-2 monoclonal antibody binding to histones

Two novel murine monoclonal antibodies that bind to intracellular antigens, designated TNT-1 and TNT-2, have been generated by immunizing mice with nuclear extracts from human lymphoma cells. The monoclonal antibodies were initially identified by indirect immunofluorescence on lymphoma cell lines and subsequently were found to stain the nucleus of all cell types from several species including plants by indirect immunofluorescence techniques. Immunoblot analysis demonstrated that TNT-1 bound to a protein of 22 kD and TNT-2 bound to two proteins of 15 and 22 kD, consistent with the known molecular weight of histones. To characterize their immunoreactivity, competition radioimmunoassays were performed using purified histone fractions H1, H2a, H2b and H3. By these assays, TNT-1 was found to bind to histone fraction H1 and TNT-2 to an epitope common to histone fractions H1 and H3. Histones are found in abundance in the nucleus of the cell and are known to play a major role in chromosome structure and gene expression. Upon cell death, histones remain tightly bound to DNA and consequently provide an abundant intracellular antigen that can be exploited in targeting necrotic cells, such as those found in tumors.