Monoclonal Antibodies that Recognize a Broad Range of Mammalian Terminal Deoxynucleotidyl Transferases

Abstract

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for lymphocyte precursors in the bone marrow and thymus and for lymphoblastic leukemia and lymphoma cells. To simplify and enhance the detection and phenotypic analysis of these cells, we sought to develop monoclonal antibodies to this enzyme. In order to obtain antibodies that bind a variety of mammalian TdTs, mice were immunized with bovine TdT and the hybridoma secretions were screened by immunofluorescence assays on cultured TdT-positive and negative human lymphoblasts. Four monoclonal antibodies which bound specifically to TdT-positive lymphoblasts were characterized in detail. All four antibodies immunoprecipitated the native 60 kd TdT molecule from extracts of TdT-positive human lymphoblasts and bound specifically in immunoblot assays to the 43.8 and 11 kd proteolytic fragments of bovine thymus TdT. To assess whether the antibodies bound to related or distinct epitopes on bovine TdT, we measured the displacement of radiolabeled antibody from the immobilized enzyme by an excess of unlabeled heterologous antibody. These studies revealed that three of the antibodies competed for the same determinant on bovine TdT, while one antibody reacted with a distinct epitope. Antibody binding to either epitope, however, partially inhibited the enzymatic activity of bovine TdT. Specificity for TdT was tested by immunofluorescence and competition radioimmunoassays. In these assays, the antibodies did not stain a variety of known TdT-negative human hematopoietic cells and cell lines. both normal and neoplastic, nor were the antibodies displaced from purified bovine TdT by extracts of these TdT-negative cells. These results confirmed the cross-reactivity of the antibodies with human and bovine TdT. To assess cross-reactivity with TdT from other species, extracts of rabbit, mouse, and rat thymus were prepared and shown to specifically displace the antibodies from bovine TdT. Thus, these antibodies bound to TdT derived from at least five mammalian species. To determine whether these antibodies could be used to detect small subpopulations of TdT-positive cells, mixtures of TdT-positive and negative cells were prepared and stained with fluorescein conjugates of the antibodies. When assayed by flow cytometry, a population of 1% TdT-positive cells was easily detectable. We conclude that these monoclonal antibodies should be useful for the enumeration and analysis of TdT-positive cells in normal and neoplastic hematopoietic tissues from several mammalian sources, including man.