Production of a monoclonal antibody (59.4) against canine lymphocyte surface antigen and its immunohistochemical application.
A monoclonal antibody was produced by immunizing BALB/c mice with freshly prepared canine thymocytes and peripheral blood leukocytes. The antibody, designated 59.4, was of the IgG1 subclass type and mainly reacted with lymphocytes. In single-color flow cytometric analysis, lymphocytes from the peripheral blood, thymus and spleen were graded into three categories according to their fluorescence intensity labeling by antibody 59.4: weakly, moderately and intensely positive cells. Two-color analysis revealed that a major population of CD8-positive cells were intensely labeled by antibody 59.4, but less than 50% of CD4-positive cells were moderately reacted with antibody 59.4. Immunohistochemically, thymocytes in the medulla showed moderately intense immunoreactivity to 59.4, but most lymphocytes in the cortex were negative in reaction. Immunostaining using antibody 59.4 demonstrated characteristic aggregations of 59.4-positive lymphocytes in the reticulum cell-free region of the thymic medulla. In the spleen, scattered lymphocytes in the outer layer of the marginal zone and in the red pulp were intensely labeled by antibody 59.4, while lymphocytes gathering in the mantle zone and periarterial lymphatic sheath (PALS) were moderately stained. Antibody 59.4 appears to recognize an antigen which is expressed by a more-differentiated T cell-lineage but not by immature T cells in the thymic cortex.