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 fres hly prepared canine thymocytes and peripheral blood leukocytes. The an tibody, designated 59.4, was of the IgG1 subclass type and mainly reac ted with lymphocytes. In single-color flow cytometric analysis, lympho cytes from the peripheral blood, thymus and spleen were graded into th ree categories according to their fluorescence intensity labeling by a ntibody 59.4: weakly, moderately and intensely positive cells. Two-col or analysis revealed that a major population of CD8-positive cells wer e intensely labeled by antibody 59.4, but less than 50% of CD4-positiv e cells were moderately reacted with antibody 59.4. Immunohistochemica lly, thymocytes in the medulla showed moderately intense immunoreactiv ity to 59.4, but most lymphocytes in the cortex were negative in react ion. Immunostaining using antibody 59.4 demonstrated characteristic ag gregations of 59.4-positive lymphocytes in the reticulum cell-free reg ion of the thymic medulla. In the spleen, scattered lymphocytes in the outer layer of the marginal zone and in the red pulp were intensely l abeled by antibody 59.4, while lymphocytes gathering in the mantle zon e and periarterial lymphatic sheath (PALS) were moderately stained. An tibody 59.4 appears to recognize an antigen which is expressed by a mo re-differentiated T cell-lineage but not by immature T cells in the th ymic cortex.