In vitro cell-mediated cytotoxicity against allogeneic erythrocytes in ginbuna crucian carp and goldfish using a non-radioactive assay

Cell-mediated cytotoxicity of clonal ginbuna crucian carp leukocytes against allogeneic erythrocytes is described using a sensitive non-radioactive in vitro assay. Hemoglobin released from target erythrocytes after cell-mediated erythrolysis was detected by tetramethylbenzidine (TMB). TMB assay showed clear correlation with a 51Cr-release assay and even exhibited higher cytotoxicity. The use of erythrocytes as target cells has several advantages over a conventional 51Cr-release assay. Erythrocytes do not have cytotoxic activity, are relatively homogeneous, are available in large numbers and erythrocyte donors need not be killed. Leukocytes from fish sensitized by erythrocyte injection or scale grafting efficiently lysed allogeneic erythrocytes, but did not kill isogeneic or autologous erythrocytes. In contrast, leukocytes from unsensitized fish did not lyse allogeneic erythrocytes and repeated sensitizations by allogeneic grafts were necessary to induce cytotoxic cells. Effector cells isolated from peripheral blood showed a higher cytotoxic effect toward allogeneic target cells than effector cells isolated from kidney. These studies support the hypothesis that fish are capable of a genetically restricted specific cell-mediated cytotoxicity.