The Sheep Erythrocyte T-Dependent Antibody Response (TDAR)

The sheep erythrocyte T-dependent antibody response (TDAR) evaluates the ability of animals sensitized in vivo to produce primary IgM antibodies to sheep erythrocytes (sRBC). The assay enumerates the number of antigen-specific IgM antibody-producing cells in the spleen. When exposure to the test material takes place in vivo, as does sensitization, the actual quantification of the number of antibody-producing cells occurs ex vivo. Following the animal being euthanized, a single-cell suspension of spleen cells is prepared. These spleen cells containing the IgM-secreting plasma cells are incubated in a semisolid matrix of agar, sheep erythrocytes, and guinea pig serum as a single-cell layer between a Petri dish and glass cover slip. After a 3-h incubation period, lysis of sRBCs around each of the IgM-secreting antigen-specific plasma cells results in the formation of a clear plaque, which can easily be counted. The TDAR has been found to be the most sensitive functional assay for evaluating effects on the immune system, particularly the humoral immune component in young adult rodents. Data suggest, however, that it may not be possible to measure the TDAR in preweaning rodent pups due to the immature status of their immune cells. Nevertheless, the TDAR to sheep erythrocytes still remains the gold standard for evaluating the potential adverse effects of xenobiotics on the immune system.