Antigens on mouse and rat lymphocytes recognized by rabbit antiserum against rat brain: the quantitative analysis of a xenogeneic antiserum

Abstract

Quantitative assays for measuring the binding of xenogeneic antiserum to dispersed cell suspensions are described. Cells were incubated with unlabeled xenogeneic antiserum and the antibody bound measured indirectly by a second binding step with 125I-labeled anti-immunoglobulin antibody. This indirect radioactive binding assay was calibrated by measuring, with a radioimmunoassay, the true amount of antibody bound in the first step. With these methods one can measure the strength of antisera, quantitate the number of antigenic sites, and partially differentiate determinants being recognized on cell surfaces. The binding of rabbit anti-rat brain antiserum to rat and mouse lymphocytes was analyzed in detail. After absorption of the antiserum with rat liver, the antibody remaining recognized lymphocyte antigens that were distributed among various rat and mouse tissues in quantities identical to Thy-1.1 antigen. Thus, at saturation, 670 000 Ig molecules from liver-absorbed rabbit antiserum were bound per rat thymocyte, and the antiserum bound to 90%, 21%, 4% and 2% of rat thymocytes, spleen, lymph node and thoracic duct lymphocytes, respectively. With mouse tissues, 90% 24% and 50% of thymocytes, spleen and lymph node cells, respectively, were labeled. In rat brain the concentration of xenoantigen increased with age, while in thymocytes the full adult amount was present at birth. Three antigenic determinants could be defined with the liver absorbed rabbit antiserum: the Thy-1.1 antigen, a rat specific antigen and an antigen cross-reacting between rat and mouse tissues. All 3 may be on the Thy-1 molecule. The anti-brain antiserum contained about 0.05 mg/ml of antibody specific to these xenoantigens.