Separation and isolation of lymphocyte subpopulations of different affinity for the antigen

In order to separate, isolate, and determine the number and distribution of the subpopulations of lymphocytes of diverse affinities that are present in an immune response toward a single hapten, anti-trinitrophenyl (TNP) lymphocytes from immunized animals were purified by cell chromatography. Non-adherent spleen cells were passed through a column consisting of TNP-substituted polyacrylamide beads. The retained cells were eluted by applying a linear concentration gradient of TNP-lysine. Elution profiles having a limited number of peaks were obtained in all cases. The avidity of the cells in each fraction was measured by inhibition of formation of immune rosettes by free hapten. Results showed that each peak was located along the gradient according to its affinity since there was a direct correlation between the affinity and the concentration of hapten needed for the elution. The cells in each peak appeared to belong to a homogeneous subpopulation as shown by the slope of the curves obtained in the determination of avidity, suggesting that each peak corresponded to one expanded clone.