In vitro immunization of murine lymphocytes using immobilized immunogens

Murine splenic lymphocytes were immunized in vitro using immobilized antigens. Immobilization was achieved by covalently linking the antigens to Sepharose beads. Tetanus toxoid (TT) was used as test antigen in soluble and immobilized forms. The outcome of in vitro immunization was assayed in terms of the number of antigen-specific antibody-forming cells (AFCs) enumerated by filter immuno plaque assay. The AFC specific to TT were significantly higher in cultures stimulated with immobilized antigen as compared with soluble antigen. The effect of various concentrations of antigen, time kinetics, effect of serum, and leucyl-leucine O-methyl ester treatment on the in vitro-immunization system has been studied. The results indicate that immobilized antigens are more potent than their soluble counterparts in vitro and hence are useful in in vitro-immunization protocols.