Requirement of the immunogen (E. coli beta-galactosidase) for the response towards a determinant responsible for antibody-mediated enzyme activation, while antibodies binding some other site can be elicited by mitogen alone.

Virgin mouse spleen cells cultured in vitro without antigen or mitogen produced a measurable amount of IgM binding E. coli beta-galactosidase (beta-gal). Lipopolysaccharide (LPS) and LPS plus antigen enhance this response, which cannot be considered truly polyclonal since it does not include the production of antibodies directed towards a conformation-dependent determinant, responsible for the activation of a mutant beta-gal, and known otherwise to be highly immunogenic. By priming in vivo with alum-treated beta-gal (unable to elicit activating antibodies), an activating response is obtained in vitro by LPS plus antigen, but not by LPS alone. These results are compatible with a two-signal requirement for the activation of B cells and may be explained as follows: (a) the mitogen, in absence of immunogen, stimulates those clones which have received a specific signal from cross-reacting structures in the environment: (b) instead, no cross-reaction are available for the conformation-controlled structure of the "activating" determinant; thus, intact immunogen is required as well as mitogen. Because of this "uniqueness", the molecule of beta-gal offers a highly specific tool to probe carrier-hapten relationships in native proteins.