The secondary anti-erythrocyte response of rabbit spleen cells stimulated in vitro.

Suspensions of spleen cells from rabbits immunized to sheep erythrocytes and stimulated by the specific antigen produced high numbers of antibody-synthesizing cells. Several factors were found to affect secondary antigenic stimulation . The extent of the immune response elicited was a function of antigen concentration. Maximum was obtained by stimulation with 10 to 10 erythrocytes/10 spleen cells; 10 erythrocytes inhibited the response. The maximum usually occurred on days 4–6; glutamine increased the rate at which antibody-synthesizing cells appeared. It was found that the source of the normal serum supplement used in the growth medium markedly affected the results. Storage of rabbit serum reduced its efficacy; after 6 months storage at –10° little or no response was obtained. The addition of specific antiserum on days 1, 2 or 3 reduced the cellular response. With cells from twenty-six rabbits tested 6–39 months after immunization, maxima of 1700–29,700 antibody-synthesizing cells/10 spleen cells were attained. No relationship was evident between the time from immunization and the extent of the response. The activity of the extracellular antibody produced by the cells correlated roughly with the number of antibody-synthesizing cells. The antibody elicited was specific and stable on storage at –10° for 2 years. On the basis of sensitivity to 2-mercaptoethanol, it was a macroglobulin. Disc electrophoresis and identification of the haemolytic component showed that the antibody synthesized migrated with the γ-globulins, at the same rate as the active component in anti-erythrocyte rabbit serum.