Partial purification of lymphoblasts after in vitro immunization increases the yield in Ig-producing hybridomas

Using in vitro immunization with a human plasma protein (apolipoprotein-A1) as antigen, we have shown that it is possible to prepare more monoclonal antibodies using a ten-fold lower concentration of antigen compared to in vivo immunization procedures (Weech et al., 1985). In addition, we can increase the number of Ig-producing hybridomas after in vitro immunization by a simple one-step separation of the lymphoblasts on a Percoll gradient before the fusion procedure. In order to apply this procedure to in vivo immunization techniques, it is necessary to expand the B-blast/plasma cell population by culturing the spleen cells for 4-6 days before fusion. Only antibodies of the IgM class were produced with the in vitro technique. However, by combining in vivo priming with in vitro immunization, it is possible to produce specific antibodies to both IgG and IgM classes.