Affinity Repertoire of Monoclonal Antibodies Obtained by Primary or SecondaryIn VitroImmunization

Abstract

The effect of the antigenic dose (1 ng-10 micrograms DNP-KLH/ml) on affinity constants of monoclonal antibodies produced by in vitro immunization was investigated. A statistically significant (p = 0.0000) trend could be demonstrated since the higher antigen concentration that was used during the five day culture the lower was the obtained Kass value. The range of affinities of the IgM monoclonal antibodies was similar to what was obtained from IgM antibodies produced by in vivo immunizations. However, the combination of a primary in vivo immunization followed by a secondary in vitro stimulation schedule resulted in an antigen dependent generation of high affinity isotype switch variants, exhibiting even higher affinities compared to monoclonal antibodies produced from only in vivo immunized animals. These results demonstrate that it is possible to elicit both an antigen-driven primary or secondary response in vitro.