AFFINITY MATURATION OF ANTIHAPTEN ANTIBODIES IN A SINGLE-MOUSE ANALYZED BY 2-DIMENSIONAL AFFINITY ELECTROPHORESIS

Affinity maturation of anti-hapten antibodies in an inbred mouse (BALB /c) was analyzed by two-dimensional affinity electrophoresis (2D-AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) gr oup were separated into a large number of IgG families according to th e differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the Ig G families consisted of several spots showing an identical affinity fo r the ligand but different pI, and the spots were specifically stained by immunostaining with an anti-murine IgG subclass antibody. These re sults lead us to the conclusion that each of the IgG families is deriv ed from a single clone of antibody-producing cells specific to DNP. Th e mass and affinity of the IgG families varied in the course of immuni zation with DNP-conjugated chicken serum albumin (DNP-CSA). The first injection of DNP-CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the m ass or affinity of the IgG families significantly. This suggests that an antigen-induced somatic mutation of the immunoglobulin gene does no t occur frequently to mature the affinity of antibodies by the additio nal injections after the second injection. 2D-AEP enables us to analyz e the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immun ization.