Immune response to a hapten of fluorescein isothiocyanate in a single mouse analyzed by two‐dimensional affinity electrophoresis

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Abstract
Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two‐dimensional affinity electrophoresis (2D‐AEP). Anti‐FITC antibodies were induced by immunization with FITC‐conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL‐D6) was separated by 2D‐AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti‐FITC antibodies in the antiserum can be generated by a single clone of anti‐FITC antibody‐producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti‐FITC antibodies with a low diversity but high specificity to FITC.