Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two‐dimensional affinity electrophoresis

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Abstract
The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 μg, 5 μg, 50 μg, 500 μg and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots. showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 μg) most of the families showed high affinity (Kd < 1 μM). When the dose was increased, not only high affinity antibodies but also intermediate (1 μM < Kd < 50 μM) and low affinity (Kd > 50 μM) antibodies were produced. The increase of FITC-BSA up to 500 μg markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgGl, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgGl, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2 and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.