Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations

Abstract

Abstract Liquid isoelectric focusing profiles of a number of conventionally raised rabbit antibodies directed against acidic, neutral, and basic haptens have been determined. All antibody preparations showed a degree of heterogeneity similar to that already reported for antibodies to the basic hapten p-azophenyltrimethylammonium. There was little difference in the degree of heterogeneity found in the focusing profiles of antibody from different rabbits directed against the same hapten. There appear to be between 10 and 30 individual antibody species in each case, but some of this complexity may be a result of variable, postsynthetic deamidation. Isoelectric focusing profiles of the antigens also showed considerable heterogeneity. The mean isoelectric point of both rabbit and bovine γ-globulins was approximately pH 6.0, and the distribution of the isoelectric point, pI, values of the focused specifically purified rabbit antibodies about this point was reciprocally related to the pI of the focused antigens. It appears that a single charge unit causes a displacement of the mean pI value by approximately 0.1 pH unit. The displacement found for various antibody preparations indicates antibodies to acidic and basic antigens may differ by as many as 19 charged residues.