Isolation and Characterization of Electrophoretically Homogeneous Rabbit Antihapten Antibody Populations

Abstract

Abstract A method is described by which liquid isoelectric focusing over a pH range 5 to 8 has been used on a preparative scale for the further purification of specifically purified antibodies directed against the positively charged p-azophenyltrimethylammonium hapten (anti-Ap antibodies). The anti-Ap antibodies separated into 4 to 8 major fractions and 10 to 16 minor fractions. More than 95% of the antibodies directed against the positively charged hapten had isoelectric points between pH 5 and 8. The charge, structural, and functional homogeneity of several of the major fractions was assessed by a number of independent criteria. The charge homogeneity of three of the electrofocused antibody fractions was demonstrated by the presence of monodisperse zones in electrophoresis, by focusing the major fractions over a narrow pH range, and also by acrylamide disc electrophoresis of fully reduced and alkylated light chains in alkaline urea gels. The structural homogeneity of two of the electrofocused antibody fractions was demonstrated by the finding of single NH2-terminal residues on the isolated, fully reduced and alkylated light chains. Sequence studies on the isolated light chains of these fractions showed each to have a single coherent sequence compared with the parent antibody which was typically heterogeneous. These antibody fractions showed only the b4 allotypic marker on their light chains and 95 to 96% of the heavy chains contained the a1 allotypic marker, although the unfractionated antibodies were heterozygous (a1, a3) with respect to the heavy chain allotypic markers. The functional homogeneity of these electrofocused antibody fractions was determined by equilibrium dialysis using the 125I-labelled p-iodophenyltrimethylammonium hapten. The results of the binding plots, using both the Scatchard and Sips analyses, demonstrated that the specifically purified anti-Ap antibodies had a heterogeneity index of 0.6 and contained 2 moles of combining sites per mole protein, while each of the electrofocused anti-Ap antibody fractions had a heterogeneity index of 1.0 and contained 2 moles of combining sites per mole protein.