Antibodies directed to singly and doubly charged forms of the p-azobenzenearsonate group

Abstract

The anti- p-azobenzenearsonate antibodies from the IgG fraction of serum of rabbits hyperimmunized with bovine γ-globulin- p-azobenzenearsonate have been separated into two fractions by successive adsorptions with a specific immunoadsorbent, first at pH 5 and then at pH 9·5. A portion of the antibodies adsorbed at pH 5 was eluted by hapten at pH 5. This fraction contained antibodies which appeared to be directed mainly against the singly ionized form of the benzenearsonate group. Antibodies adsorbed at pH 9·5 were eluted by hapten at pH 9·5 to provide a fraction of antibodies which appeared to be directed largely against the doubly ionized form of the benzenearsonate group. The differences in specificity of antibodies in the two fractions were demonstrated by measuring average binding constants ( K o ) at pH 5 and pH 9 with p-azobenzenearsonate. The pH 5 fraction bound the hapten in the singly charged form at pH 5 with a K 0 10-fold greater than that of the pH 9·5 fraction. The pH 9·5 fraction bound the hapten at pH 9 with a k 0 50-fold greater than that at pH 5, whereas the pH 5 fraction bound the hapten at pH 9 with a K 0 only 5-fold greater.