Strategy for fractionating high-affinity antibodies to steroid hormones by affinity chromatography

Abstract

A general strategy for fractionating high-affinity antibodies to steroid hormones has been developed and applied to the fractionation of an antiserum to testosterone 3-(O-carboxymethyl)oxime–bovine serum albumin. If the antibodies interacting with a stationary phase containing a low concentration of immobilized steroid are considered as monovalent binders, a simple equation can be applied to show that the affinity of the antibody–stationary phase interaction must be higher than about 2 × 10⁶ l mol^–1 in order to avoid the loss of antibodies during the loading and washing of the column. Conversely, to elute the retained antibodies, the affinity must be decreased to a value lower than about 2 × 10⁵ l mol^–1 and the dissociation rate constants of the antibody–steroid complexes must be ≫ 1 s^–1. In order to prepare an affinity column that satisfies these conditions, the ligand to be immobilized was selected on the basis of the cross-reactions of the antiserum with several testosterone derivatives. Moreover, the dissociation rate constants of several antibodies of known affinity were measured, together with the effect of acidic buffers and various organic solvents on the antiserum–testosterone interaction. Then, an affinity column, prepared by coupling testosterone 17β-acetate to AH-Sepharose 4B, was used to load the antiserum without loss of antibodies during the washing step. The retained antibodies were successfully eluted by a mixture of 30% dioxane in phosphate–citrate buffer (pH 3.4). The affinity of the eluted antibodies was in the range 7 × 10⁹–2 × 10¹¹ l mol^–1 and was linearly related to the retention volume. These results confirm that high-affinity antibodies can be fractionated to steroid hormones by a proper choice of the ligand on the stationary phase and the eluent composition.