We compare the classic method of equilibrium dialysis for achieving thermodynamic equilibrium with a method in which polyethylene glycol is used to separate antibody-bound and unbound steroid. Antisera to both polar and nonpolar steroids were studied. Comparable Scatchard plots were obtained by the two methods. Equilibrium constants of association of lo9 to IO'" litersimol of the different antisera were determined by both methods. The described method should simplify the study of steroid-antibody interaction. In addition, this method will permit investigators to characterize quickly and conveniently the antisera to be used for sensitive radioimmunoassays.
"An important requirement for unambiguous determination of ligandprotein interaction is for the system to be in a state of thermodynamic equilibrium" (I). Therefore, the quantitative study of steroid-protein interaction is greatly dependent upon the methods employed for separating bound from unbound steroid.' Although several methods for separating antibody-bound and unbound steroid have been employed for radioimmunoassays (2-6). the technique of equilibrium dialysis has remained the reference method for studying antibody-steroid interaction. This report compares the efficacy of a method in which polyethylene glycol is used to separate antibody-bound and unbound steroid with the classic method of equilibrium dialysis for achieving thermodynamic equilibrium.