Determination of antibody hapten association kinetics: a simplified experimental approach

A simplified method was developed for the determination of antibody hapten association kinetics, that permits the study of high affinity interactions with second order forward rate constants of the order of 10⁷ to 10⁸ M^-1 sec^-1. Use of tritiated haptens of high specific activity, and antibodies of high affinity allows reactions to be run at initial hapten and antibody concentrations of the order of 10^-9 to 10^-10 M, well below the level at which mixing becomes the rate limiting step. Separation of antibody bound from free hapten by the use of dextran coated charcoal can be carried out with sufficient rapidity (2 sec) for the systems under investigation to be largely undisturbed. With this technique, the association of ³H ouabain with rabbit ouabain specific antibody was found to occur with a rate constant of 0.8 x 10⁷ M^-1 sec^-1, similar to association rates of dye haptens with antibodies of substantially lower affinity. The ratio of this association rate constant to the independently determined dissociation rate constant was 5.4 x 10⁹ M^-1, in satisfactory agreement with a k(o) value of 3.5 x 10⁹ M^-1, determined by Sips analysis of data obtained under equilibrium conditions. This approach should be applicable to the direct kinetic assessment of numerous high affinity antibody hapten systems of current interest.