68.    Characterizing High Affinity Antigen/Antibody Complexes by Kinetic and Equilibrium Based Methods

Analytical Biochemistry (2004) In press

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar (nM) to picomolar (pM) equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for four hours, we were able to measure dissociation rate constants (kd) on the order of 1 X 10–5 s-1. To characterize high affinity interactions by KinExA, samples needed to be equilibrated for up to 35 hours to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution phase and solid phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high affinity interactions.