Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods.
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 to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, 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 h 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.
Drake
Andrew W
AW
Abgenix, Inc., 6701 Kaiser Drive, Fremont, CA 94555, USA.
Myszka
David G
DG
Klakamp
Scott L
SL
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D020349:Surface Plasmon Resonance / Q000379:methods*; D013816:Thermodynamics; D013997:Time Factors
D000906:Antibodies; D000941:Antigens