Characterizing High Affinity Antigen/Antibody Complexes by Kinetic and Equilibrium Based Methods
The development and application of monoclonal antibodies as drugs requires accurate and precise characterization of the antigen/antibody binding constants. Anticipating the required affinity for a therapeutically efficacious monoclonal antibody (mAb) is complex. Generally, the equilibrium dissociation constant for the antigen/mAb complex should be less than 100–1,000 picomolar (pM), depending upon the nature of the target, the desired function of the antibody, and the localized concentration of the antigen in the diseased tissue, among other factors. Measurement of the equilibrium dissociation constant (K D), association rate constant (k a), and dissociation rate constant (k d) for these high affinity antibodies is difficult because of three independent reasons: (1) the time for the antigen/antibody complex to reach equilibrium can be very long, on the order of days (2) usually, the k d for such a tight complex is extremely low, requiring long periods of data collection in order to discern enough information to predict complex stability, and (3) in cases where the k d is easily measurable (greater than 5×10−4s−1), the k a can be very fast, greater than 1×107M−1s−1.
KeywordsEquilibrium Dissociation Constant Association Rate Constant Binding Response Bead Pack Biosensor Surface
equilibrium dissociation constant
association rate constant
dissociation rate constant
- surfactant P-20
The computer algorithm for the simulation of the time to equilibrium for a 1:1 reversible interaction was graciously provided to us by Steve Lackie and Tom Glass of Sapidyne, Inc, Boise, ID.
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