Kinetic exclusion assay technology: characterization of molecular interactions

Characterization of intermolecular interactions in terms of affinity, binding kinetics, stoichiometry, specificity, and thermodynamics can facilitate the selection of lead compounds in the discovery and development of protein therapeutics. KinExA (Sapidyne Instruments, Inc., Boise, ID) is a relatively new technology that is gaining use in characterizing molecular interactions, particularly with respect to antibody therapeutics. KinExA offers a platform that allows the measurement of true equilibrium binding affinity and kinetics using unmodified molecules in solution phase. This is accomplished by using a solid-phase immobilized molecule to probe for free concentration of one interaction component after allowing sufficient time to reach equilibrium (affinity measurements), or under pre-equilibrium conditions (kinetics). In this review, the theory behind KinExA technology is discussed, and examples of applying this technology to antibody characterization are provided. Finally, a comparison among KinExA, Biacore (surface plasmon resonance), and isothermal titration calorimetry is presented, and potential future improvements and applications of KinExA are discussed.