What “Affinity Determination” Means Affinity determination is the process of quantifying how strongly two molecules bind to each other—commonly protein–protein, antibody–antigen, receptor–ligand, or protein–small molecule interactions. In most bioscience and drug discovery contexts, affinity is summarized by the equilibrium dissociation constant (KD): Lower KD = higher affinity (tighter binding). KD is an equilibrium quantity, meaning it reflects the balance between binding and unbinding at steady state. A related way to express the same concept is the association constant (KA), where KA = 1 / KD. The Core Parameters: KD, KA, kon, koff Affinity can be described in two complementary ways: 1) Equilibrium view (how much binds at steady state) KD (M): concentration at which half of binding sites are occupied in a simple 1:1 interaction model. KA (M⁻¹): binding strength as an association constant (inverse of KD). 2) Kinetic view (how fast binding happens) Many instruments determine affinity by measuring rates: kon (M⁻¹·s⁻¹): association/on-rate (how quickly complex forms) koff (s⁻¹): dissociation/off-rate (how quickly complex falls apart) For a 1:1 interaction: KD = koff / kon (at equilibrium). Surface-based biosensors often estimate affinity by extracting these rates from real-time binding curves. Why Affinity Determination…
