SPR (Biacore) assays provide a method for determining the affinity and binding kinetics of a ligand for its receptor. The technique measures the real-time binding association and dissociation rates using Surface Plasmon Resonance (SPR).
In this method, one of the binding partners (e.g. the receptor protein) is immobilised onto a biosensor surface (we use a Biacore T200). The second partner, for example the drug ligand, is then continuously flowed across the biosensor surface, where it binds to the immobilised receptor. Binding is measured as a change in resonance units (RUs) on the biosensor surface. Measuring the increase in binding over time for a given ligand concentration gives the association rate (ka or Kon). Ceasing to flow drug ligand and changing to buffer alone then allows the ligand to wash off the receptor. Measuring the decrease in bound ligand over time gives the ligand dissociation rate (kd or Koff). The affinity of the ligand for the receptor (the equilibrium dissociation constant, KD) is calculated from the kinetic association and dissociation rates (kd/ka) for several different ligand concentrations.
Binding Kinetics versus Equilibrium Binding
SPR (Biacore) measurements can be complementary to radioligand equilibrium binding affinity, providing additional binding dynamics. For instance, two ligands may have the same binding affinity, but kinetic measurements can show differences in dissociation rates that may lead to selection of a ligand with a slower off-rate as a more effective receptor blocker.
SPR is a label-free technique: it does not require either of the binding partners to be labelled with a radioisotopic or fluorescent label. The SPR technique therefore provides an alternative to a radioligand binding assay where a [3H]-radioligand is unavailable or custom radiolabelling is impractical, or where incorporating a bulky isotope (e.g. [125I]) adversely affects the ligand structure and hence the affinity of the ligand for its receptor.
Receptor-ligand kinetic affinity determinations are usually successful for receptors with larger ligands that have slower association and dissociation rates, e.g. chemokine receptors and cytokine ligands. For receptor-ligand interactions with faster association and dissociation rates, kinetic affinity analysis can be inappropriate. For these situations, equilibrium binding affinity is more appropriate.
The example shown is for the binding of the endogenous estrogen, 17β-estradiol, to human estrogen receptor α (ERα). Recombinant his-tagged human ERα was captured onto a nickel-treated NTA sensor chip surface, then estradiol from 0.1 to 30 nM was bound to the ERα for 60 seconds, followed by 180 seconds dissociation (only part is shown).
Blank-subtracted average binding responses were fitted to a saturation equilibrium binding model to generate a KD of 0.18 nM. (IUPHAR reference KD for 17β-estradiol = 0.16 nM).