Using a KinExA assay for making Kd measurements is ideal because measurements are made without perturbing the solution equilibrium and with unmodified molecules, unfettered, in solution. For an accurate Kd determination the concentration of receptor in the samples should be near or below the Kd. With the tight binding antibodies (low or sub pM) being developed now, the measurements need to be made at these very low sample concentrations. The KinExA's ability to make quantitative measurements at these low concentrations allow for accurate Kd measurements for those very tight binders.
Binding curves are dependent on Kd and receptor concentration. A binding curve can be generated by making a series of samples with constant receptor concentration and a titration of ligand. After equilibrium is reached, a KinExA measurement is made of the free receptor concentration in each sample of the titration series. Therefore the free receptor directly represents the binding curve. The binding curve generated is then analyzed to find the Kd (for low ratio curves) or receptor active concentration (for high ratio curves). Multiple curves with different receptor concentrations may be analyzed together to get both Kd and active receptor concentration.