During the assay, a fraction of the free receptor is captured by the solid phase ligand and subsequently labeled with a fluorescent secondary molecule (Figure 1). The short contact time with the solid phase does not allow significant dissociation of the pre-formed complexes in the solution. Solution dissociation is thus “kinetically excluded” from contributing to the captured receptor and the resulting signal provides a measure of the free receptor in the solution.
Measuring the free receptor as a function of total ligand in a series of equilibrated solutions enables calculation of the equilibrium dissociation constant (K). Measuring the free receptor with several points before equilibrium enables measurement of the association rate constant (k). The off rate (k) can also be directly measured, however it is usually calculated from the measured K and measured k, (k = K * k).
Kinetic exclusion assays have been used to measure K’s in the nanomolar to femtomolar range.