Competitive Radioligand Binding Assay (IC50 and Ki)
Cell or tissue homogenate containing the receptor of interest is incubated together with a radioligand for the receptor and a range of concentrations of the test (competing) drug. Typically up to 11 concentrations of the test drug are evaluated over a three log unit range and with triplicate (or quadruplicate) replicates at each concentration.
After equilibrium has been reached, receptor-bound radioligand is separated from non-bound radioligand using either.
- Filter-based Separation
- Scintillation Proximity Assay (SPA)
The filters or SPA plate are counted for radioactivity to determine the level of receptor-bound radioligand and the results are plotted to obtain the IC50 and Ki values for the drug-receptor interaction.