Antibody (Ab) Competition Binding Assay (IC50 and Ki)
Live cells containing the target of interest are incubated together with a [125I]-Ab at a single concentration along with 10 - 12 concentrations of unlabeled Ab in 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 plates are counted for radioactivity to determine the level of target-bound radioligand and the results are plotted to obtain the IC50 and Ki values for the Ab-target interaction.