Radioligand Binding Assay
Radioligand receptor binding assays can be competition, saturation or kinetic. Competitive (displacement) receptor radioligand binding assays are used to determine the relative affinities (Ki values) of test compounds for binding to a receptor site in a membrane homogenate or cells. They are performed by incubating a range of concentrations of the unlabeled test compound with a single concentration of radioligand and measuring the IC50 (nM) of the compound to competitively inhibit binding of the radiolabeled ligand to its receptor. Saturation ligand binding assays allow determination of both the number of binding sites, Bmax (fmol/mg protein or sites per cell), in the tissue or cultured cells and the dissociation constant, Kd (nM), of the radioligand and are performed by measuring levels of receptor binding of the radioligand with increasing concentrations of the radioligand alone. Kinetic assays are used to determine the association and dissociation rates of a radiolabeled ligand from a receptor and can be used to provide additional information on the receptor-ligand interaction or for optimizing assay conditions.
Radioligand binding assay protocol: filtration
The membrane homogenate or cultured cells are incubated with both the radioligand and the competing test compound. Once equilibrium has been attained, receptor-bound radioligand is separated from free (unbound) radioligand using a 96-well filtration apparatus. The receptor-bound radioactivity trapped on the filters is counted and the results plotted to obtain IC50 and Ki values for the test compound or to determine Bmax for the receptor levels.
Radioligand binding assay protocol: scintillation proximity assay
The enzyme, receptor or cultured cells are absorbed onto the surface of SPA beads. The receptor-coated beads are incubated together with a radioligand and the competing test compound. After equilibrium has been reached, light emission from the SPA beads is counted using a 96 well plate-reader and the results plotted to obtain IC50 and Ki values for competition with radiotracer binding by the test compound.
Scintillation proximity assay. Radioactive beta particles emitted on or close to the surface of the beads gives rise to light emission from the beads whereas those emitted far from the bead are absorbed by the solvent.
Standard study designs:
Competition assays: Ten concentrations of the test article
over a five-log unit range, duplicate determinations.
Saturation assays: Eight concentrations of the labeled test article over a two-log unit range, duplicate determinations.
Kinetic assays: 8 - 12 time points to obtain association or dissociation rate constants, duplicate determinations.
Competitive radioligand binding assay. Displacement of [3H]SR141716A binding to CB1 cannabinoid receptors in a brain homogenate by the cannabinoid agonists AM2233 and WIN55212-2. Displacement curves are shown fit to a two-site model with Ki values of 0.08 nM and 17 nM for AM2233 and 2 nM and 43 nM for WIN55212-2.
Saturation radioligand binding assay: Specific binding of a radioligand for the brain BZD receptor in human post-mortem brain tissue. Binding was to a single saturable site with Kd 3.2 nM and Bmax 1584 fmol/mg protein.