|
In Vitro Receptor Occupancy: Radioligand Binding Assay
Overview: A competitive radioligand
binding assay enables the IC50 and Ki
value
for the receptor-drug interaction to be determined by measuring the ability of
increasing concentations of the
(non-radioactive) test drug to compete with binding of a
radioactively-labeled drug for the targeted receptor in a
homogenate. Alternatively, the receptor density (Bmax) in the
tissue can be obtained by using a saturation radioligand binding
assay in which receptor binding is determined in the presence of increasing concentrations of the radioligand alone.
Radioligand binding assay protocol
(competition assay format): Incubate a range of concentrations of
the test drug together with a radioligand for the receptor and a
tissue homogenate or cell membrane homogenate containing the
receptor. After equilibrium has been reached, separate bound
from free radioligand using a 36-well filtration apparatus.
Count radioactivity on the filters and plot results to obtain
IC50 and Ki values for the test drug.
Testing: For competition assays, an
IC50 and Ki value is obtained for
each test drug by measuring binding over 10 drug concentrations
over a three log unit range, with triplicate
determinations at each concentration and three tubes to
determine non-specific binding (= 36 assay tubes). For protocols where the goal instead is to
determine receptor density (Bmax) in the tissue, a
hot saturation approach is used. Binding is measured using 6
concentrations of the radioligand
over a two log unit range, with triplicate determinations at each
concentration and three tubes at each concentration to determine
non-specific binding (= 36 assay tubes).
Assay information: Our receptor
binding assays are conducted on a custom basis.
Literature-based protocols for the assay
conditions, including radioligand, receptor preparation and reference drug will be used
where available and the protocol approved by the client prior to starting work.
At least one reference compound will be included for each assay
for assay validation.
Example data: |
