Radioligand Binding Assays

Radioligand Binding Assays

Radioligand binding assays are used to characterize the binding of a drug to its target receptor. They can provide information on both the affinity and mode of interaction of the drug with its receptor. For example, radioligand binding assays can provide information on whether the test drug is binding to the endogenous ligand binding site or is instead acting on another allosterically coupled binding site, such as a blocker of the receptor-coupled ion channel.

Three types of receptor binding experiments may be performed:

Kinetic Binding experiments, where the binding of one or more concentrations of radioligand is measured at an incrementing series of time points, and analysed to estimate association (kon) and dissociation (koff) rate constants.

Saturation Binding experiments, where binding of an increasing series of radioligand concentrations is measured at equilibrium and analysed to determine its binding constant (affinity constant (K), or dissociation constant (Kd)) and the concentration of specific binding sites for the radioligand (BMAX).

Competition Binding experiments, where the binding of one or more fixed concentrations of a radioligand is measured at equilibrium in the presence of an incrementing series of concentrations of a non-labelled compound and analysed to determine the half maximal inhibotory concentration (IC50) and the equilibrium inhibitor constant (Ki).

Kinetic Binding Assays (KON & KOFF)

Association: Cell or tissue homogenate containing the receptor of interest is incubated together with a single concentration of a labelled ligand with or without the presence of the non-labelled ligand (10 μM). The specific binding is measured at various times (1, 2, 5, 10, 30, 45, 60, 90 and 120 min) in triplicate (or quadruplicate) replicates at each tine point. Kinetic association constant (KON) will is calculated from this data.

Dissociation: Cell or tissue homogenate containing the receptor of interest is incubated together with a single concentration of a labelled ligand for 120 min. Then, dissociation is induced by adding 10 μM of non-labeled ligand and counted at different time points (1, 2, 5, 10, 30, 60, 90 and 120 min). Kinetic dissociation constant (KOFF) is calculated from this data.

The shape of the association and dissociation curves (monophasic vs biphasic) will is examined and the equilibrium dissociation constant (Kd) calculated from the kinetic constants.

Saturation Assay (Bmax & Kd)

Multiple concentrations (up to ten) of the radioligand are incubated in triplicate or quadruplicate with either cell, or tissue homogenate containing the target receptor of interest.

After equilibrium has been reached, receptor-bound radioligand is separated from non-bound radioligand using either

i.  Filter-based Separation
ii. Scintillation Proximity Assay (SPA)

The filters or SPA plate are counted using liquid scintillation counting to determine the level of receptor-bound radioligand and the results plotted to obtain the receptor density (Bmax) and if desired the Kd for the receptor-radioligand integration.

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 10 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.

i.  Filter-based Separation
ii. 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.

Our large selection of binding assays for assessing compound selectivity against a wide range of target classes.  If you would like to screen your compound(s) through a pre-designated or custom panels please contact us.

Competition Assays
Ref#   Receptor   Radioligand   Reference Source
RBA047   Adenosine A1   [3H]DPCPX   DPCPX T & C
RBA007   Adenosine A2A   [3H]CGS 21680   NECA T & C
RBA048   Adenosine A2B   [3H]CPX   NECA C
RBA049   Adenosine A3   [125I]AB-MECA   IB-MECA C
RBA081   Adenosine Trasnsporter   [3H]NBTI   NBTI C
RBA051   Adrenergic α1   [3H]Prazosin   Prazosin T
RBA008   Adrenergic α1A   [3H]Prazosin   WB 4101 T & C
RBA009   Adrenergic α1B   [3H]Prazosin   Phentolamine T & C
RBA052   Adrenergic α1D   [3H]Prazosin   Phentolamine C
RBA053   Adrenergic α2   [3H]RX 821002   RX 821002 T
RBA054   Adrenergic α2A   [3H]RX 821002   RX 821002 C
RBA080   Adrenergic α2C   [3H]RX 821002   RX 821002 C
RBA055   Adrenergic β   [3H]Dihydroalprenolol   Propranolol T
RBA056   Adrenergic β1   [3H]CGP 12177   Alprenolol C
RBA057   Adrenergic β2   [3H]CGP 12177   Alprenolol C
RBA058   Adrenergic β3   [125I]Cyanopindolol   Alprenolol C
RBA010   Angiotensin II AT1   [125I]Angiotensin II   Candesartan T & C
RBA011   Angiotensin II AT2   [125I]Angiotensin II   PD-123319 T
RBA084   Bradykinin BK2   [3H]Bradykinin   Bradykinin C
RBA012   Cannabinoid CB1   [3H]SR 141716A   WIN-55212-2 T & C
RBA059   Cannabinoid CB2   [3H]A836339   WIN-55212-2 C
RBA085   Cholecystokinin CCK1 (CCKA)   [125I]CCK-8   L-364718 C
RBA086   Cholecystokinin CCK2 (CCKB)   [125I]CCK-8   Sincalide C
RBA088   Corticotropin Releasing Factor CRF1   [125I](Tyr0)-CRF   Urocortin C
RBA013   Dopamine Transporter (DAT)   [3H]WIN 35428   WIN 35428
RBA014   Dopamine D1   [3H]SCH 23390   SCH 23390 T & C
RBA015   Dopamine D2   [3H]Raclopride   Haloperidol T
RBA016   Dopamine D2L   [3H]methyl-spiperone   Haloperidol C
RBA035   Dopamine D3   [3H]methyl-spiperone   Haloperidol
RBA036   Dopamine D4   [3H]methyl-spiperone   Haloperidol C
RBA037   Dopamine D5   [3H]SCH 23390   SKF38393 C
RBA082   Endothelin ETA   [125I]Endothelin-1   Endothelin-1 C
RBA083   Endothelin ETB   [125I]Endothelin-1   Endothelin-1 C
RBA087   Estrogen Receptor Non-selective   [3H]Estradiol   17β-Estradiol C
RBA017   GABAA   [3H]Muscimol   GABA T
RBA018   GABAA/BZP   [3H]Flumazenil   SKF38393 T
RBA032   GABAB   [3H]CGP 54626   CGP 52432 T
RBA089   GABA Transporter   [3H]GABA   NO-711 T
RBA090   Glutamate AMPA   [3H]AMPA   L-Glutamic acid T
RBA019   Glatamate NMDA   [3H]MK801   MK801 T
RBA033   Ion Channel Ca2+   [3H]Nitrendipine   Nifendipine T
RBA034   Ion Channel Na+ Site II   [3H]Batrachotoxinin   Veratridine T
RBA065   Histamine H1   [3H]Pyrilamine   Pyrilamine C
RBA066   Histamine H2   [3H]APT   Tiotidine C
RBA067   Histamine H3   [3H]α-Me-Histamine   α-Me-Histamine C
RBA075   Imidazoline I2   [3H]BU99008   Idazoxan T
RBA060   LRRK2   [3H]LRRK2-IN-1   LRRK2-IN-1 T
RBA076   LRRK2   [3H]GNE-9605   LRRK2-IN-1 T
RBA078   MAO-A   [3H]Harmine   Clorgyline T
RBA079   MAO-A   [3H]Ro-41-1049   Clorgyline T
RBA040   mGluR2   [3H]LY-341495   MK801 C
RBA041   mGluR5   [3H]M-MTEP   MTEP C
RBA068   Muscarinic M Non-Selectivce   [3H]QNB   Atropine T
RBA069   Muscarinic M1   [3H]QNB   Atropine C
RBA070   Muscarinic M2   [3H]QNB   Atropine T & C
RBA071   Muscarinic M3   [3H]QNB   Atropine C
RBA072   Muscarinic M4   [3H]QNB   Atropine C
RBA073   Muscarinic M5   [3H]QNB   Atropine C
RBA042   Norepinephrine Transporter (NET)   [3H]Nisoxetine   Desipramine
RBA024   Nociceptin (ORL)   [3H]Nociceptin   Nociceptin C
RBA020   Opioid Non-Selectivce   [3H]Diprenorphin   Diprenorphine T
RBA021  Opioid DOR (δ)   [125I]Deltorphin II   Naloxone C
RBA022   Opioid MOR (μ)   [3H]DAMGO   Naloxone C
RBA023   Opioid KOR (κ)   [3H]U69593   Naloxone C
RBA074   Orexin Non-Selective   [125I]Orexin-A   Orexin-A T
RBA028   Orexin Non-Selective   [3H]Almorexant   Orexin-A T
RBA029   Orexin 1 (OX1)   [3H]SB674042   SB 334867 C
RBA030   Orexin 2 (OX2)   [3H]EMPA   EMPA C
RBA061   Oxytocin   [3H]Oxytocin   Oxytocin C
RBA025   Platelet-Activating Factor (PAF)   [3H]PAF   WEB 2086 C
RBA044   Serotonin 5-HT1   [3H]Serotonin   Serotonin T
RBA001   Serotonin 5-HT1A   [3H]WAY 100635   8-OH-DPAT C
RBA002   Serotonin 5-HT1B   [3H]AZ 10519369   Ergotamine T
RBA003   Serotonin 5-HT1B   [3H]GR 125743   Ergotamine T
RBA004   Serotonin 5-HT1D   [3H]GR 125743   Ergotamine C
RBA045   Serotonin 5-HT2    [3H]Ketanserin   Ketanserin T
RBA005   Serotonin 5-HT2A   [3H]MDL 100.907   Chlorpromazine  C
RBA038   Serotonin 5-HT2B   [3H]Mesulergine   SB 206553 C
RBA039   Serotonin 5-HT2B   [125I]DOI   DOI C
RBA050   Serotonin 5-HT2C   [3H]Mesulegine   Mianserin C
RBA043   Serotonin 5-HT3   [3H]Tropisetron   MDL 72222 C
RBA077   Serotonin 5-HT6   [3H]SB258585   Serotonin C
RBA006   Serotonin Transporter (SERT)   [3H]Paroxetine   Fluoxetine
RBA031   Sigma (σ) Non-Selective   [3H]DTG   Haloperidol T
RBA026   Sigma σ1   [3H]Pentazocine   Haloperidol T
RBA027   Sigma σ2   [3H]DTG   Haloperidol C
RBA062   Vasopressin V1A   [3H]AVP   AVP C
RBA063   Vasopressin V1B   [3H]AVP   AVP C
RBA064   Vasopressin V2   [3H]AVP   AVP C

Source Key:

C: Cell, Human Recombinant
T: Tissue*

*Tissue can be, rodent, dog, monkey or human.


Equilibrium Dialysis

Equilibrium dialysis (EqD) is useful for binding studies with soluble targets and small molecules. In other binding assays, when a target is immobilized or part of membrane fragment the free ligand can be easily washed away, but this is not possible with soluble targets. However, if placed in a chamber with a dialysis membrane then bound versus free ligand can be distinguished when binding has reached equilibrium.

96-Well EqD Format

For higher throughput, we employ the 96-Well Equilibrium DIALYZER, a novel product for the simultaneous assay of up to 96 samples. Each well in this system has a separate membrane (MWCO of 5,000 or 10,000 Daltons) and thus eliminates the possibility of sample cross-contamination. Reproducibility is very high across the different wells of the Equilibrium DIALYZER and sample recovery is excellent. Wells are sealed with 8-cap strips. Thus a single row of wells or the entire plate can be used depending on the specifications of the experiment.