What is the difference between radioligand binding and SPR? Radioligand binding uses radiolabelled ligands to measure drug-receptor interactions in native membranes and tissue. Surface plasmon resonance (SPR) measures binding kinetics in real time on a sensor chip without labels. Both characterise drug-target interactions; they suit different target classes, programme stages, and biological questions.
Measuring how a drug binds to its target is not just about affinity. How quickly a compound associates with, and dissociates from, its receptor can be just as important as the equilibrium dissociation constant (KD), particularly when thinking about efficacy and duration of action. Radioligand binding and surface plasmon resonance (SPR) both help answer binding questions, but they do it in very different ways. The right choice should come from the biology of the target and the question being asked, not simply from convenience.
Radioligand Binding: The Gold Standard for Receptor Pharmacology
Radioligand binding has been the backbone of receptor pharmacology since Pert and Snyder demonstrated opiate receptor binding in nervous tissue in 1973. A ligand labelled with a radioactive isotope, typically tritium (3H) or iodine-125 (125I), is incubated with receptor-containing preparations; cell membranes, intact cells, or native tissue homogenates. After reaching equilibrium, bound and free radioligand are separated and the bound fraction quantified by scintillation counting.
The strength of radioligand binding is the flexibility. Saturation binding defines KD and Bmax (receptor density). Competition binding gives the affinity (Ki) of unlabelled compounds. Kinetic binding can resolve association (kon) and dissociation (koff) rate constants directly, as described by Motulsky and Mahan (1984). Crucially, these assays can be run on native tissue membranes, so the endogenous receptor environment is retained. That is what makes the data useful for translational pharmacology.
Radioligand binding also extends into receptor autoradiography, where binding site distribution can be mapped across tissue sections with anatomical precision. It also links to in vivo pharmacology through ex vivo receptor occupancy studies, where tissue from dosed animals is used to determine the percentage of receptors occupied at a given dose and time point.
SPR: Real-Time, Label-Free Kinetics
SPR detects changes in refractive index at a sensor chip surface as molecules bind and dissociate. The output is a sensorgram providing real-time measurement of kon, koff, and the derived KD in a single experiment. No radioactive isotope or chemical modification of the ligand is required.
This makes SPR useful in fragment-based drug discovery (FBDD), where low affinity fragments can be assessed without first making a bespoke radiolabelled tool. It is also useful for antibody-antigen interactions, biologics affinity determination, and concentration analysis in biological matrices.
The limitation is biological context. SPR typically requires purified, soluble protein immobilised on the sensor surface. For membrane-embedded receptors such as GPCRs, the protein must be solubilised and stabilised, or captured via affinity tags from detergent-solubilised membranes. In both cases, the receptor is removed from its native lipid and protein environment; pharmacology can change as a consequence.
When to Use Radioligand Binding vs SPR: A Decision Framework
It is not about which technique is better. It is about which technique answers the biological question properly.
In practice, for GPCR targets, I would not treat SPR as a replacement for a well-built competition or kinetic binding assay. It answers a different question.
Target class. For GPCRs, ion channels, and transporters, radioligand binding on native membranes remains the gold standard. These are integral membrane proteins whose pharmacology depends on the lipid bilayer, G protein coupling, and receptor reserve. For soluble targets, antibodies, and fragment screening, SPR is often an appropriate method, although practical throughput depends on the specific Biacore workflow, assay format, and available immobilised target.
Kinetic resolution. SPR provides high temporal resolution of association and dissociation in a single run. Radioligand kinetic assays, particularly competition association assays, can resolve kon and koff for unlabelled compounds, but the temporal resolution is lower. Where residence time (1/koff) is a key question, as reviewed by Liu et al. (2024), SPR can be a useful way to characterise binding kinetics, provided the assay format reflects the biology closely enough.
Throughput and workflow. SPR throughput depends heavily on instrument format, assay design, chip preparation, regeneration conditions, and data quality requirements. On a Biacore-based workflow such as ours, SPR is best positioned as a focused, label-free screening and kinetic characterisation tool for selected fragments, hits, and interaction pairs, rather than a blanket high-throughput replacement for plate-based pharmacology. Radioligand binding requires a suitable radiolabelled tool compound, but once established, competition binding is scalable and cost-effective for larger compound sets.
Biological context. If the goal is to understand compound behaviour in tissue, at physiological receptor density, alongside endogenous neurotransmitters and native signalling machinery, then native tissue radioligand binding or autoradiography is irreplaceable. SPR cannot replicate this environment.
An Integrated Approach at Gifford Bioscience
At Gifford Bioscience, we run both platforms. For receptor targets, we perform saturation, competition, and kinetic radioligand binding on cell membranes and endogenous tissue, including ethically sourced human tissue where available. Our Biacore-based SPR capability is best used for focused label-free binary screening and kinetic characterisation of selected fragments, hits, and interaction pairs.
We extend binding characterisation through receptor autoradiography for spatial mapping and ex vivo receptor occupancy to bridge in vitro binding to in vivo dosing. Together, these approaches give discovery teams a more grounded view of binding, from early hit characterisation through to tissue-level engagement.
To discuss how Gifford Bioscience can support your programme, visit www.giffordbioscience.com or contact us at info@giffordbioscience.com.
Frequently Asked Questions
What is the main difference between radioligand binding and SPR?
Radioligand binding uses a radiolabelled ligand to quantify drug-receptor interactions in cell membranes, intact cells, or tissue homogenates, with bound and free radioligand separated before scintillation counting. Surface plasmon resonance (SPR) detects changes in refractive index at a sensor chip surface as molecules bind to immobilised purified protein, generating a real time, label-free sensorgram. The practical difference is that radioligand binding can be performed on receptors in their native lipid bilayer, while SPR usually requires the target to be expressed, purified, and immobilised, which can alter the receptor environment.
Why is radioligand binding still considered the gold standard for GPCR pharmacology?
GPCR pharmacology is shaped by the surrounding lipid bilayer, by coupling to G proteins and accessory proteins, and by receptor density in the relevant tissue. Radioligand binding on cell membranes and native tissue retains those features, so KD, Ki, and Bmax values reflect the receptor in a more physiologically meaningful state. Methods that rely on detergent-solubilised, purified receptor can shift binding profiles, particularly for allosteric modulators and for compounds sensitive to receptor conformation.
What binding parameters can radioligand assays measure?
Saturation binding defines KD and Bmax for the radioligand. Competition binding returns the affinity (Ki) of unlabelled test compounds. Kinetic binding, including the Motulsky-Mahan competition association format, resolves the association (kon) and dissociation (koff) rate constants for both labelled and unlabelled ligands. Together, these formats provide affinity, receptor density, and binding kinetics from the same underlying technique.
Why is residence time important in drug discovery?
Residence time is defined as the reciprocal of the dissociation rate constant, τ = 1/koff, and reflects how long a compound remains bound to its target. As set out by Copeland (2011) and reviewed more recently by Liu et al. (2024), longer residence times can decouple pharmacodynamic effect from plasma concentration, potentially improving duration of action or apparent selectivity. Kinetic characterisation has therefore become a useful complement to equilibrium affinity during lead optimisation, particularly for slowly turning over targets.
When is SPR most useful in pharmaceutical research?
SPR is well suited to fragment-based drug discovery (FBDD), where low affinity fragments can be screened without first making a bespoke radiolabelled tool. It is also useful for antibody-antigen and protein-protein interactions, biologics characterisation including epitope binning and concentration analysis, and any setting where label-free real time kinetics are needed. SPR works best where the target can be expressed, purified, and immobilised without loss of pharmacological behaviour.
What is the advantage of performing radioligand binding in native tissue?
Native tissue preparations retain physiological receptor density, the relevant complement of G proteins and accessory proteins, and the surrounding lipid environment, all of which can be altered or lost in heterologously expressed cell lines. Pharmacology generated in native tissue therefore reflects the receptor as it exists in the relevant species and brain region. This matters for translational work, where receptor reserve and tissue level pharmacology influence the link between in vitro binding and in vivo response.
References
- Pert CB, Snyder SH. Opiate receptor: demonstration in nervous tissue. Science. 1973;179(4077):1011-1014.
- Motulsky HJ, Mahan LC. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol. 1984;25(1):1-9.
- Copeland RA. Conformational adaptation in drug-target interactions and residence time. Future Med Chem. 2011;3(12):1491-1501.
- Liu H, Zhang H, IJzerman AP, Guo D. The translational value of ligand-receptor binding kinetics in drug discovery. Br J Pharmacol. 2024;181(21):4117-4129.
- Congreve M, Rich RL, Myszka DG, Figaroa F, Siegal G, Marshall FH. Fragment screening of stabilized G protein-coupled receptors using biophysical methods. Methods Enzymol. 2011;493:115-136.
- Swinney DC. The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug Discov Devel. 2009;12(1):31-39.
- Hulme EC, Trevethick MA. Ligand binding assays at equilibrium: validation and interpretation. Br J Pharmacol. 2010;161(6):1219-1237.
- Aristotelous T, Ahn S, Shukla AK, Gawron S, Sassano MF, Kahsai AW, et al. Discovery of β2 adrenergic receptor ligands using biosensor fragment screening of tagged wild-type receptor. ACS Med Chem Lett. 2013;4(10):1005-1010. doi:10.1021/ml400312j.