In the world of biomedical research and drug discovery, having an understanding of how molecules interact is imperative to expanding our knowledge of the inner workings of complex biological systems, as well as accelerating drug discovery. One of the most powerful tools to explore these interactions is the ligand binding assay. From discovering new medications to tracking biological pathways, ligand binding assays serve as a foundation for modern molecular science.
In this post, we will take a closer look at ligand binding assays, techniques currently available, and tips to avoid pitfalls.
What is a ligand binding assay?
A ligand binding assay, or LBA, is a biophysical or biochemical test that measures interaction metrics between molecules. As its name describes, it fundamentally quantifies the binding between a ligand, a protein or nucleic acid for instance, and a target, such as a receptor, enzyme or antibody. Ligand binding assays are used widely across several biomedical fields: in drug discovery, they are critical in target validation, affinity and specificity determination, and understanding mechanisms of action and pharmacokinetics; in biomarker discovery, they are used for the identification and validation of diagnostic and disease progression markers, and in research they are important in understanding cell signalling pathways.
Available techniques for ligand binding assays
Given the wide-ranging importance of ligand binding assays, it’s no surprise that a broad spectrum of techniques has been developed to measure molecular interactions. Below are some of the most commonly used methodologies-though this list is far from exhaustive.
Surface-based Techniques: SPR, BLI, and ELISA
Many biophysical ligand binding assays rely on immobilising a ligand onto a surface, forming what are known as surface-based approaches. Examples include:
- Surface plasmon resonance (SPR) [1]
- Biolayer interferometry (BLI) [2]
- Enzyme-linked immunosorbent assays (ELISA) [3]
In each case, the ligand is tethered to a chip, biosensor, or microplate. Binding of the target molecule is then quantified using optical detection methods specific to each platform. These techniques provide real-time or end-point readouts of binding events and are widely used due to their sensitivity and versatility.
Radioligand binding assays (RBA)
Radioligand binding assays involve tagging a ligand with a radioactive isotope – commonly tritium or carbon-14 [4]. As the radioligand binds to its target in solution, changes in emitted radioactivity can be measured to determine interaction strength and kinetics. While RBAs are highly sensitive and well-validated, their use requires strict radiation safety procedures and dedicated infrastructure, which may limit accessibility for some laboratories.
Fluorescence based approaches
Several in-solution ligand binding assays rely on fluorescent labelling, including Fluorescence polarization (FP) and Förster resonance energy transfer (FRET). FP monitors molecular rotation: small, unbound fluorescent molecules rotate rapidly, producing a low polarization signal, whereas larger ligand–target complexes rotate more slowly, resulting in higher polarization [5]. FRET assays involve labelling the ligand and target with distinct fluorophores. When the two molecules come into close proximity, energy transfer occurs between the fluorophores, enabling measurement of binding events based on changes in fluorescence emission.
Microfluidic Diffusional Sizing and the Fluidity One-M
More recently, Microfluidic Diffusional Sizing (MDS) has emerged as a powerful approach for ligand binding assays. MDS measures the diffusion coefficient of a fluorescent target in a microfluidic chamber (Figure 2); given the established relationship between diffusivity and hydrodynamic radius (Rh), this can be used for size calculation.
Figure 2.The microfluidic set-up on the Fluidity One-M consumable chip plate used for size determination and ligand binding assays by Microfluidic diffusional sizing (MDS).
Tips to avoid pitfalls in ligand binding assays
Given the sensitivity of ligand binding assay approaches available, it is critical that these assays are set up effectively to maximize the quality of the data that is generated. Regardless of your approach, the tips listed below may be considered when setting up your ligand binding assays.
Understand the quality of your targets and ligands
The accuracy of affinity measurements – and even the ability to detect binding at all – depends heavily on the quality of the molecules used in the assay. Degradation or aggregation of either the target or ligand can alter the apparent affinity or completely abolish detectable binding. For this reason, rigorous quality control of all assay components is essential. Because molecular size is a sensitive indicator of aggregation (increased size) or degradation (decreased size), the Fluidity One-M includes a built-in quality-control function that evaluates the integrity of each sample before interaction analysis (Figure 3).
Figure 3. The expected hydrodynamic radius of a 150 kDa antibody, and sizes that may be expected with degradation and aggregation (theoretical data).
Optimize assay conditions
Different ligand-binding assay formats vary in how much optimization they require, but several considerations are universal. Buffer composition is critical, as specific components may be necessary for proper molecular folding, stability, or activity. Assay temperature must also be controlled, as it can significantly influence binding affinity [7] (Figure 4). This is made easy on the Fluidity One-M where assay temperatures are actively held at either 25 or 37°C.
Non-specific binding
In ligand-binding assays, few issues are more frustrating than false positives, and non-specific binding is a major cause. Such artefacts can arise when the target interacts with buffer components or with endogenous molecules present in biological matrices. Any non-specific interaction can alter the assay signal and be misinterpreted as true binding.
Approaches that report physiologically meaningful parameters – such as hydrodynamic radius in microfluidic diffusional sizing (MDS) – are advantageous in this context. Because the expected complex size at saturation can be estimated in advance, any substantial deviation from this predicted hydrodynamic radius provides a clear indication of non-specific binding.
Avoid pipetting errors
Consistent and accurate pipetting is challenging, even for highly skilled operators. Many ligand-binding assays require preparation of serial dilutions, and pipetting errors can propagate through the series, affecting not just the sample in which the error occurred but all subsequent dilutions. Indeed, in many ligand-binding assays, pipetting variability is the largest contributor to poor repeatability. For this reason, careful attention should be paid during sample preparation, and automated liquid-handling approaches should be employed whenever possible to minimize error.
Conclusion
A wide range of techniques are available for ligand-binding assays, and careful consideration should be given to their respective strengths, limitations, and suitability for the intended application. Ideally, multiple complementary approaches should be employed, incorporating both surface-based and solution-based methods. Regardless of the technique chosen, rigorous experimental optimization is essential, with particular attention paid to molecule quality, assay conditions, non-specific binding, and sample preparation.
References
[1] Pattnaik, P., 2005. Surface plasmon resonance: applications in understanding receptor-ligand interaction. Applied biochemistry and biotechnology, 126(2), pp.79-92. https://doi.org/10.1385/ABAB:126:2:079
[2] Shah, N.B. and Duncan, T.M., 2014. Bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects. Journal of Visualized Experiments (JoVE), (84), p.e51383. https://doi.org/10.3791/51383
[3] Syedbasha, M., Linnik, J., Santer, D., O’Shea, D., Barakat, K., Joyce, M., Khanna, N., Tyrrell, D.L., Houghton, M. and Egli, A., 2016. An ELISA based binding and competition method to rapidly determine ligand-receptor interactions. Journal of visualized experiments: JoVE, (109), p.53575. https://doi.org/10.3791/53575
[4] Maguire, J.J., Kuc, R.E. and Davenport, A.P., 2012. Radioligand binding assays and their analysis. In Receptor binding techniques (pp. 31-77). Totowa, NJ: Humana Press. https://doi.org/10.1007/978-1-61779-909-9_3
[5] Rossi, A.M. and Taylor, C.W., 2011. Analysis of protein-ligand interactions by fluorescence polarization. Nature protocols, 6(3), pp.365-387. https://doi.org/10.1038/nprot.2011.305
[6] De Jong, L.A., Uges, D.R., Franke, J.P. and Bischoff, R., 2005. Receptor–ligand binding assays: technologies and applications. Journal of chromatography B, 829(1-2), pp.1-25. https://doi.org/10.1016/j.jchromb.2005.10.002
[7] Truneh, A., Sharma, S., Silverman, C., Khandekar, S., Reddy, M.P., Deen, K.C., Mclaughlin, M.M., Srinivasula, S.M., Livi, G.P., Marshall, L.A. and Alnemri, E.S., 2000. Temperature-sensitive differential affinity of TRAIL for its receptors: DR5 is the highest affinity receptor. Journal of Biological Chemistry, 275(30), pp.23319-23325. https://doi.org/10.1074/jbc.M910438199