Label-free detection technologies such as Bio-Layer Interferometry (BLI) and Surface Plasmon Resonance (SPR) allow researchers to monitor the interaction between a ligand and an analyte in real time. They involve immobilizing the ligand directly onto the surface of a biosensor, over which a solution containing the analyte is then passed and a series of measurements taken. While label-free detection has historically been considered challenging, modern platforms have a low entry barrier that has opened up many different types of applications to researchers.
Biomolecular interactions serve as key triggers for countless biological processes, making them valuable targets for drug discovery. They are often studied using end-point assays such as ELISA or western blot, however a limitation of these techniques is that they capture only a snapshot in time, which can mean that critical insights are missed. By measuring real-time kinetics and affinity, researchers can obtain a more complete picture of the system in question and make better informed decisions.
As well as enabling deeper characterization of biological interactions, label-free detection offers several other advantages. First, experiments can readily be performed in complex matrices such as culture supernatants, serum, or blood, meaning that biomolecular interactions are studied under conditions closely resembling the native state. Assay design, development, and reagent preparation are also faster since there is no need to source modified components or substrates. In addition, label-free detection generates more robust assay signals than many conventional techniques since the risk of interference from secondary reagents and reporters is eliminated.
Label-free, real-time assays can answer a broad range of questions. These include whether two biomolecules are interacting, if the interaction between those biomolecules is specific, and what affinity the two binding partners have for one another. Label-free detection also makes it possible to study the kinetics of an interaction, such as through measuring the association and dissociation rate constants, as well as allows researchers to investigate why a particular biomolecular interaction is tight or fast.
Practical applications for these types of experiments include characterizing antibody-antigen interactions (e.g., measuring steady state affinities, ranking off-rates, performing epitope binning), quantifying active target concentrations (e.g., clone selection, residual analysis, glycan profiling), and assessing relative potencies (e.g., drug efficacy, comparative binding affinity, biosimilar assessment). Such assays can be performed using biomolecules of varying sizes, spanning low molecular mass drugs through to large multiprotein complexes.
Label-free assay data is typically presented in the form of a sensorgram, as shown in Figure 1. During the baseline phase, the immobilized ligand comes into contact with the assay buffer and a stable baseline is established. Following this, the analyte is introduced into the system and a corresponding increase in the response signal is seen as association between the ligand and the analyte occurs. The association phase is critical in measuring the kinetics and affinity of the interaction, and requires that the analyte concentration is known.
The dissociation phase begins when the sample is replaced with assay buffer. Because dissociation is concentration-independent, it is often used as an end-point measurement during off-rate ranking. It is followed by regeneration, which involves ensuring that all analyte molecules are removed from the ligand prior to assessing subsequent samples. By performing multiple cycles and combining the resultant association and dissociation phase data, researchers can obtain vital information about the biomolecular interaction that has taken place.
Figure 1. A typical sensorgram
Before starting a label-free detection experiment, it is important to learn as much as possible about both the ligand and the analyte that will be investigated. This could include finding out the primary amino acid sequence, molecular weight, and isoelectric point (pI) of each biomolecule, as well as determining the purity, activity, and solubility. Next, this information should be used to guide experimental design, beginning with deciding which member of the binding pair will be immobilized, before covering biosensor selection, hydration method, and optimization of the ligand density, assay buffer composition, and conditions for analyte binding. Once these factors have been established, assay robustness and performance specifications should be thoroughly evaluated prior to generating data from test samples.
Sartorius has developed an extensive selection of products and resources for label-free detection and is the only company to offer both a BLI and an SPR platform. To learn more, visit sartorius.com