Kinetic Characterization

Agile R100 is a kinetic binding data platform that can be run at your benchtop with just a 10 µL drop of sample, pipetted directly onto the biosensor surface. With no complicated systems or processes to learn, you can get to data in a matter of minutes.

What is kinetic binding data?

With a kinetic binding, or real-time assay, data is taken continuously throughout the experiment, enabling you to gain accurate affinity measurements (KD) and rate constants (kon and koff).

Why is affinity data important?

During pharmacological or biological research, biochemical processes are often examined for how they inhibit or stimulate the system. To understand the potency of a particular compound, half maximal inhibitory concentration (IC50) or half maximal effective concentration (EC50) values are often back-calculated from dose‑response curves using an end-point assay such as an ELISA. IC50 and EC50 values have limitations. They are dependent on the experimental conditions under which they’re determined, which means they can only be compared against each other when the assays conditions are identical. In contrast, the equilibrium dissociation constant (KD), a measure of affinity, is intrinsic to the interaction of the binding pair and is independent of experimental conditions. You can think of it this way: KD values indicate how strongly one thing binds to another, while IC50 and EC50 values reflect the effect of particular analytes on the biological or biochemical activities of a target and can vary depending on substrate concentration and incubation period. In this manner, kinetic characterization as a few advantages: 1) Affinity measurements provide flexibility and time savings in comparing different systems that IC50 and EC50 values can’t provide. 2) KD tends to be more reproducible because it is less dependent on experimental conditions.

What can off-rates tell me?

Drugs with slow dissociation rates or off-rates (koff) often have extended duration of action, and affinity may not be an accurate proxy to discover which compounds will provide beneficial efficacy durations. While slow dissociation is often connected with high binding affinity, this is not always the case, and without a dependable kinetic binding platform, it’s impossible to know for sure. By characterizing on- and off-rates at the same time as affinity, compounds with desirable kinetic profiles for target binding can be chosen. Many compounds can bind to numerous off-target proteins as well as the intended target. Monitoring the dissociation rates of the compound from all possible targets may be advantageous. Kinetic profiles can differentiate compounds with improved toxicity and selectivity, by enabling selection of those that have both slow off-rates for chosen targets and fast off-rates from unintended ones.


Agile R100 features:

  • Sensitive kinetic binding measurements kon, koff, and KD
  • Characterization of small molecule fragments ≥10 Da
  • Low target density requirements – just 0.5 ng of material
  • 11-log dynamic range from fM to mM provides a large working range of concentrations on a single platform
  • Easy gathering of affinity data enables fast turnarounds for structure-activity relationship (SAR) research

Protein TNFα and small molecule compound SPD304

label-free Agile R100 kinetic characterization

In the figure above, an overlay plot displays a full kinetic characterization using COOH Agile biosensors. A concentration series of small molecule SPD304 is measured against immobilized TNFα, a cell signaling protein used as a therapeutic target. These curves show the concentration dependence of the biomolecular interaction, and Agile Plus software provides kon, koff, and KD.