Binding kinetics

Biomolecular binding assays can be performed using labeled molecules (radiolabels, fluorescent labels, etc.), but appropriate, non-disruptive labeling and often elaborate washing and purification steps are required. 

In label-free quantification studies, one of the molecules to be analyzed is immobilized to a surface (the ligand) and the other molecules free in solution (the analyte). 

The sensorgram (a graphical representation of the real-time measurement) shows a typical study of analyte-ligand binding in real-time.

Image right: Typical sensorgram showing the phases baseline, association, dissociation and return to baseline.

First, as shown in the sensorgram, a baseline is determined. Simply put, this is the refractive index of only the ligand. Then, the unbound analyte in solution is added and association (binding) is measured as a change in this refractive index. After the association phase, the solution with the unbound analyte is removed and disassociation is measured. Though the signal level in a sensorgram correlates to the ligand and analyte involved and the strength of their interaction, it’s in the curvature of its association and dissociation phases where the valuable information resides. For example, the desired biological effect of a drug linked to its residence time (dissociation phase) rather than to its affinity only.

Measurements with a single analyte injection at a single concentration (single cycle kinetics) are fast but do not yield reliable estimates of all kinetic parameters. Traditionally, kinetics are measured in a multicycle kinetic experiment: reliable and detailed kinetic analysis requires data from at least four to six analyte concentrations, spanning the range of 0.1 to 10 times the expected K_D. This requires dilution series and disassociation steps in between the cycles. Therefore, multi-cycle kinetics are time intensive and not suited for screening thousands of candidate compounds. 

Regeneration-free kinetics allows faster analysis by skipping the regeneration steps. However, regeneration-free kinetic analysis can only be applied if the analyte dissociates slowly, as it is based on that little analyte dissociates during the cycles of increasing analyte concentration. We have developed a revolutionary detection method: Repeated Analyte Pulses of Increasing Duration - waveRAPID that will drastically reduce assay time and reagent consumption while improving readout to identify leads in the drug discovery process faster and with more confidence. 

waveRAPID uses pulses of analyte at a single concentration, but each pulse is applied for an increased duration. See how our methods can help you to get to kinetic data quicker and easier: the newly developed waveRAPID method and the Direct Kinetics software that provides a 1-click evaluation tool.

With the introduction of the WAVEsystem, Malvern Panalytical now also offers a optical biosensor to study real-time binding kinetics. 

The WAVEsystem can be used to provide answers to a broad range of scientific questions. These include determining whether two molecules are interacting and what the binding affinity of the molecules is (K_D), as well as confirming the biologically active concentration of a specific analyte in a sample. 

In addition, the WAVEsystem can measure the kinetic reaction parameters such as the association rate constant (k_a) and dissociation rate constant (k_d) by observing analyte-ligand binding in real-time. 

Visit our page on the GCI technique to see why the WAVEsystem with its proprietary GCI technique has superior resolution over traditional Surface Plasmon Resonance (SPR) and Bio-layer Interferometry (BLI).