Affinity and kinetics

MP-SPR is a sensitive real-time label free method that not only provides affinity but also kinetic information on molecular interactions. Even molecules of equal affinity can have very different kinetic profiles making kinetics essential selection parameter especially in drug discovery.

Terminology
What is a ligand and an analyte?

A schema of a SPR sensor surface with a ligand attached on it and analytes interacting with the ligand.

Ligand is a molecule attached on a sensor surface. Analyte is a molecule flowing freely in a gas or a liquid flow. Ligand as well as analyte can be selected based on interaction of interest. Typical examples can be protein-protein interactions, DNA-DNA interactions, biomaterial interactions. The time that an analyte is available for binding is controlled precisely (see Flow injection analysis principle) by switching between a running buffer and sample containing analyte.

What can be a sample in MP-SPR kinetic measurements?

Apart from purified samples, MP-SPR works also with crude samples such as saliva, whole serum, cell growth media or sea water for instance.

What can be an analyte in MP-SPR measurements?

Nucleotide, peptide, antibody, drug molecule, virus, nanoparticle, microvesicle, living cell, bacteria, ..

What can be a ligand in MP-SPR measurements?

Protein, nucleotide, peptide, receptor, membrane receptor, drug molecule, antibody, whole cell, biomaterials and more

How to capture a ligand on a sensor surface?

Several methods for ligand capturing are available such as direct coupling, affinity capture, or membrane anchoring depending on the molecule attached. To find out about the best strategy for your sample analysis, contact our scientific team.

How to measure affinity and kinetics of molecular interactions?

The picture shows a single injection of a sample in an MP-SPR measurement:
0-2 min: Running buffer is run over the sensor surface with an immobilized antibody (ligand)
2-6 min: Sample with analyte is running over the surface. As the analytes attaches on the surface, we can observe an association phase
6-8 min: Running buffer is run over the surface with attached ligand-analyte. The buffer will remove any unbound analyte and reveal the dissociation, if any.
8-10 min: Regeneration buffer is run over the surface to regain clean sensor surface
10-12 min: Running buffer running over the sensor

Next sample injection can follow. (Reuse of sensors is possible for most cases)

What is affinity?

Affinity determines how much ligand-analyte complexes are formed at equilibrium (when association is balance with dissociation). Terminology used in the biochemical interaction reactions has been accepted so that the KD –constant is used for determining affinity of the interaction. The smaller the KD the higher affinity of molecule A to molecule B.

What are association and dissociation constants in kinetic measurements?

Kinetic parameters give information about how fast interactions happen. According to the Law of Mass Action, the reaction rate (v) of a bimolecular “one-to-one” binding reaction (A+B↔AB), depends on the molar concentration of A, B and AB and two reaction rate constants. There is a forward (association) and reverse (dissociation) reaction. Association rate constant (ka) is number of AB formed per second and dissociation rate constant (kd) is fraction of complexes decay per second.

Dependency of affinity and kinetic parameters is shown in the following equation:

 

How to determine affinity and kinetic constants from MP-SPR measurements?

The MP-SPR data are plotted against time as sensograms. It is possible to select from a number of parameters to be plotted, including Peak minimum angle. Such sensograms are fitted with binding models in data analysis software TraceDrawer™ for MP-SPR Navi™.

See application examples for on- and off-rate calculations and plots.

 

  • SPR measurements of small molecules, interactions with lipids and biomaterials are typically hindered by bulk effect, an optical artefact. See how MP-SPR solves bulk effect with our PureKinetics™ feature.
  • What is the difference between SPR and MP-SPR? Find out here.