Biolayer Interferometry

Biolayer Interferometry – An Overview

For many molecular interactions, it is not only important to know their precise affinity, their thermodynamics, or their stoichiometry, but also to get detailed knowledge about their underlying kinetics. Two processes constitute the kinetics of a molecular interaction: Association, i.e. the formation of attractive forces between the interacting molecules, and dissociation, i.e. the disengagement of the molecules. In many pharmaceutical and biotech applications, kinetic analysis is important, for example to tailor the kinetic behavior of therapeutic or diagnostic antibodies specifically to rapid association or slow dissociation, depending on the area of application.

In order to derive the kinetics of a molecular interaction, the association and dissociation phases of the interaction have to be monitored over time. One very sensitive, robust, and precise way to do this is to detect the build-up of molecular complexes on a sensor-tip using the PALL FortéBio Octet® system. A sensor tip is used, which specifically binds one interaction partner; this can be done, for example, with the well-established biotin-streptavidin system and a biotinylated interaction partner. Initially, the sensor tip is dipped into a blank solution (e.g. buffer). Then, one interaction partner (e.g. a biotinylated antigen) is captured on the sensor tip surface and excess antigen is washed off with buffer. In order to monitor the association, the loaded sensor tip is then dipped into a solution containing the second interaction partner (e.g. an antibody). Finally, in order to monitor the dissociation, the sensor tip is dipped into blank buffer solution again, where the ligand (antibody) will dissociate over time from the coupled target (antigen). Certain sensor tips can also be regenerated and re-used for several experiments. Finally, fitting of the association and dissociation phases then provides the respective kon and koff rates.

Importantly, the Octet® system is label-free and does thus not require the modification of the interaction partners with fluorescent dyes for example. However, immobilization of one interaction partner has to be accepted in turn. The Octet® system is widely used for quantifying and analyzing the kinetics of molecular interactions involving small molecules, peptides, DNA and RNA molecules, proteins, antibodies, up to whole viruses and bacterial cells. For more information of molecular interactions that are possible to quantify with this systems, see “Typical applications”.

 

A typical Octet® experiment in order to determine the kinetics of an antibody-antigen interaction.