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Kinetic Characterization

Biomolecular Kinetics

Kinetic analysis of antibodies and other proteins is critical to both clone selection and characterization in development and production. The Octet® family of instruments accurately measures kinetic constants by bringing the detection surface directly to the sample, eliminating the need for micro-fluidics. This unique approach utilizing label-free, real-time analysis streamlines laboratory workflow and expedites assay development. It allows direct measurement of crude samples while minimizing instrument maintenance.

Key Features of Kinetics on the Octet Systems

  • Kinetic analysis: easily and accurately determine kakd, and KD
  • Kinetic screening: 96 or 384 measurements with full kinetic profiles, not just end-point
  • Increased throughput: up to 96 simultaneous reads
  • Assay directly in crude samples: no need for sample purification or preparation

Affinity Measurements — kakdKD

Unlike rough estimates of kinetic information from IC50 values obtained via ELISAs, real-time kinetic measurements offer a direct and more realistic depiction of molecular interactions. Each instrument in the Octet family is a highly capable label-free system for full affinity determination, enabling users to easily and accurately obtain kinetic constants such as kakdKD. The measurement below illustrates a full kinetic characterization using streptavidin biosensors. A titration series of the antigen is measured against an immobilized antibody. The data set is analyzed with global fitting, thereby producing a kakdKD.

Kinetic Characterization

Figure 1. Kinetic characterization of an antibody-antigen interaction, blue curves represent experimental data and red curves represent the statistical fitting of curves.

Kinetic Screening and Clone Selection

Researchers looking to select the best clones from a large pool will reap substantial benefits with the Octet family of instruments due to their throughput and crude sample compatibility. As we move into a new era of drug discovery, the number of samples or clones to be tested will continue to increase and effectively screening them will become more crucial.

Affinity Screening

In an affinity screen of receptor-ligand interactions, lysate targets were assayed against a series of amine-coupled receptors using the Octet system. A purified receptor was immobilized onto the amine reactive sensor and assayed against twelve ligands in duplicate. The receptor-ligand screen in Figure 2 illustrates good reproducibility between replicate kinetic values for all twelve ligands. An affinity isotherm plot of the association and dissociation constants is used to further discriminate interactions that have equal affinity (KD) but have different kinetic properties.

Kinetic characterization of the receptor:ligand interaction.

Figure 2: Kinetic characterization of the receptor:ligand interaction. The affinity (KD) bar chart and kd/ka isotherm plot is presented. Click image for larger view.

Off-rate Screening

Using Streptavidin biosensors on the Octet platform, a biotinylated antigen was immobilized onto the biosensor surfaces offline. Twenty-two clones were screened against the antigen for binding and subsequent off-rate analysis. The antigen was immobilized for 500 seconds, and a 300-second off-rate was subsequently assayed in a single run. Figure 3 shows the actual real-time kinetic binding charts for three sets of eight sensors sampled (N=22). The Octet system software calculates and graphs the off-rates (Kd); enabling rapid identification of the clones' rank order.

Real-time binding charts

Figure 3A: Real-time binding charts of three series of 8 biosensors, comprising a screen of 22 clones to the biotinylated antigen. Click image for larger view.


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