Kinetic Characterization
Quantitative Protein Kinetics
Kinetic analysis of antibodies and other proteins is critical to both the selection of clones for development and comprehensive kinetic characterization throughout development and production. The Octet family of BLI-based instruments accurately measures quantitative ka, kd, and KD by bringing the detection surface directly to the sample, eliminating the need for microfluidics. This unique approach in label-free, real-time kinetic analysis streamlines assay development. It allows for crude sample mixtures and minimizes maintenance, thus providing greater value than other existing methods such as SPR and ELISA, which are limited in throughput, performance, and ease-of-use.
Key Features of Kinetics on the Octet Q/QK System and the Octet RED System
- Quantitative kinetics: characterization of ka, kd, and KD without flow-cell fluidics
- Kinetic screening: 96 measurements in less than two hours
- Increased throughput: eight-channel parallel analysis, up to 96 samples fully automated
- Assay directly in crude samples: eliminate time for sample purification or preparation
Quantitative Kinetics – kobs, ka, kd, KD
Each instrument in the Octet family is a complete label-free kinetics analysis system for kobs, ka, kd, KD, and affinity determination. Using standard surface chemistries on the Octet System, eight-channel analysis provides parallel processing of continuous real-time display of biomolecular interactions. This allows the use of a variety of conditions during optimization, shortened workflow, and parallel analysis of multiple concentrations or molecule types.
As shown below, three different antigen concentrations (5 nM, 10 nM, and 20 nM) were tested in duplicate for binding to the immobilized antibody using Amine Reactive Biosensors. All concentrations including the controls were assayed in parallel on the Octet System. Figure 1 illustrates the global fitting of all concentrations and the associated residuals. Table 1 provides the affinity constants based on global fitting and the individual k observed values for each concentration.
Figure 1: Kinetic characterization of an antibody to a series of antigen. Globally fitted curves and residuals. Click image for larger view. |
Sensor |
[M] |
ka
[1/Ms] |
kaErr |
kdis
[1/s] |
kdisErr |
kobs
[1/s] |
KD
[M] |
kaRsq |
Global |
|
2.56E5 |
1.13E3 |
2.01E-5 |
8.39E-7 |
|
7.85E-11 |
0.99462 |
A12 |
2E-8 |
|
|
|
|
5.14E-3 |
|
|
B12 |
2E-8 |
|
|
|
|
5.14E-3 |
|
|
C12 |
1E-8 |
|
|
|
|
2.58E-3 |
|
|
D12 |
1E-8 |
|
|
|
|
2.58E-3 |
|
|
E12 |
5E-9 |
|
|
|
|
1.30E-3 |
|
|
F12 |
5E-9 |
|
|
|
|
1.30E-3 |
|
|
|
Table 1: Affinity constants based on global fitting and
the individual kobs values for each concentration. |
Kinetic Screening and Selection
The flexibility and throughput of BLI-based instruments lends itself readily to a variety of kinetic screening applications. A simple ranking by relative off-rate can be used to redirect resources to more promising clones without extensive purification or preparation of samples. Most methods available for determining kinetic constants and screening of antibody off-rates for rank ordering involve extensive development, long processing times, and the need for expert operators.
Figure 2A: Real-time binding charts of each series of 8 sensors which comprise the screen of
22 clones to the biotinylated antigen. Click image for larger view.
Figure 2B: Matched off-rate charts of the screen of 22 clones
to the biotinylated antigen. Click image for larger view.
Off-rate Screening
The The Octet System provides a solution for rapidly screening and identification of binding interactions between antigen:antibody pairs, sampling eight sensors in parallel for all kinetic assay steps. In a single experiment, off-rates can be determined and subsequently ranked to select promising clones for advancement in the development process.
Using Streptavidin Biosensors on the Octet QK System, a biotinylated antigen was immobilized onto the biosensor surface 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 2 is the actual real-time kinetic binding charts for three sets of eight sensors sampled, N=22. Octet System Software calculates and graphs the off-rates (kd); enabling rapid identification of the clones’ rank order.
Affinity Screening
Affinity screening using ForteBio’s Octet family of instruments is convenient, rapid, and can provide accurate determination of protein kinetics with a minimum of development time.
In an affinity screen of receptor:ligand interactions, lysates targets were assayed against a series of amine-coupled receptors, using the Octet QK System in a multiplexing strategy to accelerate the selection of which ligand to advance in the development process. A purified receptor was immobilized onto the amine reactive sensor and assayed against twelve ligands in duplicate. The receptor:ligand screen in Figure 3 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.
Figure 3: Kinetic characterization of the receptor:ligand interaction. The affinity (KD) bar chart
and kdissoc/kassoc isotherm plot is presented. Click image for larger view.
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