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Kinomics hat nutzbringende, einzigartige Erweiterungen für Affinitäts-Biosensoren definiert.

Kinomics MSK ISA Application Note

Inhibition-in-Solution Assay (ISA) utilizing Multi-Step Kinetics (MSK) on Biacore Systems

(vergrößern, 450 kB, pdf )

Determination of the inhibition constant KI (in solution) between analyte A and inhibitor I that are mixed prior to the MSK study on a Biacore system where the equilibrated solutions are checked for the free analyte A against ligand L (immobilized on the sensor) with regard to non-inhibited analyte references in order to identify inhibitors of interest. A data simulation.

· Mix analyte:inhibitor (A:I = 1:1 reaction type) at different (≥5) concentrations; especially in biosensor capture assays, prepare non-inhibited analyte samples.

· Run MSK (w/o intermediate regenerations) with L immobilized (or captured):
- Vary contact times (allow low analyte concentrations to bind longer; see 1).
- Inject the ≥5 pre-mixed inhibited samples, each followed by a dissociation.
- In the same way, inject non-inhibited reference samples (right in 1); the # of these may be less or, in case the assay is highly reproducible, skipped at all.

· Non-linear fitting and evaluation of data with MSK software incl cross-checks:
- Import affinogram (black curves in 1) and fit by a single click within 2 s (red).
- Exclude inhibited samples from evaluation (even so, they are kept calculated; shown yellow in 2) and, if indicated, omit outliers of non-inhibited references.
- Evaluated references yield KD of A:L (here 6 · 10-8 mol L-1) incl kinetics; the green traffic lights (3) indicate an excellently cross-checked reference result.

· KI from initial association rates (non-linearly extrapolated back to baseline):

- This true initial base rate is directly proportional to the analyte concentration.
In MSK from Kinomics, this rate is readily available for each single step!
- The fit of the non-inhibited samples' rate yields the reference slope (red in 4).
- From the suppressed rate of the inhibited pre-mixes (yellow dots in 4), their actual free A concentration is calculated with regard to the reference slope.
- Thus, the equilibrium concentration (ceq) of relevant compounds (A, I, AI) in the pre-mixes are known: KI is calculated according to the mass action law (5).

· Intuitive: The 50 % inhibition position of A (in 4) indicates the KI at a glance or, in other words, strong inhibitors suppress the rates more strongly.

· In comparison with others, identify the inhibitor(s) of your interest.
Done. Fast, easy, and intuitive. It's unique – it's from Kinomics.

Published online 11/25/2009 on (patent pending).
© Kinomics GmbH 2009

Kinomics MSK ISA Application Note



© Kinomics GmbH 2004-2017
Zuletzt geändert: 22.01.2017
Die Kinomics GmbH ist seit 31.12.2016 in Liquidation (i.L.). Dieser Prozess wird Ende 2017 abgeschlossen sein.