Kinomics accelarating biosensors

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

Kinomics MSK® Product Note Overview

NEW:
Version 1.1 available from Oct 2006 - includes printing and saving (and re-opening) of evaluation results.

Imagine…

you
don't need intermediate regenerations, ligand re-captures, or to wait for equilibrium…
but you've
full access to detailed kinetics and concentration in affinity biosensorics!

Unique MSK® from Kinomics provides it:


(vergrößern, 1,2 MB, pdf )

A referenced response-over-time affinogram of alternating association and dissociation steps (noisy black curves in 1 A) is simulated here as it is recordable by a standard biosensor such as provided from Biacore AB. The first two association steps were run for a longer time (240 or 180 s, resp.; all others 120 s) in order to allow for a more distinct curvature of these slow bindings at a low analyte concentration. The affinogram is easily imported into the Kinomics MSK® software and evaluated within seconds by a single button click, yielding fitted association and dissociation steps (red) between pre-set limits (vertical blue lines denote imported times of analyte concentration changes, vertical green lines denote default* fitting regions, both can be adjusted**). As indicated in the latter 3 steps, the association fitting regions have been automatically cut down (since an identified established equilibrium response doesn't contain further information). Random fitting residuals of about ± 0.5 RU are specified below (1 B), and a zoom of the middle analyte concentration step from about 1100 to 1400 s is inserted (1 C) for details. In the evaluation table (2), all steps are listed by their imported analyte concentration (in units of mol L-1), by their start time and fit region**, as well as by their determined curve parameters (exponents and equilibrium values). - In summary, the fits and residuals exhibit a perfect approximation, which "just" needs to be kinetically evaluated.

A secondary evaluation reveals a most detailed picture in terms of kinetics. Automatically with the primary fitting, six graphs (4-9) show up that are based on, or transformed to, straight-line equations (i.e., very intuitively to judge) and that evaluate the affinogram from all different points of view:
(4) Trutnau plot examines the net initial and final association rate at each concentration change time with regard to the starting response level and yields separately, the first time ever from such a single kinetics plot, the association rate constant (kass) and the maximum binding response (Rmax);
(5) Rosenthal plot investigates the ratio of bound over free analyte at the extrapolated association equilibrium with regard to the equilibrium value and yields the association equilibrium constant (KA = reciprocal dissociation equilibrium constant, KD-1) and Rmax;
(6) Base rates plot explores the association rate extrapolated to baseline with regard to the analyte concentration and yields kass · Rmax;
(7) Starting rates plot inspects the net initial association rate (also considering the following dissociation) with regard to the analyte concentration and yields kass;
(8) Exponents plot studies the association curvature with regard to the analyte concentration and yields kass and the dissociation rate constant (kdiss);
(9) Dissociation plot considers the initial dissociation rate with regard to the dissociation response and yields kdiss.

Given ideal interaction performance, the affinogram's evaluation must yield straight-line behaviour in each plot and, hence, self-consistency of the kinetics constants that are derived from different fractions of the affinogram.

Though, even without closer inspection of the partly conflicting graphs (some don't show the predicted straight-line behaviour), the results table (3) immediately discloses by its novel, intuitive traffic sign indicator* that the results are not consistent: Some graphs may show horizontally "green" = good (even if pitfalling) intra-, but vertically "red" = bad inter-plot consistency (e.g., Exponents plot in 3). These consistencies are checked in the results table (3) by a novel, automated combinatorial cross-correlation. At the level of confidence* chosen here, the horizontal (intra-plot) and vertical (inter-plot) cross-correlation yields an overall weighted value (bottom right traffic light in 3) with a % number signifying the degree of self-consistency (in this simulation case, unacceptable < 60 % "yellow").

Most significantly, mass transport limitation (MTL) conditions at low analyte concentrations, indicated in (4) by reduced initial rates (to the left of the plot), causes the low self-consistency as signified by many red lights in table (3). Deselecting the MTL-influenced steps (10) from graphical evaluation (12-17) yields an excellent self-consistency after cross-correlation as shown in table (11). It should be noted that this result has been found just by pure and applied data analysis, i.e., without any necessity to include an unknown, empirically defined MTL factor. The assessed values (last row in 11, rounded to one decimal digit) are the same as used for the simulation:
kass = 1 · 106 L mol-1 s-1, kdiss = 2.5 · 10-3 s-1, KD = 2.5 · 10-9 mol L-1 (at Rmax = 100 RU).

Since the plots depend on concentration, MSK® can also be applied, successfully, to determine unknown sample concentrations, once a standard calibration file has been recorded.

Hence, the user's benefits are massive savings in time and material as well as improved confidence of results.

*Default settings can be individually configured.
**Many imported or configured settings can be individually adjusted if desired.

Online publiziert 18.04.2006

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