Kinetic Binding Data Guide

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 Kinetic Binding Data Guide

Kinetic Characterization
If the last you learned about kinetics was during your good old college days, fear not! This section is a short refresher
on the basic principles of kinetic binding, but without all the chalk dust and sweater vests. In a nutshell, a successful
kinetic binding experiment gives you information about the on-rates and off-rates of a given reaction, and finds the
affinity of your analyte to your target. This information can be used in many biological and pharmacological settings,
from antibody screening to drug compound rank ordering. 1,2

Kinetic Characterization Compared to IC50, EC50, Affinity, and Rank Ordering

During pharmacological or biological research, biochemical processes are often examined for how they inhibit or
stimulate the system. To understand the potency of a particular compound, half maximal inhibitory concentration (IC50)
or half maximal effective concentration (EC50) values are often back-calculated from standard curves using an end point
assay such as an ELISA. Since no real-time measurement data is taken within an end point assay, but only at the end
(oh, that explains the name), no information is obtained about the rate of the reaction or the direct affinity of the
analyte for the target. During a kinetic, or real-time, assay, measurement data is taken continuously throughout the
course of the assay, and thus on-rates and off-rates can be calculated from the data.
Although it is possible to back-calculate affinity from IC50 or EC50 values, this kind of calculation is limited to certain
reactions and requires convoluted equations. Kinetic binding assays provide more reliable, accurate affinity
information. In real-time kinetic binding assays, the observed on-rate, which is dependent on analyte concentration,
and the off-rate, which is independent of analyte concentration, are measured. From these rates, a KD, or dissociation
constant, can be derived, and the inverse of this number is the affinity. Thus, the lower the KD is, the higher the affinity
the analyte has for the target. Confused? Don’t worry, we got you covered in the next section. For now, know that
these affinities can then be used to rank order the analytes (or compounds) being evaluated for their binding potential
with a particular target.
So, are you wondering whether IC50 and EC50 values can be directly compared to KD values? The short answer is no. IC50
and EC50 values can only be compared when the experiment is done in the exact same way for each binding interaction,
and this is often made to mimic the in vivo conditions under which the binding interaction would elicit a response. On
the other hand, KD values are constant values intrinsic to the specific binding interaction and are independent of
analyte concentrations. You can think of it this way: KD values indicate how strongly one thing binds to another and are
inherent to the binding interaction, while IC50 and EC50 values reflect the effect of particular analytes on the biological
or biochemical activities of a target, and are dependent on the experimental conditions. In this manner, KD provides
more flexibility when comparing interactions run in differing assay conditions, as IC 50 and EC50 values can only be
compared against each other when assay conditions are identical.
Despite the differences in the metrics, KD values are similar to IC50 and EC50 values for rank ordering! This is because the
overall ranking of the analytes in question should be in the same order when using KD, IC50, or EC50 calculations. Thus,
KD values can predict or confirm the rank order taken by IC50 or EC50 assays. Agile R100 provides KD values, and the
advantage of Agile R100 is the ability to obtain complete kinetic binding information in addition to the data an end
point assay provides. Imagine having a standard curve composed of multiple points, and also having real-time kinetics
data from each of those individual points!

Calculating kobs, kon, koff, and KD

We learned in the last section that the affinity of a binding interaction is the inverse of the dissociation constant (KD),
but how do we actually derive this from a real-time kinetics assay? First, let’s talk equations – don’t worry, there are
only 2, and they’re not too complicated.
Let’s start at the beginning and break down how we get KD. The KD value is the dissociation constant at equilibrium for a
given binding interaction expressed with units of molar concentration (M), and is independent of analyte
concentration. KD is derived from the following equation:
𝐤𝐨𝐟𝐟
𝐊𝐃 = (1)
𝐤𝐨𝐧

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where koff is the dissociation rate (analyte coming off the target) expressed in units of per seconds (s-1), and kon is the
association rate (analyte binding to the target) expressed in units of per molar concentration per second (M-1s-1).3
Though the observed binding depends on the concentration of analyte, both koff and kon are constant rate values that
are independent of concentration.
Now, let’s take it one step further and break down where we get koff and kon. During a real-time kinetics assay, koff can
be experimentally observed by calculating the rate at which a measured response is returning to baseline after removal
of excess analyte in solution (i.e. when analyte is releasing from the target when fresh buffer is added). We know that
kon is the rate at which an analyte binds to a target, but we do not actually monitor the kon experimentally. Rather, we
record the observed rate at a given concentration (kobs), where kobs depends on the analyte concentration, and is a rate
expressed in units of per second (s-1). The kobs is a combination of the on-rate and off-rate because analyte molecules
are continually binding to the target as well as releasing from the target as the real-time experiment is being recorded.
Thus, to calculate kon from the experimental koff and kobs rates, we use the following equation:
𝐤𝐨𝐛𝐬 − 𝐤𝐨𝐟𝐟
𝐤 𝐨𝐧 = (2)
[𝐀]

where A is the concentration of analyte. As you can see in Equation 2, kon remains a constant value as concentration
changes, because the kobs also changes with respect to analyte concentration. In other words, kon is dependent on
concentration, but is not variable with concentration, while kobs is both dependent on concentration and changes with
concentration. Equation 2 may be a new equation to you because it is not often reported, but this equation is true for
all kinetics. kobs is the sum of the kon and koff, and is observed during measurement. Kinetics tools use k obs to calculate
the kon and koff values.
Finally, with koff and kon in hand, we happily go back to Equation 1 and calculate our KD.

Understanding KD, kon, and koff,

But, now that you have these numbers for an experiment, what do changes in these numbers actually mean? As was
mentioned previously, affinity is the inverse of KD. Thus, when you have a high KD value, it means your analyte has a low
affinity for the target, and conversely a low KD value means your analyte has a high affinity for the target. With this in
mind, you can now rank order analytes based on their KD for a particular target, and easily determine which ones have
a higher or lower affinity. Similarly, since the koff of an analyte from a given target can be experimentally measured, you
may wish to use this to rank order. However, with high affinity binding interactions, the amount of time required to
observe complete dissociation experimentally can range from hours to days! Good thing kon and koff values can be
calculated before equilibrium is reached, and waiting hours to days is not necessary.

Under ideal conditions, KD, kon, and koff will be constants that remain independent of analyte concentration. However, it
is important to remember that even small changes in reaction conditions (i.e. temperature, target stability and/or
conformation, ionic concentration, pH, analyte degradation and/or aggregation, analyte concentration mismatch
between expected and actual, nonspecific binding, etc.) will result in KD, kon, and koff variability. Furthermore, as your
tested analyte concentrations move outside the linear range of your standard curve, variability could rise attributed to
saturation of available binding sites, and reaction conditions could play more of a role in providing variation within your
experiment.

Kinetic Analysis
Are you worried about variability within the data or variability from a KD value found in the literature? Don’t worry! A
10-fold spread of your calculated KD values can be completely normal. Variability is seen in all kinetic assays due to
differences in the user, the instrument, and the interaction of the biology with the particular technique. Figure 1 is a
collection of data from literature values that depicts the variability in KD values from 5 monoclonal antibody targets
(mAbs) across 5 common kinetics tools. 3 Pretty different, right? As you can see, kinetic characterization tools are like
snowflakes, and each one is different. Even the mAbs in Figure 1 with the least variability between systems have
~10-fold difference in KD values. So, if you see some variability within a measurement or between multiple
measurements, take a deep breath, and be assured that some variability is normal in kinetic binding measurements.

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Figure 1: Variability between 5 common biophysical characterization tools. Five different mAbs were immobilized to the biosensor
surface, and 5 different systems measured the mAb interaction with an antigen. KD values are reported here, showing greater than
10-fold difference between systems. Data collected from literature values.3

You may be thinking, but how do I know if I’ve successfully bound my target to the biosensor surface the same way
every time? We are still testing our shrink ray to send nano-scientists to the biosensor surface, but until then, the best
way to validate the efficacy of target immobilization on any platform is by running an interaction to see a sensor
response. Some platforms have methods to determine if mass has bound to the surface, but the only true way to
determine if the specific target has bound to the surface while maintaining its activity is to sense the interaction with
analyte.

Thanks for reading our Intro to Kinetic Binding Data Guide! We’re here to help you see deeper, know more, and make new
discoveries. For any additional questions, please contact us at techsupport@nanomedicaldiagnostics.com.

References
1. Lerner M, Nokes J, Locascio L, Barron F. Nonspecific Binding Is Prevented on AGILE R100.; 2016.
2. Afsahi S, Nokes J, Pan D, Barron F. Structure Activity Relationship of TNFα with Compounds SPD304, Evans Blue, and
Trypan Blue. San Diego; 2017.
3. Yang D, Singh A, Wu H, Kroe-Barrett R. Comparison of biosensor platforms in the evaluation of high affinity
antibody-antigen binding kinetics. Anal Biochem. 2016;508:78-96. doi:10.1016/j.ab.2016.06.024.

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