Kinetic Exclusion Assay Theory
As described on the Reversible Binding page, when a receptor and ligand reach equilibrium in solution three species are present, the receptor, the ligand and the receptor-ligand complex.
A Kinetic Exclusion Assay (KinExA®) measures the free concentration of either the receptor or the ligand without perturbing the equilibrium. (Note: In the following description the format of the assay is to immobilize the ligand to measure free receptor in the sample. Conversely the receptor may be immobilized to measure free ligand in the sample, depending on the materials used.)
Measurement of the free receptor is accomplished by briefly exposing the sample mixture to a solid phase on which the ligand is immobilized. The exposure time is critical because keeping the interaction time of the sample to the measurement solid phase quite short results in a situation where the only significant binding to the solid phase is from the free receptor. The Kinetic Exclusion Assay is in contrast to a competition assay in which the equilibrated solution is in contact with the solid phase long enough for the solid phase ligand to compete for the solution receptor.
The advantage of KinExA is that the signal from the captured receptor represents only the concentration that is free in the solution. Knowing the equilibrium concentration of receptor allows determination of the binding constants as described on the Reversible Binding page.
The commercially available instruments for performing KinExA assays (Sapidyne's KinExA 3200 and 3100) use a small column of particles through which the sample and other reagents are passed. The contact time of any portion of the sample with the solid phase is then the transit time through the column and can be controlled by the flow rate chosen (from around 50 milliseconds to about a second).
Systems with nM range binders or tighter will be in the kinetic exclusion assay mode (KinExA mode). Weaker binders can still be measured but may require extra care to avoid perturbation of the sample from the measurement. KinExA is particularly well suited for measuring affinity and kinetics and also works as an improved immunoassay platform. A more detailed explanation of the KinExA measurements are included below.
KinExA for Kd measurement:
Binding curves are dependent on Kd and receptor concentration (About Binding Curves). A binding curve can be generated by making a series of samples with constant receptor concentration and a titration of ligand. After equilibrium is reached, a KinExA measurement is made of the free receptor concentration in each sample of the titration series. Therefore the free receptor directly represents the binding curve. The binding curve generated is then analyzed to find the Kd (for low ratio curves) or receptor active concentration (for high ratio curves). Multiple curves with different receptor concentrations may be analyzed together to get both Kd and active receptor concentrations.
Using a KinExA assay for making Kd measurements is ideal because it can determine the free receptor concentration without perturbing the solution equilibrium that has been established with unmodified molecules, unfettered, in solution. For an accurate Kd determination the concentration of receptor in the samples should be near or below the Kd. With the tight binding antibodies routinely developed now (low or sub pM), the measurements need to be made at these very low sample concentrations. The KinExA's ability to make quantitative measurements at these low concentrations allow for accurate Kd measurements for those very tight binders.
KinExA for kon and koff measurement:
Measurement of free receptor concentration may also be done prior to the samples reaching equilibrium. Measurements taken as a system approaches equilibrium provide data for the kinetics of the reaction. Two methods can be used for kinetic measurements, the direct method and the inject method.
In the direct method, a single sample of receptor and ligand is prepared and the free fraction is repeatedly measured over time as it approaches equilibrium. The on rate, kon, can be calculated from the curve. This direct measure of the kinetic curve does require that the time the sample takes to reach equilibrium be long enough to allow several measurements to be made. The time of this reaction can be reduced by lowering the concentration of the reactants. The concentration, however, must be kept above the Kd for significant binding to occur, so some weaker binding systems cannot be slowed enough to enable use of this method.
In cases where the reaction cannot be slowed enough to use the direct method, the inject method may be used. The inject method can interrogate very short reaction times by injecting the receptor into a stream of ligand, upstream from the particle column. The incubation time is then reduced to the flow time from the injection point to the particle column. This time can be adjusted by the flow rate used, and if needed can be reduced to a few seconds.
Unlike solid phase kinetic measurements, the molecules in the reaction are unmodified and free to move about in solution. Some differences do occur between the solution phase and solid phase kinetic measurements1 with the solution phase measurement often faster2 – sometimes substantially.3
The off rate, koff, can also be directly measured, however it is usually just calculated from the measured Kd and measured kon, (koff = Kd * kon).
KinExA for immunoassay:
The ultimate sensitivity of any immunoassay depends on the properties of the antibody used. Most immunoassays do not fully achieve the potential sensitivity because either the readout method lacks sufficient sensitivity to take advantage of the antibody's ability or because competition is limiting the assay sensitivity. The kinetic exclusion assay prevents competition from interfering, and the measurement sensitivity (low to sub pM) can take full advantage of extremely tight binding antibodies.4
In practice KinExA has been shown to be 10 to 1000 fold more sensitive than ELISA using the same reagents.5 A study was conducted in Japan at the Central Research Institute of the Electric Power Industry (CRIEPI) and funded by the New Energy and Industrial Technology Development Organization (NEDO). The study compared different immunoassay systems by sending identical reagents to outside labs and analyzing the results of the most sensitive immunoassay for each set of reagents. The labs used were Biacore, for an SPR based immunoassay, Sapidyne, for a KinExA based assay, and Kyoto Electronics Manufacturing (KEM) for ELISA. For all 4 sets of reagents the KinExA assay was the most sensitive and had the widest dynamic range.6 In fact, KEM has since acquired a license to use KinExA for their commercial dioxin measurement system because of the improved sensitivity, speed, and accuracy compared to ELISA.
1. Blake II R.C., Delehanty J.B., Khosraviani M., Yu H., Jones R.M., Blake D.A. 2003. Allosteric binding properties of a monoclonal anti- body and its fab fragment. Biochem 42: 497-508. http://www.ncbi.nlm.nih.gov/pubmed/12525177
2. Razai A., Garcia-Rodriguez C., Lou J., Geren I.N., Forsyth C.M., Robles Y., Tsai R., Smith T.J., Smith L.A., Siegel R.W., Feldhaus M., Marks J.D. 2005. Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A. J Mol Biol 351: 158-169. http://www.ncbi.nlm.nih.gov/pubmed/16002090
3. Glass T.R., Ohmura N., Saiki H. 2007. Least detectable concentration and dynamic range of three immunoassay systems using the same antibody. Anal Chem 79: 1954-1960. http://www.ncbi.nlm.nih.gov/pubmed/17256970
4. Ohmura N., Lackie S.J., Saiki H. 2001. An immunoassay for small analytes with theoretical detection limits. Anal Chem 73: 3392-3399. http://www.ncbi.nlm.nih.gov/pubmed/11476240
5. Blake D.A., Jones R.M., Blake R.C., Pavlov A.R., Darwish I.A., Yu H. 2001. Antibody-based sensors for heavy metal ions. Biosens Bioelectron 16: 799-809. http://www.ncbi.nlm.nih.gov/pubmed/11679258
6. Glass T.R., Ohmura N., Saiki H. 2007. Least detectable concentration and dynamic range of three immunoassay systems using the same antibody. Anal Chem 79: 1959. Figure 4. http://www.ncbi.nlm.nih.gov/pubmed/17256970