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Nov 26, 2020 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. There are situations where weaker binding systems cannot be slowed enough to enable use of this method.
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 substantially3.
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).
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. https://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. https://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. https://www.ncbi.nlm.nih.gov/pubmed/17256970
