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First published on July 14, 2008, doi:10.1177/1087057108321086

Journal of Biomolecular Screening 2008;13:674.

A more recent version of this article appeared on August 1, 2008

Article

A Novel Method for Determination of the Affinity of Protein: Protein Interactions in Homogeneous Assays

Philip Newton*, Paula Harrison, and Stephen Clulow

Cambridge Antibody Technology

* To whom correspondence should be addressed. E-mail: NewtonP{at}medimmune.com.


  Abstract
Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the KD of the protein-protein interaction. Accurate measurement of KD during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the KD of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50 = (([Ki]/KD) x [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/KD). KD is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF®) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured KD values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed. (Journal of Biomolecular Screening XXXX:xx-xx)


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