CPC G01N 33/557 (2013.01) [G01N 33/15 (2013.01)] | 12 Claims |
1. A method for the determination of the binding affinity (KD) of a drug to its target comprising:
a)(i) determining the percentage of free drug or free target in at least two samples, each of which comprises drug, target and non-covalent drug-target complexes in the drug concentration range at a constant target concentration where the free drug percentage stays constant, by determining based on the result of an immunoassay the percentage of free drug in a said at least two samples comprising drug, target and drug-target-complexes for different drug:target ratios in each of said at least two samples, and if the determined percentages of free drug are not comparable for all drug:target ratios then the drug:target ratio in said at least two samples is lowered and the sample is re-analyzed by the same immunoassay until the percentages of free drug are comparable for all determined drug:target ratios, or
(ii) determining based on the result of an immunoassay the percentage of free target a at least two samples, each of which comprises drug, target and drug-target-complexes for different drug:target ratios in the each of said at least two samples, and if the determined percentage of free target is not comparable for all drug:target ratios then the drug:target ratio in said at least two samples is increased and the sample is re-analyzed by the same immunoassay until the determined fractions of free drug are comparable for all determined drug:target ratios, wherein for each of a)(i) and a)(ii), each sample comprises an of excess target compared to drug or an excess of drug compared to target, and then
b) calculating based on the percentage of free drug or calculating based on the percentage of free target determined in the previous step the binding affinity for the drug to its target,
wherein said percentages of free drug are comparable if the relative difference (% Diff) of two determined percentages is less than 100%, and said determined percentages of free drug are not comparable if the relative difference (% Diff) of said two determined percentages is greater than 100%, wherein % Diff is calculated with the following formula:
% Diff.=[(highest percentage)−(lowest percentage)]/(arithmetic mean of the percentages),
wherein for the calculation of KD the following equation is used:
KD=(free drug percentage)*(target concentration [nM])/(1−free drug percentage).
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