IMMUNOASSAY-BASED DETERMINATION OF IN-SOLUTION BINDING KINETICS

摘要

FIELD: pharmaceuticals.;SUBSTANCE: invention relates to a method for determining the binding affinity of a drug to its target, comprising the following steps: determining the fraction of free drug or free target in a sample, comprising a drug, target and non-covalent drug-target-complexes in the drug concentration range at a constant target concentration, where the free drug fraction remains constant, by determining, based on the result of an immunoassay, the fraction of free drug in a drug-containing sample, target and drug-target-complexes, for at least two different drug ratios: the target in the sample and, if a certain fraction of the free drug is not comparable for all used ratios of the drug: the target, then the ratio of the drug: the target in the sample is reduced, and the sample is reanalyzed by the same immunoassay, or based on the result of the immunoassay, of the proportion of the free target in the sample, containing the drug and target-drug-complexes, for at least two different drug-target ratios: the target in the sample, and, if a certain fraction of the free target is not comparable for all used ratios of the drug: target, then the drug: target ratio in the sample is increased and the sample is reanalyzed by the same immunoassay, and the calculation based on the proportion of free drug or calculation based on the proportion of the free target, determined at the previous stage, the affinity of the binding of the drug to its target, where "comparable" means the relative difference (% of differences) of two specified values of less than 100 %, and "incomparable" means the relative difference (% difference) of two defined values greater than 100 % where % is difference, calculated according to the following formula % dif. = [(highest value) – (lowest value)] / (arithmetic mean), where to calculate the binding affinity (K_D) apply the following equation: K_D = (free drug share) * (target concentration [nM] / (1 – free drug fraction).;EFFECT: technical result of the invention lies in the fact that the determination of the affinity of the binding agent and its ligand in the samples can be carried out using very limited (small) number of measurements (samples), which reduces the complexity of the method and improves its accuracy.;12 cl, 6 ex, 11 dwg