Patent ID: 7074334

Claim:
A method for determining the binding behavior of ligands which bind specifically to target molecules at at least one binding site, which method comprises: (A) preparing a first mixed phase wherein the ligands are contacted with (i) markers which bind specifically to the target molecules, and (ii) with the target molecules, wherein the ligands are present in a concentration K1, the markers are present in a concentration K2 and the target molecules are present in a concentration K3; (B) preparing a second mixed phase wherein the markers are contacted with the target molecules; (C) incubating the first and second mixed phases under identical conditions, which conditions permit a binding of the ligands and the markers to the target molecules, resulting in (a) a quantity M1 of markers GM1 that are bound to target molecules and a concentration K4 of unbound markers in the first mixed phase and (b) a quantity M2 of markers GM2 that are bound to target molecules and a concentration K5 of unbound markers in the second mixed phase; (D) separating the markers GM1 from the first mixed phase and separating the markers GM2 from the second mixed phase; (E1) determination of the concentrations K4 and KS, and calculating a concentration K6 from K5, wherein K6 is a concentration of unbound markers in the second mixed phase under the assumption that the markers have been contacted therein in the concentration K2 with the target molecules in the concentration K3, and determining the binding behavior of the ligands from the ratio of the concentrations K6 and K4, or (E2) determination of the quantities M1 and M2, and calculating a quantity M3 from M2, wherein M3 is a quantity of bound markers in the second mixed phase under the assumption that the markers have been contacted therein in the concentration K2 with the target molecules in the concentration K3, and determining the binding behavior of the ligands from the ratio of the quantities M3 and M1 ; wherein the markers are present in a native form and the determination of K4 and K5 or M1 and M2 is carried out by mass spectrometry.