Method for determining the binding behavior of ligands which specifically bind to target molecules

• US 7,074,334 B2
• Filed: 05/21/2002
• Issued: 07/11/2006
• Est. Priority Date: 05/23/2001
• Status: Expired due to Fees

• Current Assignee: Klaus Wanner
• Original Assignee: Klaus Wanner
• Inventors: Bertling, Wolf, Höfner, Georg, Wanner, Klaus
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US10/478,025
• Publication Number:
  US 20040232077A1
• Time in Patent Office: 1,512 Days
• Field of Search: 435/7.1, 436/517, 436/518, 210/635, 210/656, 210/659, 210/143, 210/198.2
• US Class Current: 210/656
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The invention relates to a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site, whereby the markers are present in a native form and the determination of the concentrations K4 and K5 or of the quantities M2 and M1 is carried out by means of mass spectrometry.

Claims

1. 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.

2. The method of claim 1, wherein in (B) the markers are present in a concentration K2 and the target molecules are present in a concentration K3.

3. The method of claim 1, wherein the markers GM1 comprise specifically bound markers SGM1 and the markers GM2 comprise specifically bound markers SGM2 and wherein SGM1 and SGM2 are liberated after (D), and quantities thereof are used in (E2) instead of M1 and M2.

4. The method of claim 1, further comprising repeating (A) to (E1) or (A) to (E2) wherein in at least one of (A) and (B) an agent which competes with the markers for a specific binding to the target molecules is added to at least one of the first and second mixed phases in an amount which substantially prevents or cancels a specific binding of the markers to the target molecules.

5. The method claim 1, further comprising repeating (A) to (E1) or (A) to (E2) at least once with a different ratio of K1 and K3.

6. The method of claim 1, wherein (D) comprises at least one of dialysis, precipitation, adsorption, binding to immobilized molecules which have an affinity to at least one of the target molecules, complexes of the target molecules and the markers and complexes of the target molecules and the ligands, centrifugation, chromatography, and filtration.

7. The method of claim 1, wherein (D) comprises ultrafiltration.

8. The method of claim 1 , wherein (D) is carried out under conditions under which at least one of(a) the concentrations of the unbound markers in the first and the second mixed phases and (b) the amounts of the bound markers GM 1 and GM2 remain constant.

9. The method of claim 1, wherein the mass spectrometry is carried out in one of the MS, MS/MS and MSⁿ modes.

10. The method of claim 9, wherein the mass spectrometry is preceded by at least one of a capillary electrophoresis, a gas chromatography and a liquid chromatography.

11. The method of claim 1, wherein the mass spectrometry is preceded by a HPLC.

12. The method of claim 11, wherein the HPLC comprises reverse phase chromatography.

13. The method of claim 1, wherein prior to the mass spectrometry a sample preparation by means of at least one of extraction, filtration, gas chromatography, capillary electrophoresis, liquid chromatography, fast preformance liquid chromatography, reverse phase chromatography, size exclusion chromatography and affinity chromatography is carried out.

14. The method of claim 1, wherein prior to the mass spectrometry a sample preparation by means of at least one of solid phase extraction, liquid-liquid extraction, ultrafiltration and HPLC is carried out.

15. The method of claim 1, wherein the first and the second mixed phases of (A) and (B) comprise at least one of more than 10 different target molecules and more than 10 different markers.

16. The method of claim 15, wherein the first and the second mixed phases of (A) and (B) comprise at least one of more than 100 different target molecules and more than 100 different markers.

17. The method of claim 1, wherein the first and the second mixed phases of (A) and (B) comprise a common marker which is specific to different target molecules.

18. The method of claim 15, wherein the different markers differ from each other with respect to at least one of the molar mass, the formation of ions or daughter ions in the mass spectrometry, the chromatographic behavior and the electrophoretic behavior.

19. The method of claim 1, wherein the first mixed phase of (A) comprises at least one of more than 10 different target molecules and more than 10 different markers.

20. The method of claim 19, wherein the first mixed phase of (A) comprises at least one of more than 100 different target molecules and more than 100 different markers.

21. The method of claim 19, wherein the ligands are generated by methods of combinatorial chemistry.

22. The method of claim 1, further comprising at least one of identifying ligands which are bound by the target molecules and determining a quantity of ligands which are bound by the target molecules.

23. The method of claim 22, wherein mass spectrometry is used for the at least one of identifying the bound ligands and determining the quantity thereof.

24. The method of claim 23, wherein prior to mass spectrometry the bound ligands are specifically liberated from the target molecules.

25. The method of claim 1, wherein the markers are used as external standard for the determination of K4 and KS or M1 and M2.

26. The method of claim 1, wherein after (D) at least one marker which comprises a marking substance is added as an internal standard to at least one the first and second mixed phases.

27. The method of claim 1, wherein the target molecules comprise target molecules that are embedded in membrane structures.

28. The method of claim 1, wherein the target molecules comprise target molecules that are not bound to a solid support.

29. The method of claim 1, further comprising a regeneration of the target molecules after one of (D) and (F2).

30. The method of claim 1, wherein at least one of the target molecules, markers and ligands are selected from peptides, proteins, hormones, nucleic acids, sugars, polymers, low molecular weight compounds of natural or synthetic origin and structures on or inside cells, cell fragments, cell homogenates, synaptosomes, liposomes, vesicles, tissue cuts, viruses, components and fragments thereof, capsides, components and fragments thereof, and mixtures of natural substances.

31. The method of claim 30, wherein at least one of the target molecules, markers and ligands are selected from receptors, prions, enzymes, transport proteins, ion channels, antibodies, synaptic plasma membrane vesicles and vesicles found inside cells.

32. A process for determining the capability of a chemical species to exhibit pharmaceutical activity, wherein the process comprises carrying out the method of claim 1.

33. The process of claim 32, wherein the ligands comprise the chemical species.