Method for detecting and/or quantifying first molecules

• US 20040038427A1
• Filed: 08/27/2003
• Published: 02/26/2004
• Est. Priority Date: 03/29/2000
• Status: Abandoned Application

• Current Assignee: November Aktiengesellschaft Gesellschaft Fur Molekulare Medizin
• Original Assignee: November Aktiengesellschaft Gesellschaft Fur Molekulare Medizin
• Inventors: Kosak, Hans, Krause, Jurgen
• Application Number:
  US10/240,205
• Publication Number:
  US 20040038427A1
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

The invention relates to a method for detecting and/or quantifying the binding of first molecules (16) to second molecules (18) affine therein. The inventive method comprises the following steps: a) providing a container (10), wherein the second molecules (18) are immobilized on a wall, b) contacting the second molecules (18) to a solution containing the first molecules (16), c) incubating the container (10) in such a way that the first molecules (16) bind to the second molecules (18) and build up on the wall and d) detecting the changes in concentration of the first molecules (16) in the solution by means of radiation without removing the solution from the container (10), whereby the solution is contained in said container (10).

Claims

1. A method for detecting and/or quantifying the binding of first molecules (16) to second molecules (18) with affinity therefor, comprising the following steps:

a) providing a vessel (10) in which the second molecules (18) are immobilized on a wall,

b) contacting the second molecules (18) with a solution containing the first molecules (16),

c) incubating the vessel (10) so that the first molecules (16) bind to the second molecules (18) and accumulate on the wall and

d) detecting the changes in concentration of the first molecules (16) in the solution contained in the vessel (10) by means of radiation, without removing solution from the vessel.

9. The method as claimed in claim 1 or 2, in which the number of the second molecules (18) is greater than the number of the first molecules (16).

17. The method as claimed in any of the preceding claims, in which a light beam (20), in particular of a defined wavelength, preferably a laser beam, is used to irradiate the solution for detection.

18. The method as claimed in claim 17, in which the light beam (20) is introduced parallel to the wall.

19. The method as claimed in claim 17 or 18, in which the light beam (20) is polarized.

20. The method as claimed in any of the preceding claims, in which a fluorescence, a diffraction, an absorption or an optical activity is measured for detection.

21. The method as claimed in any of the preceding claims, in which the solution additionally contains fifth molecules (26) which serve as internal markers and have no specific affinity for the first (16), second (18), third (23) or fourth molecules (24).

22. The method as claimed in any of the preceding claims, in which the first (16), third (23), fourth (24) and/or fifth molecules (26) or fragments of these molecules or components associated therewith are fluorescent, light-diffracting, light-absorbing or optically active.

23. The method as claimed in any of the preceding claims, in which step d is carried out by multiple or continuous determination of the concentration of the first (16), third (23) or fourth molecules (24), while carrying out step c, in particular at the start and end thereof.

24. The method as claimed in any of the preceding claims, in which free binding sites on the wall of the vessel (10) are saturated by sixth molecules bound thereto.

25. The method as claimed in any of the preceding claims, in which the solution contains at least one additive inhibiting unspecific binding, in particular a detergent, a protein, a protein mixture or a salt.

26. The method as claimed in any of the preceding claims, in which a specific change in concentration is determined by subtracting the value of a concentration change when carrying out the method without second molecules from the value of the concentration change according to step d.

27. The method as claimed in any of the preceding claims, in which the vessel (10) has the form of a cavity of a microtiter plate containing at least 96, in particular 384, cavities.

28. The method as claimed in any of the preceding claims, in which the vessel (10) has essentially no immobilized second molecules (18) at the point of entry and/or exit of the light beam (20, 22), preferably at the base of the vessel (10).

29. The method as claimed in any of the preceding claims, in which the vessel (10) is a capillary open at its ends (12, 14).

30. The method as claimed in claim 29, in which the capillary is filled with the solution by means of capillary forces.

31. The method as claimed in any of the preceding claims, in which the quotient of the wall area in mm² and the solution volume in mm³ is greater than 1 mm⁻

1, preferably greater than 3 mm<sup>−

1</sup>.

32. The method as claimed in any of the preceding claims, in which the first (16), second (18), third (23), fourth (24), fifth (26) or sixth molecules are selected from the following group:

peptides, proteins, nucleic acids, sugars, polymers, messengers, cells, cell fragments, viruses, components thereof or fragments of said components, capsids, components thereof or fragments of said components and hormones.

33. The method as claimed in any of the preceding claims, in which the method is carried out simultaneously or in quick succession in a number of vessels (10), in particular capillaries.

34. A microtiter plate for carrying out a method as claimed in any of claims 1-33, having a multiplicity of cavities which have in each case a wall and a base, said wall being activated for the binding of second molecules (18),

characterized in that

the base is not activated for the binding of second molecules (18).

35. The microtiter plate as claimed in claim 34, in which second molecules (18) are immobilized on the wall and the base has essentially no immobilized second molecules (18).

36. The microtiter plate as claimed in claim 35 or 36, in which at least sections of the wall are made of a porous material.

38. The microtiter plate as claimed in any of claims 35 to 38, in which the base is made of a transparent or translucent material.

39. The microtiter plate as claimed in claims 35 to 39, in which third (23) or fourth molecules (24) are associated with the wall or the second molecules (18).

40. The use of capillaries on whose walls second molecules (18) are immobilized for carrying out the method as claimed in any of claims 1-33.

2. A method for detecting and/or quantifying the binding of first molecules (16) to second molecules (18) with affinity therefor, comprising the following steps:

a) providing a vessel (10) in which the second molecules (18) are immobilized on a wall,

b) contacting the second molecules (18) with a solution containing the first molecules (16), with third molecules (23) having an affinity for the first molecules (16) being contained in the solution or in the vessel (10),

c) incubating the vessel (10) so that the first molecules (16) bind to the second (18) and third molecules (23) and accumulate on the wall and

d) detecting the changes in concentration of the third molecules (23) in the solution contained in the vessel (10) by means of radiation, without removing solution from the vessel.

6. The method as claimed in claim 2 or 3, in which the third (23) or fourth molecules (24) contained in the vessel (10) are associated with the second molecules (18) or the wall.

10. The method as claimed in claim 2, in which the number of the first molecules (16) is not less than the number of the third molecules (23).

3. A method for detecting and/or quantifying the binding of first molecules (16) to second molecules (18) with affinity therefor, comprising the following steps:

a) providing a vessel (10) in which the second molecules (18) are immobilized on a wall,

b) contacting the second molecules (18) with a solution containing the first molecules (16), with fourth molecules (24) having an affinity for the second molecules (18) being contained in the solution or in the vessel (10),

c) incubating the vessel (10) so that the first molecules (16) bind to the second molecules (18) and the fourth molecules (24) are, at least partially, dissolved or remain and

d) detecting the changes in concentration of the fourth molecules (24) in the solution contained in the vessel (10) by means of radiation, without removing solution from the vessel.

7. The method as claimed in claim 3, in which the affinity of the fourth molecules (24) for the second molecules (18) is not greater or not substantially greater than the affinity of the first molecules (16) for the second molecules (18).

8. The method as claimed in claim 3, in which the number of the first molecules (16) is greater than the number of the second molecules (18) and the number of the second molecules (18) is greater than the number of the fourth molecules (24).

4. A method for detecting and/or quantifying the enzymic or chemical activity of first molecules (16) toward second molecules (18) with affinity therefor, comprising the following steps:

a) providing a vessel (10) in which the second molecules (18) are immobilized on a wall,

b) contacting the second molecules (18) with a solution containing the first molecules (16),

c) incubating the vessel (10) so that the first molecules (16) convert the second molecules (18) with release of a fragment (19a) of the second molecules (18) and

d) detecting the change in concentration of the fragment (19a) in the solution contained in the vessel (10) by means of radiation, without removing solution from the vessel.

11. The method as claimed in claim 4, in which the first molecule (16) is a cleaving enzyme, preferably a protease, peptidase, nuclease, helicase or lipase, or a sugar-degrading enzyme.

12. The method as claimed in claim 4 or 11, in which the second molecule (18) forms a substrate for the first molecule (16), which is preferably derived from a protein, peptide, a nucleic acid, helicase or a monomeric or polymeric sugar.

5. A method for detecting and/or quantifying the enzymic or chemical activity of first molecules (16) toward second (18) or third molecules with affinity therefor, comprising the following steps:

a) providing a vessel (10) in which the second molecules (18) are immobilized on a wall,

b) contacting the second molecules (18) with a solution containing the first molecules (16), with third molecules having an affinity for the second molecules being contained in the solution,

c) incubating the vessel (10) so that the first molecules (16) bind to the second (18) or third molecules, whereby a conversion causing the release of a fragment (19a) of the second molecules (18) is suppressed or increased by the third molecules, and

d) detecting the change in concentration of the fragment (19a) in the solution contained in the vessel (10) by means of radiation, without removing solution from the vessel (10).

13. The method as claimed in claim 5, in which the third molecule is a cleaving enzyme, preferably a protease, peptidase, nuclease, helicase or lipase, or a sugar-degrading enzyme.

14. The method as claimed in claim 5, in which the second molecule (18) forms a substrate for the third molecule, which is preferably derived from a protein, peptide, a nucleic acid, helicase or a monomeric or polymeric sugar.

15. The method as claimed in claim 5, in which the first molecule (16) is an agonist, an antagonist or a competitor with respect to the third molecule.

16. The method as claimed in claim 5, in which binding of the second molecule to the first molecule prevents cleavage of the second molecule by the third molecule.

37. The microtiter plate as claimed in claim 37, in which the porous material is selected from the following group:

cellulose, nitrocellulose, nylon, agarose, paper, cardboard.

41. The use as claimed in claim 41, in which the capillaries have a label indicating the type of the second molecules (18).

42. The use as claimed in claim 41 or 42, in which the third (23) or fourth molecules (24) are associated with the wall or the second molecules (18).