Kinetic assay

• US 20040171069A1
• Filed: 01/14/2004
• Published: 09/02/2004
• Est. Priority Date: 05/14/2001
• Status: Abandoned Application

• Current Assignee: The New Zealand Institute For Plant and Food Research Limited
• Original Assignee: The New Zealand Institute For Plant and Food Research Limited
• Inventors: Cook, Christian John, Wu, Vinqiu, Mitchell, John Stanton
• Application Number:
  US10/477,191
• Publication Number:
  US 20040171069A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07J 33/00 Normal steroids having a sulfur-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
• CPC Class Codes: C07J 43/00 Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Abstract

A hapten-linker-large group conjugate for use in a rapid assay, wherein the assay is kinetic-based not approaching equilibrium, the hapten-linker-large group conjugate being of the general formula: X—W—Y-Z wherein: X is a hapten; W is an optional thioether or ether group;Y is a linker of 10 or more atoms in length; and Z is a large group of sufficient size to provide steric hindrance with respect to the binding of X to a ligand in the absence of Y. Also provided are processes for the production of the conjugates, assay methods and kits. The assays of the invention utilising conjugates of the invention [(5)-OVA] provide a better sensitivity than the same assays with conjugates with shorter linkers [(2)-OVA and 3-OVA].

Claims

1. A hapten-linker-large group conjugate for use in a rapid assay, wherein the assay is kinetic-based not approaching equilibrium, the hapten-linker-large group conjugate being of the general formula:

X—

W—

Y-Z

Wherein;

X is a hapten;

W is an optional thioether or ether group;

Y is a linker of 10 or more atoms in length; and

Z is a large group of sufficient size to provide steric hindrance with respect to the binding of X to a ligand in the absence of Y.

2. A hapten-linker-large group conjugate of claim 1, wherein W is a thioether bridge.

3. A hapten-linker-large group conjugate of any one the preceding claims, wherein Y is of between 10 to 50 atoms inclusive in length.

4. A hapten-linker-large group conjugate of any one of the preceding claims, wherein Y is of between 11 to 24 atoms inclusive in length.

5. A hapten-linker-large group conjugate of any one of the preceding claims, wherein Y is of between 11 to 18 atoms inclusive in length.

6. A hapten-linker-large group conjugate of any one of the preceding claims, which is:

7. A hapten-linker-large group conjugate of any one of the preceding claims wherein Z is a protein or a polypeptide.

8. A hapten-linker-large group conjugate of any one of the preceding claims wherein Z is ovalbumin.

11. A hapten-linker-large group conjugate of any one of the preceding claims, wherein X is a steroid or steroid analogue.

12. A hapten-linker-large group conjugate of any one of the preceding claims wherein X is a multi-cyclic fused-ring hapten having an A-ring structure of Formula VI:

13. A hapten-linker-large group conjugate of any one of the preceding claims, wherein X is a hapten of Formula VII:

14. A hapten-linker-large group conjugate of any one of the preceding claims, wherein X is selected from the group comprising:

15. A hapten-linker-large group conjugate of any one of the preceding claims, wherein X is progesterone.

17. A hapten-linker-large group conjugate of any one of claims 1 to 9 and 11 to 15 that is:

18. A hapten-linker-large group conjugate of any-one of claims 1 to 9 and 11 to 15 that is:

19. A hapten-linker-large group conjugate of any one of the preceding claims, wherein the ligand is an immunoglobulin molecule.

20. A hapten-linker-large group conjugate of claim 18, wherein the ligand is an antibody or an antibody fragment.

21. A rapid assay method wherein the assay is kinetic-based not approaching equilibrium, the assay being for detecting a hapten in a sample, comprising the steps of:

d) contacting a ligand capable of binding the hapten with a test sample;

e) further contacting the ligand of step a) with a hapten-linker-large group conjugate of any one of claims 1 to 20 specific for the ligand; and

f) determining the amount of unconjugated hapten bound to the ligand.

22. A rapid assay method according to claim 21, wherein the second step (b) of contacting the ligand results in contacting and binding of much of the excess unbound ligand.

23. A rapid assay method according to claim 21 or claim 22, wherein the hapten-linker-large group conjugate is immobilised.

24. A rapid assay method according to any one of claims 21 to 23, wherein the mixture of step a) is flowed over the hapten-linker-large group conjugate of step b).

25. A rapid assay method according to any one of claims 21 to 24, wherein the hapten is a steroid.

26. A rapid assay method according to any one of claims 21 to 24, wherein the hapten is progesterone.

27. A rapid assay method according to any one of claims 21 to 25, wherein the ligand is an antibody.

28. A rapid assay wherein the assay is kinetic-based not approaching equilibrium, the assay being for detecting a hapten in a sample, comprising the steps of:

a) combining hapten-linker-large group conjugate of any one of claims 1 to 20 with a test sample;

b) contacting the resultant mixture with ligand capable of binding the hapten; and

c) determining the amount of unconjugated hapten bound to the ligand.

29. A rapid assay according to claim 28, wherein the ligand is immobilised.

30. A rapid assay according to claim 28 or claim 29, wherein the step b) of contacting the resultant mixture with an immobilised ligand takes place by a flow over or flow through system.

31. A rapid assay method according to any one of claims 28 to 30, wherein the hapten is a steroid.

32. A rapid assay method according to any one of claims 28 to 31, wherein the hapten is progesterone.

33. A rapid assay method according to any one of claims 28 to 32, wherein the ligand is an antibody.

34. A rapid assay kit, wherein the assay is kinetic-based not approaching equilibrium, the kit including at least:

a) a ligand which binds to a hapten; and

b) a hapten-linker-large group conjugate of any one of claims 1 to 20.

35. A rapid assay kit of claim 34, wherein the kit further includes an indicator.

36. A rapid assay kit of claim 35, wherein the indicator is bound to the hapten-linker-large group conjugate.

37. A rapid assay kit of claim 35, wherein the indicator is bound to the ligand.

38. A rapid assay kit of any one of claims 34 to 37, which is a flow over kit.

39. A rapid assay kit of claim 38, which is a test strip.

40. A rapid assay kit of any one of claims 34 to 37, which is a flow through kit.

41. A process for binding a hapten-linker-large group conjugate of any one of claims 1 to 20 to a ligand comprising the steps of contacting the conjugate with a ligand capable of binding the hapten in the conjugate for a predetermined time where the reaction does not approach equilibrium.

42. The process according to claim 41, wherein the ligand is immobilised.

43. The process according to claim 41 or claim 42, wherein the ligand is contacted with a hapten before being contacted by the hapten-linker-large group conjugate.

44. The process according to claim 41 or claim 42, wherein the ligand is contacted with the hapten-linker-large group conjugate before or simultaneously with being contacted by a hapten.

45. A hapten-linker-large group conjugate of any one of the preceding claims, wherein the ligand is an immunoglobulin molecule.

46. A process for producing a hapten-linker-large group conjugate of any one of claims 1 to 20 is provided, including at least the steps of:

g) mixing an activated steroid hapten dissolved in an polar organic solvent with an aqueous solution comprising 1-10 molar equivalents of a heterobifunctional water-soluble linker;

h) allow the mixture to react; and

i) attach a large group to the remaining free functional linker group of the reaction hapten-linker product of step b).

47. The process according to claim 46, which includes an isolation step between steps b) and c) for isolating the hapten-linker product.

48. The process according to claim 46 or claim 47, wherein the final mixture has an aqueous content of between 2 and 30%.

49. The process according to any one of claims 46 to 48, wherein the final mixture has an aqueous content of between 5 and 15%.

50. The process according to any one of claims 46 to 49, wherein the final mixture has an aqueous content of about 10%.

51. The process according to any one of claims 46 to 50, wherein the step b) reaction time is in the order of up to 24 hours.

52. The process according to any one of claims 46 to 51, wherein the reaction in b) takes place at room temperature.

53. The process according to any one of claims 46 to 52, wherein the reaction in b) takes place at substantially neutral pH.

54. The process according to any one of claims 46 to 53, wherein the aqueous solution of step a) comprises 2-5 molar equivalents of a heterobifunctional water-soluble linker when compared with the activated steroid.

55. The process according to any one of claims 46 to 54, wherein the activated steroid is an activated ester of a steroid.

56. The process according to any one of claims 46 to 55, wherein the activated steroid is a succinimide ester of a steroid.

57. The process according to any one of claims 46 to 56, wherein the heterobifunctional linker is carboxyl at one end and amino at the other end.

58. The process according to any one of claims 46 to 57, wherein the polar organic solvent is selected from the group comprising DMF, DMSO, acetone and THF.

9. A hapten-linker-large group conjugate of any one of 1 to 0, wherein Z is an indicator group.

10. A hapten-linker-large group conjugate of any one of 1 to 0 and 9, wherein Z is bilirubin.

16. A hapten-linker-large group conjugate of claim 10 that is: