Threshold ligand-receptor assay

• US 5,028,535 A
• Filed: 01/10/1989
• Issued: 07/02/1991
• Est. Priority Date: 01/10/1989
• Status: Expired due to Term

• Current Assignee: BIOSITE INCORPORATED (Abbott Laboratories)
• Original Assignee: Biosite Diagnostics Incorporated (Abbott Laboratories)
• Inventors: Buechler, Kenneth F., Valkirs, Gunars E., Anderson, Richard R.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/295,568
• Time in Patent Office: 903 Days
• Field of Search: 435/7, 435/805, 435/810, 435/7.1, 435/7.5, 435/7.9, 435/7.92, 435/7.93, 435/7.94, 435/967, 422/55-57, 436/501, 436/514, 436/518, 436/523-531, 436/807, 436/810, 436/825
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: G01N 33/743 Steroid hormones
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

This invention is directed to a ligand-receptor assay for determining the presence or amount of at least one target ligand, capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor, said ligand analogue conjugate comprising at least one ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with ligand analogue conjugate and ligand receptor to form a reaction mixture, the relative amounts of ligand analogue conjugate and ligand receptor being such that in the absence of target ligand, and subsequent to substantially equilibrium binding, substantially all of the ligand analogue conjugate is bound to ligand receptor;

b. detecting the unbound ligand analogue conjugate;

c. relating the detectable signal to the presence or amount of target ligand in the fluid sample. In one embodiment an optional means also is employed for removing receptor from the reaction mixture. In related claimed assay formats the analyte of interest may be either ligand receptor or ligand.

Claims

1. Method for determining the presence of at least one target ligand, capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor, said ligand analogue conjugate comprising at least one ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand analogue conjugate and said ligand receptor to form a homogeneous reaction mixture, the relative amounts of said ligand analogue conjugate and said ligand receptor being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand analogue conjugate is bound to said ligand receptor such that no unbound ligand analogue conjugant is detected as a result of the assay method;

b. detecting unbound ligand analogue conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand in said fluid sample.

12. Method of claim 1 or 2 or 3 or 4 wherein said ligand receptor undergoes diffusive motion during the competition for ligand receptor binding sites which occurs between ligand and ligand analogue conjugate.

13. Method of claim 1 or 2 or 3 or 4 wherein said ligand receptor is immobilized on a non-diffusive solid phase.

14. Method of claim 1 or 2 or 3 or 4 wherein said method is an immunoassay in which the ligand receptor is an antibody.

15. Method of claim 14 wherein said antibody is monoclonal.

16. Method of claim 1 or 2 or 3 or 4 wherein the number of ligand analogues coupled to said signal development element is between 1 and 50.

17. Method of claim 1 or 2 or 3 or 4 wherein the number of ligand analogues coupled to said signal development element is between 1 and 10.

18. Method of claim 1 or 2 or 3 or 4 further comprising at least one ligand receptor immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand analogue conjugate in said reaction mixture.

19. Method of claim 18 wherein said solid phase is selected from the group consisting of porous and non-porous matrices.

23. Method of claim 1 or 2 or 3 or 4 wherein said signal development element is detected by non-instrumental means.

24. Method of claim 23 wherein said signal development element is detected by visual means.

25. Method of claim 1 or 2 or 3 or 4 wherein the signal development element is an enzyme.

69. Method of claims 1 or 2 or 3 or 4 or 26 or 27 or 28 or 29 or 48 or 49 or 50 or 51 wherein said reaction mixture also contains a negative control ligand conjugate and a negative control ligand receptor such that subsequent to substantially equilibrium binding in said reaction mixture, substantially all of the negative control ligand conjugate is bound to negative control ligand receptor such that no unbound negative control ligand conjugate is detected as a result of the assay method and wherein the detection of unbound negative control ligand conjugate in the reaction mixture as a result of the assay method is an indication of an invalid method result.

2. Method for determining the presence of at least one target ligand, capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor, said ligand analogue conjugate comprising at least one ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand analogue conjugate and said ligand receptor to form a homogeneous reaction mixture, the relative amounts of said ligand analogue conjugate and said ligand receptor being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand analogue conjugate is bound to said ligand receptor such that no unbound ligand analogue conjugate is detected as a result of the assay method;

b. removing ligand receptor from said reaction mixture;

c. detecting unbound ligand analogue conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand in said fluid sample.

5. Method of claim 2 or 4 wherein a solid support means is used in step (b) to remove ligand receptor from said reaction mixture.

6. Method of claim 5 wherein at least one receptor is immobilized on said solid support.

7. Method of claim 5 wherein said solid support means is selected from the group consisting of diffusible beads and porous matrices.

8. Method of claim 2 or 4 wherein in step (b) ligand receptor is removed from said reaction mixture by precipitation.

9. Method of claim 8 wherein at least one receptor is used to precipitate ligand receptor.

10. Method of claim 2 or 4 wherein at least one of said ligand receptors in said reaction mixture is a ligand complement-ligand receptor molecule and said reaction mixture is contacted with at least one ligand complement receptor capable of binding thereto, and wherein said ligand complement receptor is used to remove said ligand complement-ligand receptor molecule from said reaction mixture.

11. Method of claim 10 wherein said ligand complement receptor is avidin and said ligand complement-ligand receptor molecule is biotin covalently bound to said ligand receptor.

20. Method of claim 2 or 4 wherein said reaction mixture contains at least one ligand complement-ligand analogue conjugate and wherein at least one ligand complement receptor is immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand complement-ligand analogue conjugate in said reaction mixture.

21. Method of claim 2 or 4 wherein said reaction mixture contains at least one ligand complement-ligand analogue conjugate and wherein at least one ligand receptor and at least one ligand complement receptor are immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand analogue conjugate and at least one unbound ligand complement-ligand analogue conjugate in said reaction mixture.

3. Method for determining the presence of at least one target ligand at or above at least one predetermined threshold ligand concentration, said target ligand capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor, said ligand analogue conjugate comprising at least one ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand analogue conjugate and said ligand receptor to form a homogeneous reaction mixture;

(i) the relative amounts of said ligand analogue conjugate and said ligand receptor being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand analogue conjugate is bound to said ligand receptor such that no unbound ligand analogue conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of ligand analogue conjugate and ligand receptor being selected such that at least one of said predetermined threshold ligand concentration(s) is established below which no unbound ligand analogue conjugate is detected as a result of the assay method;

b. detecting unbound ligand analogue conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand at or above said predetermined threshold ligand concentration in said fluid sample.

22. Method of claim 3 or 4 further comprising at least one ligand receptor immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand analogue conjugate in said reaction mixture, wherein a detectable signal at each locus signifies the presence of ligand at or above a predetermined threshold ligand concentration specific for that locus.

4. Method for determining the presence of at least one target ligand at or above at least one predetermined threshold ligand concentration, said target ligand capable of competing with a ligand analogue conjugate for binding sites available on a ligand receptor, said ligand analogue conjugate comprising at least ligand analogue coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand analogue conjugate and said ligand receptor to form a homogeneous reaction mixture;

(i) the relative amounts of said ligand analogue conjugate and said ligand receptor being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand analogue conjugate is bound to said ligand receptor such that no unbound ligand analogue conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of ligand analogue conjugate and ligand receptor being selected such that at least one of said predetermined threshold ligand concentration is established below which no unbound ligand analogue conjugate is detected as a result of the assay method;

b. removing ligand receptor from said reaction mixture;

c. detecting unbound ligand analogue conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand at or above said predetermined threshold ligand concentration in said fluid sample.

26. Method for determining the presence of at least one target ligand receptor, capable of competing with a ligand receptor conjugate for binding sites available on a ligand analogue construct, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand receptor, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture, the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand receptor, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method;

b. detecting unbound ligand receptor conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand receptor in said fluid sample.

37. Method of claim 26 or 27 or 28 or 29 wherein said ligand analogue construct undergoes diffusive motion during the competition for ligand analogue binding sites which occurs between said target ligand receptor and said ligand receptor conjugate.

38. Method of claim 26 or 27 or 28 or 29 wherein said ligand analogue construct is immobilized on a non-diffusive solid phase.

39. Method of claim 26 or 27 or 28 or 29 wherein said method is an immunoassay in which the ligand receptor is an antibody.

40. Method of claim 26 or 27 or 28 or 29 further comprising at least one ligand analogue immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate in said reaction mixture.

41. Method of claim 40 wherein said solid phase is selected from the group consisting of porous and non-porous matrices.

45. Method of claim 26 or 27 or 28 or 29 wherein said signal development element is detected by non-instrumental means.

46. Method of claim 45 wherein said signal development element is detected by visual means.

47. Method of claim 26 or 27 or 28 or 29 wherein the signal development element is an enzyme.

27. Method for determining the presence of at least one target ligand receptor, capable of competing with a ligand receptor conjugate for binding sites available on a ligand analogue construct, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand receptor, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture, the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand receptor, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method;

b. removing ligand analogue construct from said reaction mixture;

c. detecting unbound ligand receptor conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand receptor in said fluid sample.

30. Method of claim 27 or 29 wherein a solid support means is used in step (b) to remove ligand analogue construct from said reaction mixture.

31. Method of claim 30 wherein at least one receptor is immobilized on said solid support.

32. Method of claim 30 wherein said solid support means is selected from the group consisting of diffusible beads and porous matrices.

33. Method of claim 27 or 29 wherein in step (b) ligand analogue construct is removed from said reaction mixture by precipitation.

34. Method of claim 33 wherein at least one (ligand analogue construct) receptor is used to precipitate ligand analogue construct.

35. Method of claim 27 or 29 wherein at least one of said ligand analogue constructs in said reaction mixture is a ligand complement-ligand analogue construct and said reaction mixture is contacted with at least one ligand complement receptor capable of binding thereto, and wherein said ligand complement receptor is used to remove said ligand complement-ligand analogue construct from said reaction mixture.

36. Method of claim 35 wherein said ligand complement-ligand analogue construct is biotin covalently bound to said ligand analogue construct and said ligand complement receptor is avidin.

42. Method of claim 27 or 29 wherein said reaction mixture contains at least one ligand complement-ligand receptor conjugate molecule and wherein at least one ligand complement receptor is immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand complement-ligand receptor conjugate molecule in said reaction mixture.

43. Method of claim 27 or 29 wherein the reaction mixture also contains at least one ligand complement-ligand receptor conjugate molecule and wherein at least one ligand analogue and at least one ligand complement receptor are immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate and at least one unbound ligand complement-ligand receptor conjugate molecule in said reaction mixture.

28. Method for determining the presence of at least one target ligand receptor at or above at least one predetermined threshold ligand receptor concentration, said target ligand receptor capable of competing with a ligand receptor conjugate for binding sites available on said ligand analogue construct, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand receptor, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture;

(i) the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand receptor, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of ligand receptor conjugate and ligand analogue construct being selected such that at least one of said predetermined threshold ligand receptor concentration(s) is established below which no unbound ligand receptor conjugate is detected as a result of the assay method;

b. detecting unbound ligand receptor conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand receptor at or above said predetermined threshold ligand receptor concentration(s) in said fluid sample.

44. Method of claim 28 or 29 further comprising at least one ligand analogue immobilized son a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate in said reaction mixture, wherein a detectable signal at each locus signifies the presence of ligand receptor at or above a predetermined threshold ligand receptor concentration specific for that locus.

70. Method of claims 3 or 4 or 28 or 29 or 50 or 51 wherein detection is by visual means and wherein said reaction mixture contains at least one reference ligand conjugate wherein the detection of reference ligand conjugate defines at least one reference concentration for at least one target ligand which is used in conjunction with said predetermined threshold concentration to relate said assay response to a range of target concentration(s).

29. Method for determining the presence of at least one target ligand receptor at or above at least one predetermined threshold ligand receptor concentration, said target ligand receptor capable of competing with a ligand receptor conjugate for binding sites available on a ligand analogue construct, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand receptor, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture;

(i) the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand receptor, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of said ligand receptor conjugate and ligand analogue construct being selected such that at least one of said predetermined threshold ligand receptor concentration(s) is established below which no unbound ligand receptor conjugate is detected as a result of the assay method;

b. removing ligand analogue construct from said reaction mixture;

c. detecting unbound ligand receptor conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand receptor at or above said predetermined threshold ligand receptor concentration(s) in said fluid sample.

48. Method for determining the presence of at least one target ligand, capable of competing with a ligand analogue construct for binding sites available on a ligand receptor conjugate, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture, the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method;

b. detecting unbound ligand receptor conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand in said fluid sample.

58. Method of claim 48 or 49 or 50 or 51 wherein the ligand analogue construct is immobilized on a non-diffusive solid phase.

59. Method of claim 48 or 49 or 50 or 51 wherein said method is an immunoassay in which the ligand receptor is an antibody.

60. Method of claim 59 wherein said antibody is monoclonal.

61. Method of claim 48 or 49 or 50 or 51 further comprising at least one ligand analogue immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate in said reaction mixture.

62. Method of claim 61 wherein said solid phase is selected from the group consisting of porous and non-porous matrices.

66. Method of claim 48 or 49 or 50 or 51 wherein said signal development element is detected by non-instrumental means.

67. Method of claim 66 wherein said signal development element is detected by visual means.

68. Method of claim 48 or 49 or 50 or 51 wherein the signal development element is an enzyme.

49. Method for determining the presence of at least one target ligand, capable of competing with a ligand analogue construct for binding sites available on a ligand receptor conjugate, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogenous reaction mixture, the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method;

b. removing ligand analogue construct from said reaction mixture;

c. detecting unbound ligand receptor conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand in said fluid sample.

52. Method of claim 49 or 51 wherein a solid support means is used in step (b) to remove ligand analogue construct from said reaction mixture.

53. Method of claim 52 wherein said solid support means is selected from the group consisting of diffusible beads and porous matrices.

54. Method of claim 49 or 51 wherein in step (b) ligand analogue construct is removed from said reaction mixture by precipitation.

55. Method of claim 49 or 51 wherein at least one of said ligand analogue constructs in said reaction mixture is a ligand complement-ligand analogue construct and said reaction mixture is contacted with at least one ligand complement receptor capable of binding thereto, and wherein said ligand complement receptor is used to remove said ligand complement-ligand analogue construct from said reaction mixture.

56. Method of claim 55 wherein said ligand complement-ligand analogue construct is biotin covalently bound to said ligand analogue construct and said ligand complement receptor is avidin.

57. Method of claim 52 or 53 or 54 or 55 wherein said ligand analogue construct undergoes diffusive motion during the competition for ligand receptor conjugate binding sites which occurs between ligand and ligand analogue construct.

63. Method of claim 49 or 51 wherein the reaction mixture contains at least one ligand complement-ligand receptor conjugate molecule and wherein at least one ligand complement receptor is immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand complement-ligand receptor conjugate molecule in said reaction mixture.

64. Method of claim 49 or 51 wherein the reaction mixture also contains at least one ligand complement-ligand receptor conjugate molecule and wherein at least one ligand analogue and at least one ligand complement receptor are immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate and at least one unbound ligand complement-ligand receptor conjugate molecule in said reaction mixture.

50. Method for determining the presence of at least one target ligand at or above at least one predetermined threshold ligand concentration, said target ligand capable of competing with a ligand analogue construct for binding sites available on a ligand receptor conjugate, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogeneous reaction mixture;

(i) the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of ligand receptor conjugate and ligand analogue construct being selected such that at least one of said predetermined threshold ligand concentration(s) is established below which no unbound ligand receptor conjugate is detected as a result of the assay method;

b. detecting unbound ligand receptor conjugate in said reaction mixture;

c. relating the detectable signal to the presence of said target ligand at or above said predetermined threshold ligand concentration(s) in said fluid sample.

65. Method of claim 50 or 51 further comprising at least one ligand analogue immobilized on a solid phase in at least one distinct locus for the detection of at least one unbound ligand receptor conjugate in said reaction mixture wherein a detectable signal at each locus signifies the presence of ligand at or above a predetermined threshold ligand concentration specific for that locus.

51. Method for determining the presence of at least one target ligand at or above at least one predetermined threshold ligand concentration, said target ligand capable of competing with a ligand analogue construct for binding sites available on a ligand receptor conjugate, said ligand receptor conjugate comprising at least one ligand receptor coupled to a signal development element capable of emitting a detectable signal, in a fluid sample suspected of containing said target ligand, comprising the steps of:

a. contacting said fluid sample with said ligand receptor conjugate and said ligand analogue construct to form a homogenous reaction mixture;

(i) the relative amounts of said ligand receptor conjugate and said ligand analogue construct being selected such that in the absence of said target ligand, and subsequent to substantially equilibrium binding in said reaction mixture, substantially all of said ligand receptor conjugate is bound to said ligand analogue construct such that no unbound ligand receptor conjugate is detected as a result of the assay method; and

(ii) the relative and absolute amounts of ligand receptor conjugate and ligand analogue construct being selected such that at least one of said predetermined threshold ligand concentration(s) is established below which no unbound ligand receptor conjugate is detected as a result of the assay method;

b. removing ligand analogue construct from said reaction mixture;

c. detecting unbound ligand receptor conjugate in said reaction mixture;

d. relating the detectable signal to the presence of said target ligand at or above said predetermined threshold ligand concentration(s) in said fluid sample.