Threshold ligand-receptor assay

Claim 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 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.

Claim 3

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.

Claim 4

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.

Claim 5

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.

Claim 6

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.

Claim 7

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.

Claim 8

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.

Claim 9

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

Claim 10

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

Claim 11

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

Claim 12

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

Claim 13

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

Claim 14

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.

Claim 15

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.

Claim 16

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.

Claim 17

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

Claim 18

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

Claim 19

Method of claim 14 wherein said antibody is monoclonal.

Claim 20

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.

Claim 21

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.

Claim 22

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.

Claim 23

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

Claim 24

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.

Claim 25

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.

Claim 26

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.

Claim 27

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

Claim 28

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

Claim 29

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

Claim 30

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.

Claim 31

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.

Claim 32

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.

Claim 33

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.

Claim 34

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.

Claim 35

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.

Claim 36

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.

Claim 37

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.

Claim 38

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.

Claim 39

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

Claim 40

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

Claim 41

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

Claim 42

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

Claim 43

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.

Claim 44

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.

Claim 45

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.

Claim 46

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

Claim 47

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

Claim 48

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.

Claim 49

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

Claim 50

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.

Claim 51

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.

Claim 52

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.

Claim 53

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

Claim 54

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

Claim 55

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

Claim 56

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.

Claim 57

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.

Claim 58

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.

Claim 59

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.

Claim 60

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.

Claim 61

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.

Claim 62

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.

Claim 63

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.

Claim 64

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.

Claim 65

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

Claim 66

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

Claim 67

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.

Claim 68

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.

Claim 69

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.

Claim 70

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

Claim 71

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

Claim 72

Method of claim 59 wherein said antibody is monoclonal.

Claim 73

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.

Claim 74

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

Claim 75

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.

Claim 76

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.

Claim 77

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.

Claim 78

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

Claim 79

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

Claim 80

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

Claim 81

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.

Claim 82

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).