US4769321A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Assay method and reagent therefor
Assignee: Individual
•
Filing: 1986-04-21
•
Granted: 1988-09-06
•
Expires: 2006-04-21
Abstract
Abstract Enzymatic assays are described which enable the detection of inter alia enzymes, cofactors and substrates in small quantity with the ability to produce a visually detectable change.
Inventors
- 👤 Colin H. Self
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 02:23
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/581; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/966; Y10S435/975
Claims Analysis
Total Claims: 130
Claim 1
Independent Claim
174 words
In a competitive binding assay in which competition between an unknown amount of a first component and a standard amount of the same component which has been labelled for a standard amount of its complimentary component takes place, the improvement which comprises: (a) employing as the labelled component a conjugate of the first component and an enzyme, (b) contacting the conjugate with a precursor for a modulator for a cyclic chemical reaction whereby by said modulator is produced as the result of reaction of the enzyme and precursor for the modulator, and (c) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 2
Dependent Claim
174 words
In a competitive binding assay in which competition between an unknown amount of a first component and a standard amount of the same component which has been labelled for a standard amount of its complimentary component takes place, the improvement which comprises: (a) employing as the labelled component a conjugate of the first component and an enzyme, (b) contacting the conjugate with a precursor for a modulator for a cyclic chemical reaction whereby by said modulator is produced as the result of reaction of the enzyme and precursor for the modulator, and (c) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 3
Dependent Claim
17 words
The improvement according to claim 1 wherein one of the components is bound to a solid matrix.
Claim 4
Dependent Claim
22 words
The improvement according to claim 1 wherein the modulator is a cofactor which the cyclic chemical reaction interconverts with its reduced form.
Claim 5
Dependent Claim
14 words
The improvement according to claim 1 wherein the cofactor is a nicotinamide adenine dinucleotide.
Claim 6
Dependent Claim
15 words
The improvement according to claim 1 wherein NAD is converted to NADH by a dehydrogenase.
Claim 7
Dependent Claim
13 words
The improvement according to claim 5 wherein the dehydrogenase is an alcohol dehydrogenase.
Claim 8
Dependent Claim
25 words
The improvement according to claim 5 wherein the conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a color change is produced.
Claim 9
Dependent Claim
14 words
The improvement according to claim 5 wherein cyclic chemical reaction comprises two enzymatic reactions.
Claim 10
Dependent Claim
15 words
The improvement according to claim 1 wherein the first component is an antibody or antigen.
Claim 11
Dependent Claim
15 words
The improvement according to claim 1 wherein the first component and conjugate are added sequentially.
Claim 12
Dependent Claim
15 words
The improvement according to claim 10 wherein the first components is added before the conjugate.
Claim 13
Dependent Claim
20 words
The improvement according to claim 1 wherein the labelled component is a conjugate of the first component and a phosphatase.
Claim 14
Dependent Claim
20 words
The improvement according to claim 5 wherein the labelled component is a conjugate of the first components and a phosphatase.
Claim 15
Dependent Claim
13 words
The improvement according to claim 13 wherein the phosphatase is an alkaline phosphatase.
Claim 16
Dependent Claim
34 words
The improvement according to claim 14 wherein the NADP is converted to NAD by the conjugate and the NAD starts a cyclic chemical reaction which proceeds at pH 8-10.5 to interconvert NAD and NADH.
Claim 17
Dependent Claim
18 words
The improvement according to claim 15 wherein the reduction of NAD to NADH is effected by a dehydrogenase.
Claim 18
Dependent Claim
12 words
The improvement according to claim 16 wherein the dehydrogenase is alcohol dehydrogenase.
Claim 19
Dependent Claim
13 words
The improvement according to claim 1 wherein the complimentary component is an antibody.
Claim 20
Dependent Claim
13 words
The improvement according to claim 18 wherein the antibody is a monoclonal antibody.
Claim 21
Dependent Claim
22 words
The improvement according to claim 1 wherein the modulator is a cofactor which the cyclic chemical reaction interconverts with its oxidized form.
Claim 22
Dependent Claim
14 words
The improvement according to claim 20 wherein the cofactor is a nicotinamide adenine dinucleotide.
Claim 23
Dependent Claim
15 words
The improvement according to claim 20 wherein NAD is converted to NADH by a dehydrogenase.
Claim 24
Dependent Claim
13 words
The improvement according to claim 22 wherein the dehydrogenase is an alcohol dehydrogenase.
Claim 25
Dependent Claim
24 words
The improvement according to claim 22 wherein conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 26
Dependent Claim
15 words
The improvement according to claim 22 wherein the cyclic chemical reaction comprises two enzymatic reactions.
Claim 27
Dependent Claim
20 words
The improvement according to claim 20 wherein the labelled component is a conjugate of the first component and a phosphatase.
Claim 28
Dependent Claim
13 words
The improvement according to claim 26 wherein the phosphatase is an alkaline phosphatase.
Claim 29
Dependent Claim
31 words
The improvement according to claim 27 wherein the NADPH is converted to NADH by the conjugate and the NADH starts a cyclic chemical reaction which proceeds to interconvert NAD and NADH.
Claim 30
Dependent Claim
18 words
The improvement according to claim 28 wherein the reduction of NAD to NADH is effected by a dehydrogenase.
Claim 31
Dependent Claim
12 words
The improvement according to claim 29 wherein the dehydrogenase is alcohol dehydrogenase.
Claim 32
Dependent Claim
14 words
The improvement according to claim 1 wherein the determinable change is a conductance change.
Claim 33
Dependent Claim
15 words
The improvement according to claim 1 wherein the determinable change is measured by an electrode.
Claim 34
Dependent Claim
174 words
In a competitive binding assay in which competition between an unknown amount of a first component and a standard amount of the same component which has been labelled for a standard amount of its complimentary component takes place, the improvement which comprises: (a) employing as the labelled component a conjugate of the first component and an enzyme, (b) contacting the conjugate with a precursor for a modulator for a cyclic chemical reaction whereby by said modulator is produced as the result of reaction of the enzyme and precursor for the modulator, and (c) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 35
Dependent Claim
174 words
In a competitive binding assay in which competition between an unknown amount of a first component and a standard amount of the same component which has been labelled for a standard amount of its complimentary component takes place, the improvement which comprises: (a) employing as the labelled component a conjugate of the first component and an enzyme, (b) contacting the conjugate with a precursor for a modulator for a cyclic chemical reaction whereby by said modulator is produced as the result of reaction of the enzyme and precursor for the modulator, and (c) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 36
Dependent Claim
18 words
The improvement according to claim 33 wherein the modulator has one less phosphate group than the modified modulator.
Claim 37
Dependent Claim
17 words
The improvement according to claim 34 wherein the modulator is fructose-6-phosphate and the modified modulator is fructose-6-diphosphate.
Claim 38
Dependent Claim
17 words
The improvement according to claim 34 wherein the modulator is ATP and the modified modulator is ADP.
Claim 39
Dependent Claim
18 words
The improvement according to claim 33 wherein the modulator has one more phosphate group than the modified modulator.
Claim 40
Dependent Claim
12 words
The improvement to claim 33 wherein the determinable product is inorganic phosphate.
Claim 41
Dependent Claim
13 words
The improvement according to claim 33 wherein the complimentary component is an antibody.
Claim 42
Dependent Claim
13 words
The improvement according to claim 39 wherein the antibody is a monoclonal antibody.
Claim 43
Dependent Claim
246 words
In a method for determining a member of a ligand and receptor pair which comprises an assay wherein: (a) a sample of a first member of the ligand and receptor pair is contacted with a matrix bound second member of the pair whereby said first member becomes bound to said second member; (b) the thus produced matrix bound first member is contacted with a conjugate which binds specifically to the matrix bound first member which conjugate comprises a phosphatase whereby said phosphatase is bound to the matrix and (c) the matrix bound and unbound conjugate are separated and the matrix bound or the unbound conjugate is assayed; the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for NADH whereby NADH is produced or with a precursor for NAD whereby NAD is produced, and (e) contacting the thus produced NADH or NAD with the components of a secondary system which is activated by the NADH or NAD, said secondary system comprising a cyclic chemical reaction which interconverts NADH and NAD, neither of which is present until the production of NADH or NAD by the conjugate, and which secondary system maintains the increase in concentration of NADH or NAD produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product at each turn of the cycle whereby amplification of the determinable change occurs.
Claim 44
Dependent Claim
246 words
In a method for determining a member of a ligand and receptor pair which comprises an assay wherein: (a) a sample of a first member of the ligand and receptor pair is contacted with a matrix bound second member of the pair whereby said first member becomes bound to said second member; (b) the thus produced matrix bound first member is contacted with a conjugate which binds specifically to the matrix bound first member which conjugate comprises a phosphatase whereby said phosphatase is bound to the matrix and (c) the matrix bound and unbound conjugate are separated and the matrix bound or the unbound conjugate is assayed; the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for NADH whereby NADH is produced or with a precursor for NAD whereby NAD is produced, and (e) contacting the thus produced NADH or NAD with the components of a secondary system which is activated by the NADH or NAD, said secondary system comprising a cyclic chemical reaction which interconverts NADH and NAD, neither of which is present until the production of NADH or NAD by the conjugate, and which secondary system maintains the increase in concentration of NADH or NAD produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product at each turn of the cycle whereby amplification of the determinable change occurs.
Claim 45
Dependent Claim
24 words
The improvement according to claim 41 in which the determinable change results from the production of a determinable product and the conjugate produces NADH.
Claim 46
Dependent Claim
16 words
The improvement according to claim 42 wherein the conjugate is a conjugate of an alkaline phosphatase.
Claim 47
Dependent Claim
16 words
The improvement according to claim 42 wherein the conjugate is a conjugate of an acid phosphatase.
Claim 48
Dependent Claim
18 words
The improvement according to claim 42 wherein the cyclic chemical reaction is allowed to proceed at pH 8-10.5.
Claim 49
Dependent Claim
17 words
The improvement according to claim 45 wherein the pH is maintained by the presence of a buffer.
Claim 50
Dependent Claim
13 words
The improvement according to claim 42 wherein the NADH is produced from NADPH.
Claim 51
Dependent Claim
15 words
The improvement according to claim 42 wherein the assay is carried out at ambient temperature.
Claim 52
Dependent Claim
26 words
The improvement according to claim 42 wherein the phosphatase is bound to the ligand or receptor in the conjugate by reaction with a bifunctional conjugating reagent.
Claim 53
Dependent Claim
16 words
The improvement according to claim 41 wherein the immunoassay is for the detection of an antigen.
Claim 54
Dependent Claim
16 words
The improvement according to claim 41 wherein the immunoassay is for the detection of an antibody.
Claim 55
Dependent Claim
26 words
The improvement according to claim 41 wherein the cyclic chemical reaction which interconverts NADH and NAD comprises one enzyme catalyzed reaction and one non-enzyme catalyzed reaction.
Claim 56
Dependent Claim
15 words
The improvement according to claim 52 wherein NAD is converted to NADH by a dehydrogenase.
Claim 57
Dependent Claim
12 words
The improvement according to claim 53 wherein the dehydrogenase is alcohol dehydrogenase.
Claim 58
Dependent Claim
24 words
The improvement according to claim 54 wherein the conversion of NADH to NAD accompanies reduction of tetrazolium compound whereby a colour change is produced.
Claim 59
Dependent Claim
25 words
The improvement according to claim 53 wherein the conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 60
Dependent Claim
15 words
The improvement according to claim 41 wherein the cyclic reaction comprises two enzyme catalyzed reactions.
Claim 61
Dependent Claim
15 words
The improvement according to claim 57 wherein NAD is converted to NADH by a dehydrogenase.
Claim 62
Dependent Claim
25 words
The improvement according to claim 57 wherein the conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 63
Dependent Claim
12 words
The improvement according to claim 58 wherein the dehydrogenase is alcohol dehydrogenase.
Claim 64
Dependent Claim
22 words
The improvement according to claim 57 wherein the conversion of NADH accompanies reduction of tetrazolium compound whereby a colour change is produced.
Claim 65
Dependent Claim
14 words
The improvement according to claim 41 wherein the determinable change is a conductance change.
Claim 66
Dependent Claim
15 words
The improvement according to claim 41 wherein the determinable change is measured by an electrode.
Claim 67
Dependent Claim
204 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) the presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separate from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, (e) said modulator is a cofactor which by chemical reaction interconnects with its oxidised form; and (f) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its oxidised form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 68
Dependent Claim
204 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) the presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separate from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, (e) said modulator is a cofactor which by chemical reaction interconnects with its oxidised form; and (f) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its oxidised form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 69
Dependent Claim
14 words
The improvement according to claim 64 wherein the cofactor is a nicotinamide adenine dinucleotide.
Claim 70
Dependent Claim
15 words
The improvement according to claim 64 wherein NAD is converted to NADH by a dehydrogenase.
Claim 71
Dependent Claim
13 words
The improvement according to claim 66 wherein the dehydrogenase is an alcohol dehydrogenase.
Claim 72
Dependent Claim
25 words
The improvement according to claim 66 wherein the conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 73
Dependent Claim
15 words
The improvement according to claim 66 wherein the cyclic chemical reaction comprises two enzymatic reactions.
Claim 74
Dependent Claim
14 words
The improvement according to claim 64 wherein the ligand is an antibody or antigen.
Claim 75
Dependent Claim
16 words
The improvement according to claim 70 wherein the ligand in solution and conjugate are added sequentially.
Claim 76
Dependent Claim
16 words
The improvement according to claim 71 wherein the ligand in solution is added before the conjugate.
Claim 77
Dependent Claim
14 words
The improvement according to claim 64 wherein the determinable change is a conductance change.
Claim 78
Dependent Claim
15 words
The improvement according to claim 64 wherein the determinable change is measured by an electrode.
Claim 79
Dependent Claim
206 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matrix to which is bound a ligand or receptor is contacted with a conjugated with a conjugate and with the solution, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, (e) said modulator is a cofactor which by chemical reaction interconnects with its oxidised form; and (f) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its oxidised form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 80
Dependent Claim
206 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matrix to which is bound a ligand or receptor is contacted with a conjugated with a conjugate and with the solution, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, (e) said modulator is a cofactor which by chemical reaction interconnects with its oxidised form; and (f) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its oxidised form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 81
Dependent Claim
14 words
The improvement according to claim 75 wherein the cofactor is a nicotinamide adenine dinucleotide.
Claim 82
Dependent Claim
16 words
The improvement according to claim 75 wherein the NAD is converted to NADH by a dehydrogenase.
Claim 83
Dependent Claim
13 words
The improvement according to claim 77 wherein the dehydrogenase is an alcohol dehydrogenase.
Claim 84
Dependent Claim
24 words
The improvement according to claim 77 wherein the conversion of NADH to NAD accompanies reduction of tetrazolium compound whereby a colour change is produced.
Claim 85
Dependent Claim
15 words
The improvement according to claim 77 wherein the cyclic chemical reaction comprises two enzymatic reactions.
Claim 86
Dependent Claim
14 words
The improvement according to claim 75 wherein the ligand is an antibody or antigen.
Claim 87
Dependent Claim
14 words
The improvement according to claim 75 wherein the determinable change is a conductance change.
Claim 88
Dependent Claim
15 words
The improvement according to claim 75 wherein the determinable change is measured by an electrode.
Claim 89
Dependent Claim
139 words
In an immunoassay in which a conjugate of a phosphatase is contacted with a phosphate of an organic compound which is thereby dephosphorylated causing a determinable change which is determined, the improvement which comprises employing as the phosphate of the organic compound a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced by the conjugate of a phosphatase which is contacted with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and thereby regenerates said nicotinamide adenine dinucleotide and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 90
Dependent Claim
139 words
In an immunoassay in which a conjugate of a phosphatase is contacted with a phosphate of an organic compound which is thereby dephosphorylated causing a determinable change which is determined, the improvement which comprises employing as the phosphate of the organic compound a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced by the conjugate of a phosphatase which is contacted with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and thereby regenerates said nicotinamide adenine dinucleotide and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 91
Dependent Claim
29 words
The improvement according to claim 84 in which the immunoassay is an enzyme linked immunoabsorbent assay in which the determinable change results from the production of a determinable product.
Claim 92
Dependent Claim
11 words
The improvement according to claim 85 which employs a monoclonal antibody.
Claim 93
Dependent Claim
13 words
The improvement according to claim 86 wherein the conjugate comprises a monoclonal antibody.
Claim 94
Dependent Claim
30 words
The improvement according to claim 84 in which the determinable change results from the production of a determinable product and the nicotinamide adenine dinucleotide is interconverted with its oxidized form.
Claim 95
Dependent Claim
16 words
The improvement according to claim 84 wherein the conjugate is a conjugate of an alkaline phosphatase.
Claim 96
Dependent Claim
30 words
The improvement according to claim 84 wherein NADPH is converted to NADH by the conjugate and the NADH starts a cyclic chemical reaction which proceeds to interconvert NAD and NADH.
Claim 97
Dependent Claim
19 words
The improvement according to claim 90 in which the reduction of NAD to NADH is effected by a dehydrogenase.
Claim 98
Dependent Claim
13 words
The improvement according to claim 91 wherein the reductase is an alcohol dehydrogenase.
Claim 99
Dependent Claim
25 words
The improvement according to claim 92 wherein the oxidation of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 100
Dependent Claim
18 words
The improvement according to claim 84 wherein the immunoassay is for the determination of an antibody or antigen.
Claim 101
Dependent Claim
14 words
The improvement according to claim 84 wherein the determinable change is a conductance change.
Claim 102
Dependent Claim
15 words
The improvement according to claim 84 wherein the determinable change is measured by an electrode.
Claim 103
Dependent Claim
183 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) The presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with a phosphatase; (b) The matrix bound conjugate is separated from unbound conjugate; and (c) The matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) Contacting the conjugate to be assayed with a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced; and (e) Contacting the thus produced nicotinamide adenine dinucleotide with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and which thereby regenerates said nicotinamide adenine dinucleotide produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 104
Dependent Claim
183 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) The presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with a phosphatase; (b) The matrix bound conjugate is separated from unbound conjugate; and (c) The matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) Contacting the conjugate to be assayed with a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced; and (e) Contacting the thus produced nicotinamide adenine dinucleotide with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and which thereby regenerates said nicotinamide adenine dinucleotide produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 105
Dependent Claim
30 words
The improvement according to claim 97 in which the determinable change results from the production of a determinable product and the nicotinamide adenine dinucleotide is interconverted with its oxidized form.
Claim 106
Dependent Claim
11 words
The improvement according to claim 98 which employs a monoclonal antibody.
Claim 107
Dependent Claim
12 words
The improvement to claim 99 wherein the conjugate comprises a monoclonal antibody.
Claim 108
Dependent Claim
16 words
The improvement according to claim 100 wherein the conjugate is a conjugate of an alkaline phosphatase.
Claim 109
Dependent Claim
33 words
The improvement according to claim 101 wherein NADPH is converted to NADH by the conjugate and the NADH starts a cyclic chemical reaction which proceeds at pH 8.10-5 to interconvert NAD and NADH.
Claim 110
Dependent Claim
19 words
The improvement according to claim 102 in which the reduction of NAD to NADH is effected by a dehydrogenase.
Claim 111
Dependent Claim
12 words
The improvement according to claim 102 wherein the reductase is alcohol dehydrogenase.
Claim 112
Dependent Claim
25 words
The improvement according to claim 102 wherein the oxidation of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 113
Dependent Claim
16 words
The improvement according to claim 97 wherein the ligand or receptor is an antibody or antigen.
Claim 114
Dependent Claim
14 words
The improvement according to claim 97 wherein the determinable change is an conductance change.
Claim 115
Dependent Claim
15 words
The improvement according to claim 97 wherein the determinable change is measured by an electrode.
Claim 116
Dependent Claim
182 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) A matrix to which is bound a ligand or receptor is contacted with a conjugate and with the solution, said conjugate comprising a ligand or receptor labelled with a phosphatase; (b) The matrix bound conjugate is separated from unbound conjugate; and (c) The matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) Contacting the conjugate to be assayed with a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced; and (e) Contacting the thus produced nicotinamide adenine dinucleotide with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and which thereby regenerates said nicotinamide adenine dinucleotide produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 117
Dependent Claim
182 words
In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) A matrix to which is bound a ligand or receptor is contacted with a conjugate and with the solution, said conjugate comprising a ligand or receptor labelled with a phosphatase; (b) The matrix bound conjugate is separated from unbound conjugate; and (c) The matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) Contacting the conjugate to be assayed with a phosphate of a nicotinamide adenine dinucleotide whereby a nicotinamide adenine dinucleotide is produced; and (e) Contacting the thus produced nicotinamide adenine dinucleotide with the components of a cyclic chemical reaction which is activated by the nicotinamide adenine dinucleotide, which cyclic chemical reaction interconverts said nicotinamide adenine dinucleotide and its oxidized or reduced form and which thereby regenerates said nicotinamide adenine dinucleotide produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable change as the cyclic chemical reaction proceeds, whereby amplification occurs.
Claim 118
Dependent Claim
30 words
The improvement according to claim 109 in which the determinable change results from the production of a determinable product and the nicotinamide adenine dinucleotide is interconverted with its oxidized form.
Claim 119
Dependent Claim
11 words
The improvement according to claim 110 which employs a monoclonal antibody.
Claim 120
Dependent Claim
13 words
The improvement according to claim 111 wherein the conjugate comprises a monoclonal antibody.
Claim 121
Dependent Claim
33 words
The improvement according to claim 111 wherein NADPH is converted to NADH by the conjugate and the NADH starts a cyclic chemical reaction which proceeds at pH 8-10.5 to interconvert NAD to NADH.
Claim 122
Dependent Claim
16 words
The improvement according to claim 109 wherein the conjugate is a conjugate of an alkaline phosphatase.
Claim 123
Dependent Claim
19 words
The improvement according to claim 114 in which the reduction of NAD to NADH is effected by a dehydrogenase.
Claim 124
Dependent Claim
12 words
The improvement according to claim 115 wherein the reductase is alcohol dehydrogenase.
Claim 125
Dependent Claim
25 words
The improvement according to claim 116 wherein the oxidation of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.
Claim 126
Dependent Claim
18 words
The improvement according to claim 114 wherein the ligand or receptor in solution and conjugate are added sequentially.
Claim 127
Dependent Claim
18 words
The improvement according to claim 118 wherein the ligand or receptor in solution is added before the conjugate.
Claim 128
Dependent Claim
16 words
The improvement according to claim 109 wherein the ligand or receptor is an antibody or antigen.
Claim 129
Dependent Claim
14 words
The improvement according to claim 109 wherein the determinable change is a conductance change.
Claim 130
Dependent Claim
15 words
The improvement according to claim 109 wherein the determinable change is measured by an electrode.
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Cited
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-09-06 | 1992-03-07 | 1992-09-06 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-09-06 | 1996-03-07 | 1996-09-06 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-09-06 | 2000-03-07 | 2000-09-06 | $7700.00 | Window Closed / Lapsed |