US4629688A
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Homogeneous specific binding assay method
Assignee: Bayer Corp
•
Filing: 1978-04-10
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Granted: 1986-12-16
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Expires: 1998-04-10
Abstract
Abstract A test composition, device, and method for their use in a homogeneous specific binding assay which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The test composition and device comprise a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction is affected by reaction between the specific binding substance in the conjugate and a specific binding counterpart thereto. The presence of a ligand in a liquid medium may be determined using competitive or displacement binding or sequential saturation techniques wherein the specific binding substance in the conjugate is the ligand or a specific binding analog thereof, or using a direct binding technique wherein the specific binding substance is a specific binding partner of the ligand. The effect of the specific binding reaction on the activity of the conjugated reactant is related to the presence or amount of the ligand in the liquid medium tested.
Inventors
- 👤 Robert C. Bolguslaski
- 👤 Robert J. Carrico
- 👤 James E. Christner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 21:09
CPC Classifications
C; C07; C07D; C07D209/00; C07D209/02; C07D209/44; C07D209/48; C07D237/00; C07D237/26; C07D237/30; C07D237/32; C07D493/00; C07D493/02; C07D493/10; C07D495/00; C07D495/02; C07D495/04; C07H; C07H21/00; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; G01N33/58; G01N33/581; G01N33/582; Y; Y10; Y10S; Y10S435/00; Y10S435/966
Claims Analysis
Total Claims: 45
Claim 1
Independent Claim
117 words
A method for determining a ligand in a medium suspected of containing the ligand which comprises: combining in an aqueous solution said medium, a reagent and a specific binding partner for the ligand in the medium, said reagent being a conjugate of said ligand or a specific binding analog thereof and a low molecular weight label compound having a bond enzymatically cleavable to produce a product distinguishable by a detectable signal and said enzymatic cleavage being substantially altered by binding of said reagent to said binding partner; adding an enzyme capable of cleavage of said enzymatically cleavable bond; and analyzing for the effect of said medium on enzymatic cleavage of said bond by measuring said detectable signal.
Claim 2
Independent Claim
117 words
A method for determining a ligand in a medium suspected of containing the ligand which comprises: combining in an aqueous solution said medium, a reagent and a specific binding partner for the ligand in the medium, said reagent being a conjugate of said ligand or a specific binding analog thereof and a low molecular weight label compound having a bond enzymatically cleavable to produce a product distinguishable by a detectable signal and said enzymatic cleavage being substantially altered by binding of said reagent to said binding partner; adding an enzyme capable of cleavage of said enzymatically cleavable bond; and analyzing for the effect of said medium on enzymatic cleavage of said bond by measuring said detectable signal.
Claim 3
Dependent Claim
15 words
The method of claim 1 wherein the label compound is a substrate for the enzyme.
Claim 4
Dependent Claim
14 words
The method of claim 2 wherein the enzymatic cleavage of the substrate is hydrolytic.
Claim 5
Dependent Claim
19 words
The method of claim 2 wherein the product formed by enzymatic cleavage of the substrate label compound is fluorescent.
Claim 6
Dependent Claim
45 words
The method of claim 2 wherein the substrate label compound includes a bridge group through which it is coupled to the ligand or analog thereof in the reagent and wherein the enzyme cleaves said bridge group to release said product distinguishable by a detectable signal.
Claim 7
Dependent Claim
12 words
The method of claim 5 wherein the released detectable molecule is fluorescent.
Claim 8
Dependent Claim
31 words
The method of claim 6 wherein the bridge group is an ester group, the enzyme is an esterase, and the released fluorescent molecule is umbelliferone or fluorescein, or a derivative thereof.
Claim 9
Dependent Claim
20 words
The method of claim 2 wherein the enzymatic cleavage of the substrate label compound is part of a cyclic reaction.
Claim 10
Dependent Claim
18 words
The method of claim 8 wherein the substrate label compound is a cycled reagent in the cyclic reaction.
Claim 11
Dependent Claim
11 words
The method of claim 8 wherein the cyclic reaction is exponential.
Claim 12
Dependent Claim
18 words
The method of claim 10 wherein the substrate label compound is a cycled reagent in the cyclic reaction.
Claim 13
Dependent Claim
15 words
The method of claim 1 wherein the label compound is a coenzyme for the enzyme.
Claim 14
Dependent Claim
12 words
The method of claim 12 wherein the coenzyme is a nucleotide coenzyme.
Claim 15
Dependent Claim
26 words
The method of claim 12 wherein the coenzyme is selected from the group consisting of the adenosine phosphates, nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate.
Claim 16
Dependent Claim
12 words
The method of claim 12 wherein the coenzyme is nicotinamide adenine dinucleotide.
Claim 17
Dependent Claim
11 words
The method of claim 12 wherein the coenzyme is adenosine triphosphate.
Claim 18
Dependent Claim
12 words
The method of claim 12 wherein the coenzyme is flavin adenine dinucleotide.
Claim 19
Dependent Claim
41 words
The method of claim 12 wherein the enzymatic cleavage of the coenzyme label compound is part of a cyclic reaction as follows: ##STR50## wherein the coenzyme label compound is comprised in one of reactants A, reactants B, and the cycled reagent.
Claim 20
Dependent Claim
14 words
The method of claim 18 wherein the coenzyme label compound is the cycled reagent.
Claim 21
Dependent Claim
16 words
The method of claim 19 wherein reactions A and B are catalyzed by the same enzyme.
Claim 22
Dependent Claim
17 words
The method of claim 20 wherein the cycled coenzyme label compound cycles between oxidized and reduced forms.
Claim 23
Dependent Claim
12 words
The method of claim 21 wherein the coenzyme is flavin adenine dinucleotide.
Claim 24
Dependent Claim
11 words
The method of claim 18 wherein the cyclic reaction is exponential.
Claim 25
Dependent Claim
14 words
The method of claim 23 wherein the coenzyme label compound is the cycled reagent.
Claim 26
Dependent Claim
21 words
The method of claim 1 wherein the reagent, the binding partner, and the enzyme are combined with the medium substantially simultaneously.
Claim 27
Dependent Claim
22 words
The method of claim 1 wherein the medium is combined with the binding partner and the enzyme and thereafter with the reagent.
Claim 28
Dependent Claim
17 words
The method of claim 1 wherein the label compound has a molecular weight of less than 9000.
Claim 29
Dependent Claim
36 words
The method of claim 1 wherein the ligand is selected from the group consisting of antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites, and pharmacological agents, and their receptors and binding substances.
Claim 30
Dependent Claim
23 words
The method of claim 1 wherein the ligand is a hapten or an antigen and the specific binding partner is an antibody thereto.
Claim 31
Dependent Claim
21 words
The method of claim 1 wherein said enzymatic cleavage is substantially inhibited by binding of said reagent to said binding partner.
Claim 32
Dependent Claim
14 words
The method of claim 1 wherein the medium under assay is a biological fluid.
Claim 33
Independent Claim
104 words
A method for determining a ligand in a medium suspected of containing the ligand which comprises: combining in an aqueous solution said medium and a reagent which is a conjugate of a specific binding partner for the ligand and a low molecular weight label compound having a bond enzymatically cleavable to produce a product distinguishable by a detectable signal and said enzymatic cleavage being substantially altered by binding of said reagent to said ligand; adding an enzyme capable of cleavage of said enzyamtically cleavable bond; and analyzing for the effect of said medium an enzymatic cleavage of said bond by measuring said detectable signal.
Claim 34
Independent Claim
104 words
A method for determining a ligand in a medium suspected of containing the ligand which comprises: combining in an aqueous solution said medium and a reagent which is a conjugate of a specific binding partner for the ligand and a low molecular weight label compound having a bond enzymatically cleavable to produce a product distinguishable by a detectable signal and said enzymatic cleavage being substantially altered by binding of said reagent to said ligand; adding an enzyme capable of cleavage of said enzyamtically cleavable bond; and analyzing for the effect of said medium an enzymatic cleavage of said bond by measuring said detectable signal.
Claim 35
Dependent Claim
15 words
The method of claim 32 wherein the label compound is a substrate for the enzyme.
Claim 36
Dependent Claim
14 words
The method of claim 33 wherein the enzymatic cleavage of the substrate is hydrolytic.
Claim 37
Dependent Claim
15 words
The method of claim 32 wherein the label compound is a coenzyme for the enzyme.
Claim 38
Dependent Claim
17 words
The method of claim 32 wherein the label compound has a molecular weight of less than 9000.
Claim 39
Dependent Claim
11 words
The method of claim 33 wherein the ligand is an antibody.
Claim 40
Dependent Claim
20 words
The method of claim 32 wherein said enzymatic cleavage is substantially inhibited by binding of said reagent to said ligand.
Claim 41
Independent Claim
115 words
A method for determining the presence of a protein in a medium suspected of containing the protein which comprises: combining in an aqueous solution said medium, a reagent and an antibody for the protein in the medium, said reagent being a conjugate of said protein and a low molecular weight compound having a bond enzymatically cleavable by hydrolysis to produce a product distinguishable by a detectable signal and said enzymatic cleavage by hydrolysis being substantially inhibited by binding of said reagent to said antibody; adding an enzyme capable of hydrolytic cleavage of said enzymatically cleavable bond; and analyzing for the effect of said medium on enzyme cleavage of said bond by measuring said detectable signal.
Claim 42
Independent Claim
115 words
A method for determining the presence of a protein in a medium suspected of containing the protein which comprises: combining in an aqueous solution said medium, a reagent and an antibody for the protein in the medium, said reagent being a conjugate of said protein and a low molecular weight compound having a bond enzymatically cleavable by hydrolysis to produce a product distinguishable by a detectable signal and said enzymatic cleavage by hydrolysis being substantially inhibited by binding of said reagent to said antibody; adding an enzyme capable of hydrolytic cleavage of said enzymatically cleavable bond; and analyzing for the effect of said medium on enzyme cleavage of said bond by measuring said detectable signal.
Claim 43
Dependent Claim
10 words
The method of claim 39 wherein said product is fluorescent.
Claim 44
Dependent Claim
22 words
The method of claim 39 wherein said bond is a bridge group that undergoes said hydrolytic enzyme cleavage to produce said product.
Claim 45
Dependent Claim
19 words
The method of claim 41 wherein said bridge group is an ester group and said enzyme is an esterase.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-12-15 | 1990-06-16 | 1990-12-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-12-15 | 1994-06-16 | 1994-12-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-12-15 | 1998-06-16 | 1998-12-16 | $7700.00 | Window Closed / Lapsed |