US4745072A
Expired / Lapsed
US
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Immunoassay and immunometric assay of free ligand concentrations in biological fluids
Assignee: Individual
•
Filing: 1984-06-22
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Granted: 1988-05-17
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Expires: 2004-06-22
Abstract
Abstract A method of measuring the concentration of a free ligand in a biological fluid containing the free ligand and ligand bound to endogenous binding agent, by the steps of (a) mixing a sample of the fluid with an analogue of the ligand, a specific binder with which the free ligand and the ligand analogue bind, and an exogenous binding agent which binds the ligand analogue but not the ligand, either the ligand analogue or the specific binder being labelled, (b) incubating the resulting mixture, (c) determining either the amount of the labelled analogue bound or the amount of labelled specific binder bound, or not bound, to the ligand analogue, and (d) correlating the determined amount to the amount of free ligand present in the sample. The method is useful to measure concentration of free thyroid hormones and other hormones in body fluids, employing antibodies specific to the ligand analogue as the exogenous binding agents.
Inventors
- 👤 Roger P. Ekins
- 👤 Thomas M. Jackson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 16:21
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/74; G01N33/78; G01N33/53; Y; Y10; Y10S; Y10S436/00; Y10S436/804; Y10S436/815; Y10S436/817
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
158 words
A method of measuring the concentration of free ligand in a biological fluid containing the ligand both as a free ligand and as ligand reversibly bound to an endogenous binding agent, comprising (a) mixing a sample of the biological fluid with (1) an analogue of the ligand, (2) a specific binder with which the free ligand and the ligand analogue reversibly bind, and (3) an exogenous binding agent which reversibly binds the ligand analogue but not the ligand, either the ligand analogue or the specific binder being labelled, (b) incubating the resulting mixture so that the ligand and ligand analogue compete for the specific binder, (c) determining either (1) the amount of the labelled analogue bound to the specific binder or the exogenous binding agent; or (2) the amount of labelled specific binder bound, or not bound, to the ligand analogue, and (d) correlating the determined amount to the amount of free ligand present in the biological fluid.
Claim 2
Independent Claim
158 words
A method of measuring the concentration of free ligand in a biological fluid containing the ligand both as a free ligand and as ligand reversibly bound to an endogenous binding agent, comprising (a) mixing a sample of the biological fluid with (1) an analogue of the ligand, (2) a specific binder with which the free ligand and the ligand analogue reversibly bind, and (3) an exogenous binding agent which reversibly binds the ligand analogue but not the ligand, either the ligand analogue or the specific binder being labelled, (b) incubating the resulting mixture so that the ligand and ligand analogue compete for the specific binder, (c) determining either (1) the amount of the labelled analogue bound to the specific binder or the exogenous binding agent; or (2) the amount of labelled specific binder bound, or not bound, to the ligand analogue, and (d) correlating the determined amount to the amount of free ligand present in the biological fluid.
Claim 3
Dependent Claim
95 words
A method as claimed in claim 1 wherein the contribution from the endogenous binding agents to S, wherein S is defined as one plus the sum of the products of the concentrations of each of the binding agents in the sample other than the specific binder and the respective equilibrium constants for the reactions between those binding agents and the ligand analogue, is zero or constant for all samples and the exogenous binding agent and its concentration are selected so that its contribution to S is at least 10, S being not more than 500.
Claim 4
Dependent Claim
112 words
A method as claimed in claim 1 wherein the contribution from the endogenous binding agents to S, wherein S is defined as one plus the sum of the products of the concentrations of each of the binding agents in the sample other than the specific binder and the respective equilibrium constants for the reactions between those binding agents and the ligand analogue and S varies significantly from sample to sample and the exogenous binding agent and its concentration are chosen so that its contribution to S is at least half the value of S in any sample and is comparable with or greater than the variation in S from sample to sample.
Claim 5
Dependent Claim
28 words
A method as claimed in claim 1 wherein the exogenous binding agent is a chemical reagent which binds the ligand analogue but does not substantially bind the ligand.
Claim 6
Dependent Claim
35 words
A method as claimed in claim 4 wherein the exogenous binding agent is an antibody having an affinity for the ligand analogue at least two orders of magnitude higher than its affinity for the ligand.
Claim 7
Dependent Claim
13 words
A method as claimed in claim 1 wherein the ligand analogue is labelled.
Claim 8
Dependent Claim
13 words
A method as claimed in claim 1 wherein the specific binder is labelled.
Claim 9
Dependent Claim
14 words
A method as claimed in claim 1 wherein the label is a radioactive label.
Claim 10
Dependent Claim
15 words
A method as claimed in claim 1 wherein the free ligand is a free hormone.
Claim 11
Dependent Claim
15 words
A method as claimed in claim 9 wherein the free ligand is free thyroid hormone.
Claim 12
Dependent Claim
96 words
A method as claimed in claim 1 wherein P 1 , P 2 , . . . , P n are the endogenous binding agents in the biological fluid samples, [P 1 ], [P 2 ], . . . [P n ] are the concentrations of the endogenous binding agents in the biological samples, K P , K P2 . . . K Pn are the equilibrium constants between the free ligand analogue and the ligand analogue bound to the endogenous binding agents in the biological fluid samples, P X is the exogenous binding agent X,
Claim 13
Dependent Claim
101 words
[P X ] is the concentration of the binding agent X in the biological fluid samples, K PX is the equilibrium constant between the free ligand analogue and the ligand analogue bound to the binding agent X in the biological fluid samples, and K PX [P X ] is at least 10 and S is not more than 500, where S is 1+ΣK P [P]+K PX [px], ΣK P [P] is K P1 [P 1 ]+K P2 [P 2 ]+. . . =K Pn [P n ], and is zero or substantially constant in all biological fluid samples to be assayed.
Claim 14
Dependent Claim
101 words
[P X ] is the concentration of the binding agent X in the biological fluid samples, K PX is the equilibrium constant between the free ligand analogue and the ligand analogue bound to the binding agent X in the biological fluid samples, and K PX [P X ] is at least 10 and S is not more than 500, where S is 1+ΣK P [P]+K PX [px], ΣK P [P] is K P1 [P 1 ]+K P2 [P 2 ]+. . . =K Pn [P n ], and is zero or substantially constant in all biological fluid samples to be assayed.
Claim 15
Dependent Claim
9 words
The method of claim 11 wherein S is 50-500.
Claim 16
Dependent Claim
206 words
A method as claimed in claim 1 wherein P 1 , P 2 , . . . , P n are the endogenous binding agents in the biological fluid samples, [P 1 ], [P 2 ], . . . [P n ] are the concentrations of the binding agents in each of the biological fluid samples, K P1 , K P2 . . . K Pn are the equilibrium constants between the free ligand analogue and the ligand analogue bound to the endogenous binding agents in the biological fluid samples, P x is the exogenous binding agent X, [P x ] is the concentration of the binding agent X in the biological fluid samples, K Px is the equilibrium constant between the free ligand analogue and the ligand analogue bound to the binding agent X in the biological fluid samples, and K Px [P x ] is at least s /2, where S is 1+ΣK P [P]+K PX [px], ΣK P [P] is K P1 [P 1 ]+K P2 [P 2 ]+ . . . =K Pn [P n ], and varies significantly from sample to sample of the biological fluid samples to be assayed, K px [px] being comparable with or greater than that variation.
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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) | 1991-05-17 | 1991-11-16 | 1992-05-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-17 | 1995-11-16 | 1996-05-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-05-17 | 1999-11-16 | 2000-05-17 | $7700.00 | Window Closed / Lapsed |