US5631169A
Expired / Lapsed
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Fluorescent energy transfer immunoassay
Assignee: LAKOWICZ JOSEPH R PHD
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Filing: 1994-01-19
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Granted: 1997-05-20
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Expires: 2014-01-19
Abstract
Abstract A fluorometric luminescence immunoassay method includes forming a sample by exposing a first immune reaction reactant to a second immune reaction reactant capable of reacting with the first reactant, one of the first and second immune reaction reactants being labelled with a photoluminescent energy transfer donor and the other being labelled with a photoluminescent energy transfer acceptor complementary to the photoluminescent donor. At least the photoluminescent donor has the property of photoluminescence, and the photoluminescent donor and acceptor are chosen so that when the first immune reaction reactant reacts with the second immune reaction reactant, the donor and the acceptor are capable of interacting to produce a detectable luminescence lifetime change. The sample is excited with radiation, and the resulting emission is detected. The apparent luminescent lifetime is then calculated to determine the presence of a reaction product of the first and second immune reaction reactants.
Inventors
- 👤 Joseph R. Lakowicz
- 👤 Badri P. Maliwal
- 👤 Richard Thompson
- 👤 Alvydas Ozinskas
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 17:19
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/582; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6408; G01N21/6428; G01N33/53; G01N33/536; G01N33/542; G01N2021/6439; G01N2021/6441; Y; Y10; Y10S; Y10S436/00; Y10S436/80
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
159 words
A competitive method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner competes with the analyte for binding to said second binding partner, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the first binding partner binds to the second binding partner, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparant luminescence lifetime of the donor to quantify binding of the first binding partner to the second binding partner, thereby inversely quantifying the analyte.
Claim 2
Dependent Claim
159 words
A competitive method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner competes with the analyte for binding to said second binding partner, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the first binding partner binds to the second binding partner, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparant luminescence lifetime of the donor to quantify binding of the first binding partner to the second binding partner, thereby inversely quantifying the analyte.
Claim 3
Dependent Claim
36 words
The method of claim 1, wherein the photoluminescent donor is selected from the group consisting of cyanines, oxazines, thiazines, porphyrins, phthalocyanines, fluorescent infrared-emitting polynuclear aromatic hydrocarbons, phycobiliproteins, sulfonyl chlorides, carbodiimides, haloacetyl derivatives, squaraines and organo-metallic complexes.
Claim 4
Dependent Claim
35 words
The method of claim 1, wherein the photoluminescent acceptor is selected from the group consisting of cyanines, oxazines, thiazines, porphyrins, phthalocyanines, polynuclear aromatic hydrocarbons, phycobiliproteins, squaraines, organo-metallic complexes, carbodiimides, sulfonyl chlorides, haloacetyls, and azo dyes.
Claim 5
Dependent Claim
14 words
The method of claim 1, wherein the apparent lifetime is calculated using phase-modulation fluorometry.
Claim 6
Dependent Claim
14 words
The method of claim 1, wherein the apparent lifetime is calculated using time-resolved fluorometry.
Claim 7
Dependent Claim
29 words
The method of claim 2, wherein the photoluminescent donor is selected from the group consisting of: fluoresceinisothiocyanate, dichlorotriazinylaminofluorescein, 1,3- bis-(2-dialkylamino-5-thienyl)-substituted squaraines, and compounds having the following structural formulas: ##STR3##
Claim 8
Dependent Claim
31 words
The method of claim 3, wherein the photoluminescent acceptor is selected from the group consisting of: eosin isothiocyanate, tetramethylrhodamine isothiocyanate, 1,3-bis- (2-dialkylamino-5-thienyl)-substituted squaraines, and compounds having the following structural formulas: ##STR4##
Claim 9
Dependent Claim
12 words
The method of claim 1, wherein the sample is formed in vitro.
Claim 10
Dependent Claim
32 words
The method of claim 1, wherein the sample is excited using a Ti-sapphire laser, a laser diode or a continuous wave frequency-double mode-locked argon ion or Nd:YAG laser synch-pumping a dye laser.
Claim 11
Dependent Claim
16 words
The method of claim 9, wherein the sample is excited at a wavelength of 380 nm.
Claim 12
Dependent Claim
12 words
The method of claim 1, wherein the lifetime is about 5 ns.
Claim 13
Dependent Claim
17 words
The method of claim 1, wherein the sample is excited with a HeNe laser or laser diode.
Claim 14
Dependent Claim
31 words
The method of claim 1, wherein the donor or acceptor is selected from the group consisting of succinimidyl esters of: 5- or 6- carboxyfluorescein; 5- or 6- carboxytetra-methylrhodamine; and 7-amino-4-methylcoumarin-3-acetic acid.
Claim 15
Dependent Claim
14 words
The method of claim 1, wherein the radiation is modulated light or pulsed light.
Claim 16
Dependent Claim
18 words
The method of claim 14, wherein the sample is excited with sinusoidally modulated light or square wave light.
Claim 17
Dependent Claim
14 words
The method of claim 14, wherein the sample is excited using a helium-cadmium laser.
Claim 18
Dependent Claim
16 words
The method of claim 16, wherein the sample is excited at a wavelength of 442 nm.
Claim 19
Dependent Claim
18 words
The method of claim 1, wherein the donor has a lifetime of about 0.1 ns to 400 ns.
Claim 20
Dependent Claim
16 words
The method of claim 4, wherein there is a phase angle difference of about 45 degrees.
Claim 21
Dependent Claim
165 words
A competitive method of quantifying an analyte in a sample, comprising the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner competes with the analyte for binding to said second binding partner, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the first binding partner binds to the second binding partner, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculation the apparent luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify binding of the first binding partner to the second binding partner, thereby inversely quantifying the analyte.
Claim 22
Dependent Claim
165 words
A competitive method of quantifying an analyte in a sample, comprising the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner competes with the analyte for binding to said second binding partner, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the first binding partner binds to the second binding partner, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculation the apparent luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify binding of the first binding partner to the second binding partner, thereby inversely quantifying the analyte.
Claim 23
Dependent Claim
156 words
A sandwich method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner and said second binding partner bind to the analyte to form an immune complex, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the immune complex is formed, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparant luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify the immune complex, thereby quantifying the analyte.
Claim 24
Dependent Claim
156 words
A sandwich method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner and said second binding partner bind to the analyte to form an immune complex, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when the immune complex is formed, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparant luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify the immune complex, thereby quantifying the analyte.
Claim 25
Dependent Claim
156 words
A sandwich method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner and said second binding partner bind to the analyte to form an immune complex, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when said immune complex is formed, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparent luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify the immune complex, thereby quantifying the analyte.
Claim 26
Dependent Claim
156 words
A sandwich method of quantifying an analyte in a sample, consisting essentially of the steps of: adding to said sample a first binding partner and a second binding partner, wherein said first binding partner and said second binding partner bind to the analyte to form an immune complex, wherein one of said first and second binding partners is labelled with a photoluminescent energy transfer donor and the other is labelled with a photoluminescent energy transfer acceptor, wherein the photoluminescent energy transfer donor and acceptor are chosen such that when said immune complex is formed, the donor and the acceptor are brought into interacting proximity, producing a detectable luminescence lifetime change in the photoluminescence lifetime of the donor; exposing the sample to an exciting amount of radiation; detecting the resulting emission; and calculating the apparent luminescence lifetime of the donor without the use of a fluorescence intensity measurement to quantify the immune complex, thereby quantifying the analyte.
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) | 2000-05-19 | 2000-11-18 | 2001-05-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-05-19 | 2004-11-18 | 2005-05-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-05-19 | 2008-11-18 | 2009-05-20 | $7700.00 | Window Closed / Lapsed |