US4894348A
Expired / Lapsed
US
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Fluorescein-conjugated proteins with enhanced fluorescence
Assignee: Sclavo Inc
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Filing: 1987-07-01
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Granted: 1990-01-16
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Expires: 2007-07-01
Abstract
Abstract A method for conducting assays for an analyte, usually in a biological sample, utilizes reagents which contain biological ligands linked to fluorescein or a derivative of fluorescein through a linkage to the fluorescein nucleus selected from acetamido and thioacetamido. In these reagents, the fluorescence efficiency is improved over that obtained in the commonly used FITC-labeled reagents.
Inventors
- 👤 Ronald Robert C.
- 👤 Phuc H. Nguyen
- 👤 Gerald L. Rowley
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 11:12
CPC Classifications
C; C07; C07D; C07D311/00; C07D311/02; C07D311/78; C07D311/80; C07D311/82; C09; C09B; C09B11/00; C09B11/04; C09B11/06; C09B11/08; C09B49/00; C09B49/12; C09B49/126; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; Y; Y10; Y10S; Y10S436/00; Y10S436/80; Y10S436/805; Y10S436/807; Y10S436/808; Y10S530/00; Y10S530/802; Y10S530/866
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
181 words
In a method for the determination of analyte in a sample by means of a specific binding assay, said assay comprising the step of measuring the presence or amount of analyte by detecting or quantitating a fluorescent label present in one of the phases of a two-phase system, the improvement wherein said label is supplied as a compound of the formula: (4-Fl)--NHCOCR.sub.2 XL'--Y' or (5-Fl)--NHCOCR.sub.2 XL'--Y' wherein 4-Fl and 5-Fl represent a fluoroscein residue or a substituted fluorescein residue conjugated to the adjacent NH through the 4 or 5 position of the fluorescein nucleus respectively; each R is independently H or lower alkyl (1-4C); X is --S--S--(CH 2 ) n --, wherein n is 1-4; L' is the residue of L, wherein L is a functional group capable of forming a covalent bond, L being selected from the group consisting of carboxyl, sulfhydryl, amino, halomethyl, epoxide, aldehyde, hydroxy, derivatized carboxyl and vinyl; and Y' is the residue of Y, wherein Y is a biological ligand or a biological ligand conjugated to linker wherein the linker is capable of reactivity with L.
Claim 2
Dependent Claim
181 words
In a method for the determination of analyte in a sample by means of a specific binding assay, said assay comprising the step of measuring the presence or amount of analyte by detecting or quantitating a fluorescent label present in one of the phases of a two-phase system, the improvement wherein said label is supplied as a compound of the formula: (4-Fl)--NHCOCR.sub.2 XL'--Y' or (5-Fl)--NHCOCR.sub.2 XL'--Y' wherein 4-Fl and 5-Fl represent a fluoroscein residue or a substituted fluorescein residue conjugated to the adjacent NH through the 4 or 5 position of the fluorescein nucleus respectively; each R is independently H or lower alkyl (1-4C); X is --S--S--(CH 2 ) n --, wherein n is 1-4; L' is the residue of L, wherein L is a functional group capable of forming a covalent bond, L being selected from the group consisting of carboxyl, sulfhydryl, amino, halomethyl, epoxide, aldehyde, hydroxy, derivatized carboxyl and vinyl; and Y' is the residue of Y, wherein Y is a biological ligand or a biological ligand conjugated to linker wherein the linker is capable of reactivity with L.
Claim 3
Dependent Claim
11 words
The method of claim 1 wherein Fl is fluorescein or eosin.
Claim 4
Dependent Claim
10 words
The method of claim 1 wherein each R is H.
Claim 5
Dependent Claim
19 words
The method of claim 1 wherein Y' is a protein residue or a protein residue conjugated to a linker.
Claim 6
Dependent Claim
17 words
The method of claim 4 wherein the protein residue is an immunoglobulin or an immunoreactive fragment thereof.
Claim 7
Dependent Claim
136 words
A compound useful as a labeling reagent in a specific binding assay, which has the formula: (4-Fl)--NHCOCR.sub.2 XL'--Y' or (5-Fl)--NHCOCR.sub.2 XL'--Y' wherein 4-Fl and 5-Fl represent a fluoroscein residue or a substituted fluorescein residue conjugated to the adjacent NH through the 4 or 5 position of the fluorescein nucleus respectively; each R is independently H or lower alkyl (1-4C); X is --S--S--(CH 2 ) n --, wherein n is 1-4; L' is the residue of L, wherein L is a functional group capable of forming a covalent bond, L being selected from the group consisting of carboxyl, sulfhydryl, amino, halomethyl, epoxide, aldehyde, hydroxy, derivatized carboxyl annd vinyl; and Y' is the residue of Y, wherein Y is a biological ligand or a biological ligand conjugated to linker wherein the linker is capable of reactivity with L.
Claim 8
Dependent Claim
136 words
A compound useful as a labeling reagent in a specific binding assay, which has the formula: (4-Fl)--NHCOCR.sub.2 XL'--Y' or (5-Fl)--NHCOCR.sub.2 XL'--Y' wherein 4-Fl and 5-Fl represent a fluoroscein residue or a substituted fluorescein residue conjugated to the adjacent NH through the 4 or 5 position of the fluorescein nucleus respectively; each R is independently H or lower alkyl (1-4C); X is --S--S--(CH 2 ) n --, wherein n is 1-4; L' is the residue of L, wherein L is a functional group capable of forming a covalent bond, L being selected from the group consisting of carboxyl, sulfhydryl, amino, halomethyl, epoxide, aldehyde, hydroxy, derivatized carboxyl annd vinyl; and Y' is the residue of Y, wherein Y is a biological ligand or a biological ligand conjugated to linker wherein the linker is capable of reactivity with L.
Claim 9
Dependent Claim
11 words
The compound of claim 6 wherein Fl is fluorescein or eosin.
Claim 10
Dependent Claim
10 words
The compound of claim 6 wherein each R is H.
Claim 11
Dependent Claim
19 words
The compound of claim 6 wherein Y' is a protein residue or a protein residue conjugated to a linker.
Claim 12
Dependent Claim
17 words
The compound of claim 9 wherein the protein residue is an immunoglobulin or an immunoreactive fragment thereof.
Claim 13
Dependent Claim
31 words
A kit for the conduct of a specific binding assay which comprises the compound of claim 6 and additional reagents required for the conduct of the assay along with instructions therefor.
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) | 1993-01-15 | 1993-07-17 | 1994-01-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-01-15 | 1997-07-17 | 1998-01-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-01-15 | 2001-07-17 | 2002-01-16 | $7700.00 | Window Closed / Lapsed |