Fluorescein-conjugated proteins with enhanced fluorescence
OwnerRonald Robert C., Phuc H. Nguyen +1inventors
InventorsRonald Robert C., Phuc H. Nguyen, Gerald L. Rowley
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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.
Claims11
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.sub.2).sub.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.
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.sub.2).sub.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.
Dependencies are inferred from the claim text.