EP0087564A1
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EP
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Specific binding assay method, reagent system and labelled conjugate for use in this method
Assignee: Bayer Corp
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Filing: 1983-01-19
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Granted: —
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Expires: 2003-01-19
Abstract
Abstract An improved specific binding assay, e.g., homogeneous or heterogeneous immunoassay, for determining an analyte employing a labeled conjugate of said analyte and a photogenic, e.g., fluorescent, label. Where the analyte in the analyte-photophor conjugate whose light emission is measured in the assay as a function of the analyte is capable of significantly reducing such light emission upon coming into quenching orientation with the photophor, the present invention employs a labeled conjugate in which the analyte is joined to the photogenic label by a linking group which substantially hinders such quenching orientation. In this manner, ten percent or more of the photogenicity of the unconjugated photophor can be preserved in the measured analyte-photophor conjugate. The use of conventional flexible linking groups in the labeled conjugate allows quenching to occur with attendant large losses in assay sensitivity, whereas sensitivity is increased in the present invention by the use of relatively rigid linking groups. The invention is applicable to a wide variety of photogenic, e.g., fluorescent and chemiluminescent, binding assay techniques.
Inventors
- 👤 Robert Thomas Buckler
- 👤 Thomas M. Li
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-02 15:25
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; G01N33/536; G01N33/542; G01N33/58; G01N33/582
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
187 words
A specific binding assay method for determining an analyte in a test sample, wherein said test sample is combined with a reagent system including a labeled conjugate comprising said analyte, or a binding analog thereof, and a photogenic label, thereby forming a reaction mixture containing a bound-species and a free-species of said labeled conjugate, said free-species yielding an analyte-photophor conjugate comprising said analyte or analog linked to a photophor capable of emitting light upon excitation, the analyte or analog portion of said analyte-photophor conjugate capable of significantly reducing such light emission upon coming into quenching orientation with the photophor, and wherein said photophor in said bound-species or said free-species is excited and resulting light emitted is measured as a function of the analyte in the sample, characterized in that in said labeled conjugate said analyte or analog is joined to said photogenic label by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene, bivalent-carbocyclic and heterocyclic fused-rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod, and bivalent spiro-linked ring structures.
Claim 2
Dependent Claim
187 words
A specific binding assay method for determining an analyte in a test sample, wherein said test sample is combined with a reagent system including a labeled conjugate comprising said analyte, or a binding analog thereof, and a photogenic label, thereby forming a reaction mixture containing a bound-species and a free-species of said labeled conjugate, said free-species yielding an analyte-photophor conjugate comprising said analyte or analog linked to a photophor capable of emitting light upon excitation, the analyte or analog portion of said analyte-photophor conjugate capable of significantly reducing such light emission upon coming into quenching orientation with the photophor, and wherein said photophor in said bound-species or said free-species is excited and resulting light emitted is measured as a function of the analyte in the sample, characterized in that in said labeled conjugate said analyte or analog is joined to said photogenic label by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene, bivalent-carbocyclic and heterocyclic fused-rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod, and bivalent spiro-linked ring structures.
Claim 3
Dependent Claim
14 words
A method according to claim 1, characterized in that said linking group comprises N,N'-piperazinylene.
Claim 4
Dependent Claim
16 words
A method according to claim 1 or 2, characterized in that said photophor is a fluoroescer.
Claim 5
Independent Claim
64 words
A method according to any of claims 1 to 3, characterized in that it is of the homogenous type wherein the photogenicity of said photophor is measurably different when said labeled conjugate is in said bound-species compared to in said free-species, and wherein said photophor is excited in said reaction mixture and the resulting emission is a function of the analyte in said sample.
Claim 6
Dependent Claim
176 words
A reagent system for the specific binding assay determination of an analyte in a test sample, comprising (a) a binding partner for said analyte, and (b) a labeled conjugate comprising said analyte, or a binding analog thereof, and a photogenic label, wherein combination of said reagent system with said sample in a liquid reaction mixture results in formation of a binding partner-bound-species and a free-species of said labeled conjugate, said free-species yielding an analyte-photophor conjugate comprising said analyte or analog linked to a photophor capable of emitting light upon excitation, the analyte or analog portion of said analyte photophor conjugate capable of significantly reducing such light emission upon coming into.quenching orientation with the photophor, characterized in that said analyte or analog is joined to said photogenic label in said labeled conjugate by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene, bivalent carboxyclic and heterocyclic fused-rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod, and bivalent spiro-linked ring structures.
Claim 7
Dependent Claim
176 words
A reagent system for the specific binding assay determination of an analyte in a test sample, comprising (a) a binding partner for said analyte, and (b) a labeled conjugate comprising said analyte, or a binding analog thereof, and a photogenic label, wherein combination of said reagent system with said sample in a liquid reaction mixture results in formation of a binding partner-bound-species and a free-species of said labeled conjugate, said free-species yielding an analyte-photophor conjugate comprising said analyte or analog linked to a photophor capable of emitting light upon excitation, the analyte or analog portion of said analyte photophor conjugate capable of significantly reducing such light emission upon coming into.quenching orientation with the photophor, characterized in that said analyte or analog is joined to said photogenic label in said labeled conjugate by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene, bivalent carboxyclic and heterocyclic fused-rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod, and bivalent spiro-linked ring structures.
Claim 8
Dependent Claim
15 words
A reagent system according to claim 5, characterized In that said linking group comprises N,N'-piperazinylene.
Claim 9
Dependent Claim
17 words
A reagent system according to claim 5 or 6, characterized in that said photophor is a fluorescer.
Claim 10
Dependent Claim
115 words
A labeled conjugate for use in the specific assay determination of an analyte in a test sample, comprising said analyte, or a binding analog thereof, chemically joined to a photophor capable of emitting light upon excitation, such light emission being significantly reduced if the analyte or analog portion of the conjugates comes into quenching orientation with the photophor, characterized in that said analyte or analog is joined to said photophor by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene. bivalent-carbocyclic and heterocyclic fused- rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod; and bivalent spiro-linked ring structures.
Claim 11
Dependent Claim
115 words
A labeled conjugate for use in the specific assay determination of an analyte in a test sample, comprising said analyte, or a binding analog thereof, chemically joined to a photophor capable of emitting light upon excitation, such light emission being significantly reduced if the analyte or analog portion of the conjugates comes into quenching orientation with the photophor, characterized in that said analyte or analog is joined to said photophor by a linking group which substantially hinders quenching orientation of said photophor with said analyte or analog portion, such linking group comprising a residue selected from piperazinylene, phenylene, cyclohexylene. bivalent-carbocyclic and heterocyclic fused- rings, acetylene, bis-substituted monosaccharides, bivalent bicyclooctane rod; and bivalent spiro-linked ring structures.
Claim 12
Dependent Claim
41 words
A labeled conjuage according to claim 8 of the formula: wherein m is an integer from 2 through 9, n is an integer from 2 through 9, R is a linking group, and L is an analyte or an analog thereof.
Claim 13
Dependent Claim
19 words
A conjugate according to claim 9, characterized in that L is a hapten of molecular weight less than 1500.
References Cited (Backward Citations)
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