EP0146815A2
Expired / Lapsed
EP
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Nucleic acid hybridization assay employing antibodies to intercalation complexes
Assignee: Bayer Corp
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Filing: 1984-11-29
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Granted: —
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Expires: 2004-11-29
Abstract
Abstract Nucleic acid hybridization assay methods and reagent systems for detecting a particular polynucleotide sequence in a test medium. An aggregate is formed in the assay reaction mixture comprising intercalation complexes between a nucleic acid intercalator and double stranded nucleic acid associated with the hybridization product of the sequence to be detected and a nucleic acid probe sequence. Hybridization of the probe with the sequence to be detected can then be determined by addition of an antibody, or a fragment thereof, capable of binding with the intercalation complexes in the formed aggregate and measuring the antibody or fragment thereof which becomes bound to such intercalation complexes associated with hybridized probe. In one preferred embodiment, this method eliminates the need to chemically modify the probe in order to form a labeled reagent. In another embodiment, the method provides an advantageous method for labeling the probe by chemical modification.
Inventors
- 👤 James P. Albarella
- 👤 Leslie H. Anderson (Deriemer)
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 06:48
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C07; C07K; C07K16/00; C07K16/18; C12Q1/6804; C12Q1/6818; C12Q1/70; C12Q1/701; C12Q1/705
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
137 words
A method for detecting a particular polynucleotide sequence in a test medium containing single stranded nucleic acids, comprising the steps of combining the test medium with (i) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be determined in the test medium, under conditions favorable to hybridization between the sequence to be detected and the complementary sequence in the probe, and determining hybridized probe, characterized in that there is also added a nucleic acid intercalator capable of binding to double stranded nucleic acid in the form of intercalation complexes, and hybridized probe is detected by adding an antibody, or a fragment thereof, capable of binding with intercalation complexes in the resulting hybridization product, and determining the antibody or fragment thereof which becomes bound to such complexes.
Claim 2
Independent Claim
137 words
A method for detecting a particular polynucleotide sequence in a test medium containing single stranded nucleic acids, comprising the steps of combining the test medium with (i) a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be determined in the test medium, under conditions favorable to hybridization between the sequence to be detected and the complementary sequence in the probe, and determining hybridized probe, characterized in that there is also added a nucleic acid intercalator capable of binding to double stranded nucleic acid in the form of intercalation complexes, and hybridized probe is detected by adding an antibody, or a fragment thereof, capable of binding with intercalation complexes in the resulting hybridization product, and determining the antibody or fragment thereof which becomes bound to such complexes.
Claim 3
Dependent Claim
31 words
The method of Claim 1 wherein the intercalator is combined with the test medium as a separate, free compound and noncovalently binds with double stranded nucleic acid to form intercalation complexes.
Claim 4
Dependent Claim
34 words
The method of Claim 1 wherein the intercalator is chemically linked to the probe in the single stranded complementary region of the probe, whereby upon hybridization said intercalation complexes are formed in such region.
Claim 5
Dependent Claim
38 words
The method of Claim 1 wherein the antibody or fragment thereof is labeled with a detectable chemical group which preferably is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
Claim 6
Dependent Claim
44 words
The method of Claim 1 according to a solid phase hybridization technique wherein one of the probe and the single stranded nucleic acids from the test medium is immobilized on a solid support and wherein the antibody associated with the solid support is determined.
Claim 7
Dependent Claim
87 words
The method of Claim 1 according to a solid phase sandwich hybridization technique wherein the test medium is combined with first and second nucleic acid probes each comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected and wherein one of the probes is immobilized on a solid support or comprises a binding site for a binding substance and is thereafter rendered immobilized by the presence of an immobilized form of such binding substance.
Claim 8
Dependent Claim
39 words
The method of Claim 1 according to a solution phase hybridization technique wherein the probe comprises a binding site for a binding substance and wherein after the hybridization step there is added an immobilized form of such binding substance.
Claim 9
Dependent Claim
19 words
The method of Claim 1 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 10
Dependent Claim
27 words
The method of Claim 1 wherein the test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.
Claim 11
Dependent Claim
66 words
A reagent system for detecting a particular polynucleotide sequence in a test medium, comprising a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, characterized by comprising additionally a nucleic acid intercalator, and an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator.
Claim 12
Dependent Claim
66 words
A reagent system for detecting a particular polynucleotide sequence in a test medium, comprising a nucleic acid probe comprising at least one single stranded base sequence which is substantially complementary to the sequence to be detected, characterized by comprising additionally a nucleic acid intercalator, and an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator.
Claim 13
Dependent Claim
39 words
The reagent system of Claim 10 wherein the antibody or fragment thereof is labeled with a detectable chemical group, which preferably is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
Claim 14
Dependent Claim
22 words
The reagent system of Claim 10 which additionally comprises a solid support for immobilizing single stranded nucleic acids from the test medium.
Claim 15
Dependent Claim
15 words
The reagent system of Claim 10 wherein the probe is immobilized on a solid support.
Claim 16
Dependent Claim
30 words
The reagent system of Claim 10 wherein the probe comprises a binding site for a binding substance and the reagent system additionally comprises an immobilized form of such binding substance.
Claim 17
Dependent Claim
19 words
The reagent system of Claim 10 wherein the intercalator is a separate, free compound, substantially uncomplexed with nucleic acids.
Claim 18
Dependent Claim
34 words
The reagent system of Claim 10 wherein the intercalator is chemically linked to a single stranded region of the probe such that duplex formation in such region results in the formation of intercalation complexes.
Claim 19
Dependent Claim
104 words
The reagent system of Claim 10 for use in a sandwich hybridization format which comprises a second nucleic acid probe, the first and second probes respectively comprising at least one single stranded base sequence which is substantially complementary to a mutually exclusive portion of the sequence to be detected, and wherein either one of the probes is labeled with a detectable chemical group and the other is immobilized, or one of the probes is labeled with a detectable chemical group and the other comprises a binding site for a binding substance and the reagent system additionally comprises an immobilized form of such binding substance.
Claim 20
Dependent Claim
20 words
The reagent system of Claim 10 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
Claim 21
Independent Claim
63 words
A method for detecting double stranded nucleic acid in a liquid medium, comprising the steps of: (a) adding to said medium (i) a nucleic acid intercalator and (ii) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (b) detecting the binding of said antibody or fragment thereof to said complex.
Claim 22
Independent Claim
63 words
A method for detecting double stranded nucleic acid in a liquid medium, comprising the steps of: (a) adding to said medium (i) a nucleic acid intercalator and (ii) an antibody, or a fragment thereof, capable of binding with intercalation complexes comprising double stranded nucleic acid complexed with the intercalator, and (b) detecting the binding of said antibody or fragment thereof to said complex.
Claim 23
Dependent Claim
38 words
The method of Claim 19 wherein the antibody or fragment thereof is labeled with a detectable chemical group, which preferably is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
Claim 24
Dependent Claim
19 words
The method of Claim 19 wherein the intercalator is selected from acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines and quinolines.
References Cited (Backward Citations)
No citation records indexed.
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