Patent US5661677A successfully refreshed from Google Patents.
EP0144913A2
Expired / Lapsed
EP
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Hybridization assay employing labeled probe and anti-hybrid
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 A nucleic acid hybridization assay involving a labeled probe and formation of a hybrid having epitopes for an antibody reagent. The label provides a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid. Particularly useful antibody reagents are antibodies such as anti-DNA'RNA, anti-RNA'RNA and antibodies to intercalated duplexes which do not bind substantially to single stranded nucleic acids. Modulation of the label response can be accomplished in a variety of ways such as by steric inactivation or hindrance or by labeling the antibody reagent with a second label which interacts with the first label. The assay is particularly advantageous because no separation of hybridized and unhybridized probe is required.
Inventors
- 👤 James P. Albarella
- 👤 Leslie H. Anderson Deriemer
- 👤 Robert J. Carrico
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 14:34
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C07; C07K; C07K16/00; C07K16/18; C12Q1/6804
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
154 words
A method for detecting a particular polynucleotide sequence in a test sample comprising single stranded nucleic acids, comprising the steps of contacting the test sample with a labeled nucleic acid probe comprising a label and at least one single stranded base sequence that is substantially complementary to the sequence to be detected, and determining the resulting hybrids, characterized in that the hybrids are formed to have the hybrids epitopes for an antibody reagent which does not bind substantially to single stranded nucleic acids, any hybrid that is formed is contacted with the antitibody reagent, the label in the labeled probe providing a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid, and the detectable response is measured as a function of the presence of the sequence to be detected in the sample.
Claim 2
Independent Claim
154 words
A method for detecting a particular polynucleotide sequence in a test sample comprising single stranded nucleic acids, comprising the steps of contacting the test sample with a labeled nucleic acid probe comprising a label and at least one single stranded base sequence that is substantially complementary to the sequence to be detected, and determining the resulting hybrids, characterized in that the hybrids are formed to have the hybrids epitopes for an antibody reagent which does not bind substantially to single stranded nucleic acids, any hybrid that is formed is contacted with the antitibody reagent, the label in the labeled probe providing a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid, and the detectable response is measured as a function of the presence of the sequence to be detected in the sample.
Claim 3
Dependent Claim
120 words
The method of Claim 1 wherein the antibody reagent is (i) selective for binding DNA * RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA, (ii) selective for binding RNA'RNA hybrids wherein both the probe and the sequence to be detected are RNA, or (iii) selective for binding intercalation complexes wherein the duplexes formed in the assay comprise a nucleic acid intercalator bound thereto in the form of intercalation complexes, preferably the labeled probe comprising the intercalator chemically linked to the probe in the single stranded complementary region of the probe whereby upon hyridization with the sequence to be detected said intercalation complexes are formed in the resulting hybrid.
Claim 4
Dependent Claim
100 words
The method of Claim 1 wherein the label interacts with a reagent member of the label detection system to provide the detectable response and the detectable response is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid due to steric hindrance of access of such detection system member to the label, the label preferably being a substrate, cofactor, or inhibitor of an enzyme which is the member of the label detection system with which the label interacts to provide the detectable response.
Claim 5
Dependent Claim
67 words
The method of Claim 1 wherein the probe is labeled with one of a first label and a second label and the antibody reagent is labeled with the other, interaction between the first and second labels providing a detectable response which is measurably different when the labeled probe and labeled antibody reagent are both bound to the same hybrid compared to when they are not so bound.
Claim 6
Dependent Claim
58 words
The method of Claim 4 wherein the first label participates in a first chemical reaction that produces a diffusible product which is a participant in a second chemical reaction with the second label to produce a product that provides the detectable response, the first and second labels preferably being catalysts for the first and second chemical reactions, respectively.
Claim 7
Dependent Claim
17 words
The method of Claim 4 wherein the first and second labels participate in an energy transfer interaction.
Claim 8
Dependent Claim
27 words
The method of Claim 1 wherein the test sample comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.
Claim 9
Dependent Claim
115 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising a labeled nucleic acid probe comprising a label and at least one single stranded base sequence that is substantially complementary to the sequence to be detected, characterized by comprising additionally an antibody reagent capable of binding to hybrids formed between any of the particular polynucleotide sequence to be detected in the sample and the labeled probe, but incapable of binding substantially to single stranded nucleic acids, the label providing a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid.
Claim 10
Dependent Claim
115 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising a labeled nucleic acid probe comprising a label and at least one single stranded base sequence that is substantially complementary to the sequence to be detected, characterized by comprising additionally an antibody reagent capable of binding to hybrids formed between any of the particular polynucleotide sequence to be detected in the sample and the labeled probe, but incapable of binding substantially to single stranded nucleic acids, the label providing a detectable response which is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid.
Claim 11
Dependent Claim
119 words
The reagent system of Claim 8 wherein the antibody reagent is (i) selective for binding DNA'RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA, (ii) selective for binding RNA'RNA hybrids wherein both the probe and the sequence to be detected are RNA, or (iii) selective for binding intercalation complexes wherein the duplexes formed in the assay comprise a nucleic acid intercalator bound thereto in the form of intercalation complexes, preferably the labeled probe comprising the intercalator chemically linked to the probe in the single stranded complementary region of the probe whereby upon hybridization with the sequence to be detected said intercalation complexes are formed in the resulting hyrid.
Claim 12
Dependent Claim
101 words
The reagent system of Claim 8 wherein the label interacts with a reagent member of the label detection system to provide the detectable response and the detectable response is measurably different when the labeled probe is comprised in a hybrid that is bound by the antibody reagent compared to when not comprised in such a hybrid due to steric hindrance of access of such detection system member to the label, the label preferably being a substrate, cofactor, or inhibitor of an enzyme which is the member of the label detection system with which the label interacts to provide the detectable response.
Claim 13
Dependent Claim
68 words
The reagent system of Claim 8 wherein the probe is labeled with one of a first label and a second label and the antibody reagent is labeled with the other, interaction between the first and second labels providing a detectable response which is measurably different when the labeled probe and labeled antibody reagent are both bound to the same hybrid compared to when they are not so bound.
Claim 14
Dependent Claim
59 words
The reagent system of Claim 11 wherein the first label participates in a first chemical reaction that produces a diffusible product which is a participant in a -second chemical reaction with the second label to produce a product that provides the detectable response, the first and second labels preferably being catalysts for the first and second chemical reactions, respectively.
Claim 15
Dependent Claim
18 words
The reagent system of Claim 11 wherein the first and second labels participate in an energy transfer interaction.
References Cited (Backward Citations)
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