EP0146039A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Hybridization assay with immobilization of hybrids by antihybrid binding
Assignee: Bayer Corp
•
Filing: 1984-11-29
•
Granted: —
•
Expires: 2004-11-29
Abstract
Abstract A nucleic acid hybridization assay wherein the hybrid formed between the polynucleotide sequence to be detected and the probe is immobilized by binding of an immobilized or immobilizable form of an antibody reagent selective for binding such hybrid. Preferred antibody reagents are selective for DNA·RNA or RNA·RNA hybrids or for intercalated duplexes. The immobilized hybrid is detected preferably by use of a labeled form of a second such antibody reagent or a labeled form of the probe. The method dispenses with the need to immobilize either the probe or sample nucleic acids and permits hybridization to proceed between the sequence to be detected and the probe in solution, resulting in rapid rates of hybridization.
Inventors
- 👤 James P. Albarella
- 👤 Leslie H. Anderson Deriemer
- 👤 Robert J. Carrico
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 10:35
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6804; C07; C07K; C07K16/00; C07K16/18; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/70; C12Q1/701; C12Q1/705
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
122 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 nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, under conditions favorable to hybridization between the probe and the sequence to be detected, and determining the resulting duplexes, characterized in that the duplexes formed by hybridization between the complementary probe sequence and the sequence to be detected are contacted with an antibody reagent which is selective for binding such duplexes, the antibody reagent being immobilized or thereafter is rendered immobilized, and said duplexes that become bound to the immobilized antibody reagent are determined.
Claim 2
Independent Claim
122 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 nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, under conditions favorable to hybridization between the probe and the sequence to be detected, and determining the resulting duplexes, characterized in that the duplexes formed by hybridization between the complementary probe sequence and the sequence to be detected are contacted with an antibody reagent which is selective for binding such duplexes, the antibody reagent being immobilized or thereafter is rendered immobilized, and said duplexes that become bound to the immobilized antibody reagent are determined.
Claim 3
Dependent Claim
72 words
The method of Claim 1 wherein said antibody reagent is selective for binding (i) DNA'RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA, (ii) RNA'RNA hybrids wherein both the probe and the sequence to be detected are RNA, or (iii) intercalation complexes wherein the duplexes formed in the assaycomprise a nucleic acid intercalator bound thereto in the form of intercalation complexes.
Claim 4
Dependent Claim
58 words
The method of Claim 1 wherein the duplexes that become bound to the immobilized antibody reagent are determined by addition of a labeled form of a second antibody reagent capable of binding said duplexes, wherein the first and second antibody reagents are preferably the same substance, and the label that becomes associated with the immobilized reagent is measured.
Claim 5
Dependent Claim
35 words
The method of Claim 1 wherein the probe is labeled and the duplexes that become bound to the immobilized antibody reagent are determined by measuring the label that has become associated with the immobilized reagent.
Claim 6
Independent Claim
45 words
The method of any one of Claims 1-4 wherein said antibody reagent is added in a soluble form and is linked to a specifically bindable ligand and wherein said resulting duplexes are additionally contacted with an immobilized form of a binding partner for said ligand.
Claim 7
Dependent Claim
24 words
The method of Claim 5 wherein said ligand is biotin or a hapten and said binding partner is avidin or an anti-hapten antibody, respectively.
Claim 8
Independent Claim
29 words
The method of any one of Claims 1-4 wherein the label is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
Claim 9
Dependent Claim
27 words
The method of Claim 1 wherein the test sample is a biological sample that has been subjected to conditions to release and denature nucleic acids present therein.
Claim 10
Dependent Claim
93 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, characterized by comprising additionally an antibody reagent which is selective for binding duplexes formed by hybridization between the complementary probe sequence and the sequence to be detected, said antibody reagent either being immobilized or being linked to a specifically bindable ligand, and if said binding is not immobilized, an immobilized form of a binding partner for said ligand.
Claim 11
Dependent Claim
93 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, characterized by comprising additionally an antibody reagent which is selective for binding duplexes formed by hybridization between the complementary probe sequence and the sequence to be detected, said antibody reagent either being immobilized or being linked to a specifically bindable ligand, and if said binding is not immobilized, an immobilized form of a binding partner for said ligand.
Claim 12
Dependent Claim
75 words
The reagent system of Claim 9 wherein said antibody reagent is selective for binding (i) DNA'RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA, (ii) RNA'RNA hybrids wherein both the probe and the sequence to be detected are R NA, or (iii) intercalation complexes wherein the duplexes formed in the assay comprise a nucleic acid intercalator bound thereto in the form of intercalation complexes.
Claim 13
Dependent Claim
36 words
The reagent system of Claim 9 which additionally comprises a labeled form of a second antibody reagent which is selective for binding said duplexes, wherein the first and second antibody reagents are preferably the same substance.
Claim 14
Dependent Claim
12 words
The reagent system of Claim 9 wherein the probe comprises a label.
Claim 15
Dependent Claim
31 words
The reagent system of either of Claims 11 or 12 wherein the label is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
Claim 16
Dependent Claim
25 words
The reagent system of Claim 9 wherein said ligand is biotin or a hapten and said binding partner is avidin or an anti-hapten antibody, respectively.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.