Patent US4743535A successfully refreshed from Google Patents.
US4743535A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Hybridization assay employing labeled probe and anti-hybrid
Assignee: Bayer Corp
•
Filing: 1984-11-07
•
Granted: 1988-05-10
•
Expires: 2004-11-07
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
- 👤 Robert J. Carrico
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 16:38
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C07; C07K; C07K16/00; C07K16/18; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/805; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
154 words
A method for detecting a particular polynucleotide sequence in a test sample containing single stranded nucleic acids, comprising the steps of: (a) forming a hybrid between any of the particular polynucleotide sequence to be detected in the sample and 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, the hybrid having epitopes for an antibody reagent which does not bind substantially to single stranded nucleic acids, (b) contacting any hybrid that is formed with the antibody 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 (c) measuring the detectable response 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 containing single stranded nucleic acids, comprising the steps of: (a) forming a hybrid between any of the particular polynucleotide sequence to be detected in the sample and 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, the hybrid having epitopes for an antibody reagent which does not bind substantially to single stranded nucleic acids, (b) contacting any hybrid that is formed with the antibody 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 (c) measuring the detectable response as a function of the presence of the sequence to be detected in the sample.
Claim 3
Dependent Claim
81 words
The method of claim 1 wherein the antibody reagent is: (i) selective for binding DNA.sup.. 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.sup.. 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.
Claim 4
Dependent Claim
69 words
The method of claim 1 wherein the label interacts with a reagent member of a 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.
Claim 5
Dependent Claim
36 words
The method of claim 3 wherein the label is 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 6
Dependent Claim
26 words
The method of claim 4 wherein the label is a substrate which is acted on by the enzyme to produce a colorimetric, fluorescent or luminescent signal.
Claim 7
Dependent Claim
41 words
The method of claim 4 wherein the label is a prosthetic group of an enzyme and wherein the apoenzyme of such enzyme is the member of the label detection system with which the label interacts to produce the catalytically active holoenzyme.
Claim 8
Dependent Claim
17 words
The method of claim 6 wherein the prosthetic group is FAD and the apoenzyme is apo(glucose oxidase).
Claim 9
Dependent Claim
36 words
The method of claim 3 wherein the label is a specifically bindable ligand for a binding substance which is the member of the label detection system with which the label interacts to provide the detectable response.
Claim 10
Dependent Claim
20 words
The method of claim 8 wherein the label is a hapten and its binding substance is a second antibody reagent.
Claim 11
Dependent Claim
24 words
The method of claim 9 wherein the label is fluorescent and wherein binding of the second antibody reagent thereto results in quenching of fluorescence.
Claim 12
Dependent Claim
41 words
The method of claim 9 wherein the detection system additionally comprises a conjugate of the hapten label, or a binding analog thereof, with an enzyme, the enzymatic activity of which conjugate is altered upon binding of the second antibody reagent thereto.
Claim 13
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 14
Dependent Claim
42 words
The method of claim 12 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.
Claim 15
Dependent Claim
21 words
The method of claim 13 wherein the first and second labels are catalysts for the first and second chemical reactions, respectively.
Claim 16
Dependent Claim
14 words
The method of claim 14 wherein the antibody reagent is labeled with an enzyme.
Claim 17
Dependent Claim
14 words
The method of claim 15 wherein the probe is labeled with a nonenzyme catalyst.
Claim 18
Dependent Claim
17 words
The method of claim 12 wherein the first and second labels participate in an energy transfer interaction.
Claim 19
Dependent Claim
21 words
The method of claim 17 wherein the first label is a fluorescer or luminescer and the second label is a quencher.
Claim 20
Dependent Claim
61 words
The method of claim 1 wherein the probe is labeled with a fluorescer or luminescer, energy transfer interaction between the label and native groups on the antibody reagent 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 21
Dependent Claim
58 words
The method of claim 1 wherein the antibody reagent is selective for binding intercalation complexes and wherein the labeled probe also comprises a nucleic acid 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 hybrid.
Claim 22
Dependent Claim
25 words
The method of claim 1 wherein the label comprised in the labeled probe is chemically linked to the probe in its single stranded complementary region.
Claim 23
Dependent Claim
29 words
The method of claim 1 wherein the label comprised in the labeled probe is chemically linked to the probe in a region other than its complementary single stranded region.
Claim 24
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 25
Dependent Claim
114 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising: (1) 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 (2) 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 26
Dependent Claim
114 words
A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising: (1) 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 (2) 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 27
Dependent Claim
82 words
The reagent system of claim 24 wherein the antibody reagent is: (i) selective for binding DNA.sup.. 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.sup.. 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.
Claim 28
Dependent Claim
70 words
The reagent system of claim 24 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.
Claim 29
Dependent Claim
37 words
The reagent system of claim 26 wherein the label is 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 30
Dependent Claim
26 words
The reagent system of claim 27 wherein the label is a substrate which is acted on by the enzyme to produce a fluorescent or luminescent signal.
Claim 31
Dependent Claim
42 words
The reagent system of claim 27 wherein the label is a prosthetic group of an enzyme and wherein the apoenzyme of such enzyme is the member of the label detection system with which the label interacts to produce the catalytically active holoenzyme.
Claim 32
Dependent Claim
18 words
The reagent system of claim 29 wherein the prosthetic group is FAD and the apoenzyme is apo(glucose oxidase).
Claim 33
Dependent Claim
37 words
The reagent system of claim 26 wherein the label is a specifically bindable ligand for a binding substance which is the member of the label detection system with which the label interacts to provide the detectable response.
Claim 34
Dependent Claim
21 words
The reagent system of claim 31 wherein the label is a hapten and its binding substance is a second antibody reagent.
Claim 35
Dependent Claim
25 words
The reagent system of claim 32 wherein the label is fluorescent and wherein binding of the second antibody reagent thereto results in quenching of fluorescence.
Claim 36
Dependent Claim
42 words
The reagent system of claim 32 wherein the detection system additionally comprises a conjugate of the hapten label, or a binding analog thereof, with an enzyme, the enzymatic activity of which conjugate is altered upon binding of the second antibody reagent thereto.
Claim 37
Dependent Claim
68 words
The reagent system of claim 24 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 38
Dependent Claim
43 words
The reagent system of claim 24 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.
Claim 39
Dependent Claim
22 words
The reagent system of claim 36 wherein the first and second labels are catalysts for the first and second chemical reactions, respectively.
Claim 40
Dependent Claim
15 words
The reagent system of claim 37 wherein the antibody reagent is labeled with an enzyme.
Claim 41
Dependent Claim
15 words
The reagent system of claim 38 wherein the probe is labeled with a nonenzyme catalyst.
Claim 42
Dependent Claim
18 words
The reagent system of claim 35 wherein the first and second labels participate in an energy transfer interaction.
Claim 43
Dependent Claim
22 words
The reagent system of claim 40 wherein the first label is a fluorescer or luminescer and the second label is a quencher.
Claim 44
Dependent Claim
62 words
The reagent system of claim 24 wherein the probe is labeled with a fluorescer or luminescer, energy transfer interaction between the label and native groups on the antibody reagent 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 45
Dependent Claim
59 words
The reagent system of claim 24 wherein the antibody reagent is selective for binding intercalation complexes and wherein the labeled probe also comprises a nucleic acid 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 hybrid.
Claim 46
Dependent Claim
26 words
The reagent system of claim 24 wherein the label comprised in the labeled probe is chemically linked to the probe in its single stranded complementary region.
Claim 47
Dependent Claim
30 words
The reagent system of claim 24 wherein the label comprised in the labeled probe is chemically linked to the probe in a region other than its complementary single stranded region.
Claim 48
Dependent Claim
27 words
The reagent system of claim 24 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.
References Cited (Backward Citations)
US3935074A
Cited
US4581333A
Cited
US4238565A
Cited
US4358535A
Cited
US4358535B1
Cited
EP0063879A2
Cited
EP0070685A2
Cited
US4463090A
Cited
US4493899A
Cited
EP0087564A1
Cited
EP0093613A1
Cited
EP0095089A1
Cited
US4547569A
Cited
EP0135159A2
Cited
US4623627A
Cited
EP0144914A2
Cited
US4563417A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-05-10 | 1991-11-09 | 1992-05-10 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-05-10 | 1995-11-09 | 1996-05-10 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-05-10 | 1999-11-09 | 2000-05-10 | $7700.00 | Window Closed / Lapsed |