EP0309230A2
Expired / Lapsed
EP
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Homogeneous protection assay
Assignee: Gen Probe Inc
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Filing: 1988-09-21
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Granted: —
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Expires: 2008-09-21
Abstract
Abstract Improved homogenous diagnostic assay methods and labels for detecting an analyte in a medium when the analyte is a member of a specific binding pair. The methods and labels provide procedures for reducing background and increasing sensitivity. The binding partner of the analyte is labeled with a substance, the stability of which detectably changes whenever said analyte is bound as a member of the specific binding pair. In a closely related system, the analyte is labeled with a substance susceptible to differential degradation depending on whether or not the analyte is bound as a member of its specific binding pair. After incubation and selective degradation or chemical or biochemical alteration, the amount of analyte bound is detected by measuring either the stability change or the extent of degradation of the label. In a particular system, chemiluminescent acridinium ester labeled probes are used in a homogenous hybridization assay format for sensitively detecting the presence of complement any target polynucleotide sequences.
Inventors
- 👤 Lyle John Arnold
- 👤 Norman C. Nelson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 05:26
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/582; C12Q1/6813; C12Q1/6816; C12Q1/6827; C12Q1/6876; C12Q1/6883; C12Q1/6886; C12Q1/6888; C12Q1/689; G01N33/53; G01N33/5306; G01N33/531; G01N33/532; G01N33/533; G01N33/536; G01N33/542; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
141 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising the binding partner, the analyte or an analog of the analyte which forms a binding pair with the binding partner, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) if said moiety is said analyte or the analog, combining in the medium said binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 2
Independent Claim
141 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising the binding partner, the analyte or an analog of the analyte which forms a binding pair with the binding partner, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) if said moiety is said analyte or the analog, combining in the medium said binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 3
Independent Claim
102 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, said binding partner which is conjugated to a substance whose stability differs when said binding partner is a member of a binding pair as compared to its stability in an unbound form; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 4
Independent Claim
102 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, said binding partner which is conjugated to a substance whose stability differs when said binding partner is a member of a binding pair as compared to its stability in an unbound form; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 5
Independent Claim
125 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising the analyte or an analog of the analyte which forms a binding pair with the binding partner, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) said binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 6
Independent Claim
125 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising the analyte or an analog of the analyte which forms a binding pair with the binding partner, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) said binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 7
Independent Claim
126 words
A method for detemining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, a first binding partner to said analyte, said first binding partner being conjugated to a substance whose stability differs when said first binding partner is a member of a binding pair as compared to its stability in an unbound form; and (2) a second binding partner to said analyte being different from said first binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 8
Independent Claim
126 words
A method for detemining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, a first binding partner to said analyte, said first binding partner being conjugated to a substance whose stability differs when said first binding partner is a member of a binding pair as compared to its stability in an unbound form; and (2) a second binding partner to said analyte being different from said first binding partner; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 9
Independent Claim
91 words
A method for detecting a nucleotide sequence in medium, which comprises: a) combining with said medium, or a portion thereof, said nucleotide sequence and a probe substantially complementary thereto, said probe being conjugated to a substance whose stability differs when said probe is hybridized to said nucleotide sequence as compared to its stability in an unhybridized form; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of nucleotide sequence in a medium.
Claim 10
Independent Claim
91 words
A method for detecting a nucleotide sequence in medium, which comprises: a) combining with said medium, or a portion thereof, said nucleotide sequence and a probe substantially complementary thereto, said probe being conjugated to a substance whose stability differs when said probe is hybridized to said nucleotide sequence as compared to its stability in an unhybridized form; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of nucleotide sequence in a medium.
Claim 11
Independent Claim
148 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising a first binding partner to said analyte, or an analog of said first binding partner which forms a binding pair which participates in a binding reaction, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) as second binding partner to said analyte different from said first binding partner in said binding reaction; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 12
Independent Claim
148 words
A method for determining in a medium the presence or amount of an analyte which combines with a binding partner to form a binding pair, which method comprises: a) combining with said medium, or a portion thereof, (1) a moiety comprising a first binding partner to said analyte, or an analog of said first binding partner which forms a binding pair which participates in a binding reaction, said moiety being conjugated to a substance whose stability differs when the moiety is a member of a binding pair as compared to its stability in an unbound form; and (2) as second binding partner to said analyte different from said first binding partner in said binding reaction; b) selectively degrading the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium.
Claim 13
Independent Claim
16 words
A method as in one of Claims 1-6 in which the substance is an acridinium ester.
Claim 14
Independent Claim
186 words
A method as in one of Claims 1-6 in which the substance is a member selected from the group consisting of: wherein X comprises a member of the group consisting of O, N, S, halogen, substituted phosphorous, substituted sulfur, substituted boron, or substituted arsenic; wherein Y comprises a member of the group consisting of O, S, or NH; wherein R₁ comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy, or is absent when X is a halogen; wherein R₂ comprises a member of the group consisting of H, alkyl, alkenyl, aryl, substituted alkenyl, substituted aryl, alkoxy or aryloxy if and only if X is N; wherein R₃ comprises a member of the group consisting of H, amino, hydroxy, thiol, halogen, nitro, amido, acetyl, alkyl, alkenyl, aryl, substituted acetyl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, or aryloxy; wherein R₄ comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl or substituted aryl; and wherein at least one of R₁, R₂, R₃ or R₄ contains a reactive site which allows chemical conjugation.
Claim 15
Dependent Claim
35 words
The method of claim 1 or 2 or 3 or 4 or 5 or 6 having the further step of separation wherein the homogeneous component is used to improve the stringency and/or to reduce backgrounds.
Claim 16
Dependent Claim
30 words
The method of claim 9 wherein the separation step is selected from the group consisting of gel filtration, hydroxyapatite, magnetic microspheres, cationic separation supports, separation membranes and antibody-based separation supports.
Claim 17
Dependent Claim
23 words
The method of claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 performed at room temperature.
Claim 18
Dependent Claim
29 words
Use of the method of claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 for detection of chimeric target nucleic acid sequences.
Claim 19
Dependent Claim
52 words
Use of the method of claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 with a probe capable of discriminating between target nucleic acid sequences and non-target sequences which differ from said target nucleic acid sequences by a single nucleotide within the region of hybridization.
Claim 20
Dependent Claim
53 words
A single stranded polymeric sequence for use in a homogeneous assay, comprising at least two nucleotide monomers or at least one nucleotide monomer and one non-nucleotide monomer, said sequence having at least one sterically tolerant label attachment site capable of permitting hybridization of said polymeric sequence to a substantially complementary nucleic acid sequence.
Claim 21
Dependent Claim
53 words
A single stranded polymeric sequence for use in a homogeneous assay, comprising at least two nucleotide monomers or at least one nucleotide monomer and one non-nucleotide monomer, said sequence having at least one sterically tolerant label attachment site capable of permitting hybridization of said polymeric sequence to a substantially complementary nucleic acid sequence.
Claim 22
Dependent Claim
23 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located at the 3′ terminus of said polymeric sequence.
Claim 23
Dependent Claim
23 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located at the 5′ terminus of said polymeric sequence.
Claim 24
Dependent Claim
29 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located on the nucleotide base of one of the nucleotide monomers comprising said polymeric sequence.
Claim 25
Dependent Claim
29 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located on the phosphate backbone of one of the nucleotide monomers comprising said polymeric sequence.
Claim 26
Dependent Claim
30 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located on one of the sugars on one of the nucleotide monomers comprising said polymeric sequence.
Claim 27
Dependent Claim
25 words
The single stranded polymeric sequence of claim 14 wherein said sterically tolerant site is located on one of the non-nucleotide monomers comprising said polymeric sequence.
Claim 28
Dependent Claim
29 words
The single stranded polymeric sequence of claim 14 or 15 or 16 or 17 or 18 or 19 or 20 wherein said site is labeled with an acridinium ester.
Claim 29
Dependent Claim
199 words
The single stranded polymeric sequence of claim 14 or 15 or 16 or 17 or 18 or 19 or 20 wherein said site is labeled with a member selected from the group consisting of: wherein X comprises a member of the group consisting of O, N, S, halogen, substituted phosphorous, substituted sulfur, substituted boron, or substituted arsenic; wherein Y comprises a member of the group consisting of O, S, or NH; wherein R1 comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy, or is absent when X is a halogen; wherein R2 comprises a member of the group consisting of H, alkyl, alkenyl, aryl, substituted alkenyl, substituted aryl, alkoxy or arlyoxy if and only if X is N; wherein R3 comprises a member of the group consisting of H, amino, hydroxy, thiol, halogen, nitro, amido, acetyl, alkyl, alkenyl, aryl, substituted acetyl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy or aryloxy; wherein R4 comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl or substituted aryl; and wherein at least one of R1, R2, R3 or R4 contains a reactive site which allows chemical conjugation.
Claim 30
Dependent Claim
44 words
The single stranded polymeric sequence for use in a homogeneous assay comprising monomeric units which may be nucleotide monomeric units or non-nucleotide monomeric units or a mixture thereof, wherein a label is attached to at least one monomeric unit at a serically tolerant site.
Claim 31
Dependent Claim
44 words
The single stranded polymeric sequence for use in a homogeneous assay comprising monomeric units which may be nucleotide monomeric units or non-nucleotide monomeric units or a mixture thereof, wherein a label is attached to at least one monomeric unit at a serically tolerant site.
Claim 32
Dependent Claim
15 words
The single stranded polymeric sequence of claim 23 wherein the label is an acridinium ester.
Claim 33
Dependent Claim
185 words
The single stranded polymeric sequence of claim 23 wherein the label is a member selected from the group consisting of: wherein X comprises a member of the group consisting of O, N, S, halogen, substituted phosphorous, substituted sulfur, substituted boron, or substituted arsenic; wherein Y comprises a member of the group consisting of O, S, or NH; wherein R1 comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy, or is absent when X is a halogen; wherein R2 comprises a member of the group consisting of H, alkyl, alkenyl, aryl, substituted alkenyl, substituted aryl, alkoxy or aryloxy if and only if X is N; wherein R3 comprises a member of the group consisting of H, amino, hydroxy, thiol, halogen, nitro, amido, acetyl, alkyl, alkenyl, aryl, substituted acetyl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, or aryloxy; wherein R4 comprises a member of the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl or substituted aryl; and wherein at least one of R1, R2, R3 or R4 contains a reactive site which allows chemical conjugation.
Claim 34
Dependent Claim
25 words
The single stranded polymeric sequence of claim 24 wherein the acridinium ester is attached to at least one non-nucleotide monomeric unit of said polymeric sequence.
Claim 35
Dependent Claim
24 words
The single stranded polymeric sequence of claim 24 wherein the label is attached to at least one non-nucleotide monomeric unit of said polymeric sequence.
Claim 36
Dependent Claim
34 words
The single stranded polymeric sequence of claim 24 wherein the acridinium ester is attached to at least one non-nucleotide monomeric unit which replaces at least one originally present monomeric unit of said polymeric sequence.
Claim 37
Dependent Claim
33 words
The single stranded polymeric sequence of claim 24 wherein the label is attached to at least one non-nucleotide monomeric unit which replaces at least one originally present monomeric unit of said polymeric sequence.
Claim 38
Dependent Claim
35 words
The single stranded polymeric sequence of claim 24 wherein the acridinium ester is attached to at least one non-nucleotide monomeric unit which is inserted between two originally present adjacent nucleotide monomers of said polymeric sequence.
Claim 39
Dependent Claim
34 words
The single stranded polymeric sequence of claim 25 wherein the label is attached to at least one non-nucleotide monomeric unit which is inserted between two originally present adjacent nucleotide monomers of said polymeric sequence.
Claim 40
Dependent Claim
31 words
Method of using the polymeric sequences of claim 26 or 27 or 28 or 29 or 30 or 31 to detect local hybrid stability within a probe-target duplex using differential degradation.
Claim 41
Dependent Claim
12 words
The method of claim 12 for the detection of chronic myelogenous leukemia.
References Cited (Backward Citations)
No citation records indexed.
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