Patent EP0288737A1 successfully refreshed from Google Patents.
EP0288737A1
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Rapid hybridization assay using latex-immobilized probe
Assignee: Bayer Corp
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Filing: 1988-03-23
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Granted: —
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Expires: 2008-03-23
Abstract
Abstract Nucleic acid hybridization assays are performed by using probes that are immobilized on discrete, water suspensible microparticles (latex). Assay times under 30 minutes from the initiation of hybridization through measurement of the generated signal are obtained. The methods are based on detection of hybrids through the use of labeled anti-hybrid antibody reagents or by labeling the sample nucleic acids. The assay formats are convenient as well as rapid. The use of latex supports for the probe eliminates the problems of immobilizing sample nucleic acids, allows the use of excess probe, and enables rapid and simple separation of hybrids for detection purposes.
Inventors
- 👤 Robert J. Carrico
- 👤 William L. Patterson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 15:58
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54313; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6804; C12Q1/6813; C12Q1/6834
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
144 words
A method for determining a particular polynucleotide sequence in a test medium containing single stranded nucleic acids. wherein either (i) the test medium is combined under hybridizing conditions with an immobilized form of a polynucleotide probe, the resulting immobilized phase is separated, and hybridized probe detected by binding of a reagent that selectively binds hybrids of the probe and the sequence to be determined but substantially not single stranded nucleic acids, or (ii) the test medium is treated to label the single stranded nucleic acids therein, an immobilized form of a polynucleotide probe is added under hybridizing conditions, the resulting immobilized phase is separated, and hybridized probe detected by measuring the label, characterized in that the immobilized form of the polynucleotide probe comprises said probe attached to discrete, water suspensible microparticles whereby the hybridization and separation steps can be completed within about 30 minutes.
Claim 2
Independent Claim
144 words
A method for determining a particular polynucleotide sequence in a test medium containing single stranded nucleic acids. wherein either (i) the test medium is combined under hybridizing conditions with an immobilized form of a polynucleotide probe, the resulting immobilized phase is separated, and hybridized probe detected by binding of a reagent that selectively binds hybrids of the probe and the sequence to be determined but substantially not single stranded nucleic acids, or (ii) the test medium is treated to label the single stranded nucleic acids therein, an immobilized form of a polynucleotide probe is added under hybridizing conditions, the resulting immobilized phase is separated, and hybridized probe detected by measuring the label, characterized in that the immobilized form of the polynucleotide probe comprises said probe attached to discrete, water suspensible microparticles whereby the hybridization and separation steps can be completed within about 30 minutes.
Claim 3
Dependent Claim
29 words
The method of claim 1 wherein a dispersion of said microparticles in the hybridization medium yields a fifty percent decrease in turbidity in no less than about 30 minutes.
Claim 4
Dependent Claim
33 words
The method of claim 1 wherein said microparticles have a specific gravity of from about 1.03 to about 1.08 grams/mL and have an average diameter of from about 0.1 to about 20 microns.
Claim 5
Dependent Claim
33 words
The method of claim 1 wherein the microparticle immobilized phase is separated by filtration or the microparticles are magnetic or magnetizable and the microparticle-immobilized phase is separated by application of a magnetic field.
Claim 6
Dependent Claim
79 words
The method of claim 1 wherein the hybrid-selective binding reagent is labeled with an enzymatically active group, a fluorescer, a chromophore, a luminescer, or a metal sol, or the label introduced to the sample nucleic acids is a ligand and is detectable by binding of a binding partner labeled with an enzymatically active group, a fluorescer, a chromophore, a luminescer, or a metal sol, whereby the hybridization, separation, and detection steps can all be completed within about 30 minutes.
Claim 7
Dependent Claim
35 words
The method of claim 1 wherein said anti-hybrid antibody reagent is selective for binding DNA · RNA hybrids, one of the probe and the sequence to be determined being DNA and the other being RNA.
Claim 8
Dependent Claim
64 words
A reagent system for determining a particular polynucleotide sequence in a test medium by nucleic acid hybridization, comprising: (1) an immobilized form of a polynucleotide probe having a single stranded base sequence to be determined, said probe being attached to discrete, water suspensible microparticles, and (2) an anti-hybrid antibody reagent selective for binding hybrids formed between said probe and the sequence to be determined.
Claim 9
Dependent Claim
64 words
A reagent system for determining a particular polynucleotide sequence in a test medium by nucleic acid hybridization, comprising: (1) an immobilized form of a polynucleotide probe having a single stranded base sequence to be determined, said probe being attached to discrete, water suspensible microparticles, and (2) an anti-hybrid antibody reagent selective for binding hybrids formed between said probe and the sequence to be determined.
Claim 10
Dependent Claim
34 words
The reagent system of claim 7 wherein said microparticles have a specific gravity of from about 1.03 to about 1.08 grams/mL and have an average diameter of from about 0.1 to about 20 microns.
Claim 11
Dependent Claim
36 words
The reagent system of claim 7 wherein said anti-hybrid antibody reagent is selective for binding DNA · RNA hybrids, one of the probe and the sequence to be determined being DNA and the other being RNA.
Claim 12
Dependent Claim
22 words
Use of the reagent system of claims 7 to 9 for the determination of a particular polynucleotide sequence in a test medium.
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