Method for the detection of an analyte by means of a nucleic acid reporter
First Claim
1. A method for the detection of an analyte comprising:
- (i) contacting at least one analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising;
a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte;
b) a nucleic acid label;
wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;
(ii) contacting said analyte dependent reporter complex with a enzyme composition wherein the nucleic acid labels on said reporter conjugates are joined to form an analyte specific amplicon;
(iii) contacting the analyte dependent amplicon with an replication composition wherein amplification products are produced; and
(iv) detecting said amplification products.
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Abstract
A process is disclosed for the detection of an analyte utilizing a nucleic acid label as a reporter. The analyte is detected by the binding of at least two reporter conjugates, each conjugate comprising a member of a binding pair and a nucleic acid label. The binding of the reporter conjugates to the analyte facilitates the juxtaposition of the nucleic acid labels, forming a single nucleic acid amplicon. The amplicon may then be detected directly, or may be used as a template of the generation of amplification products. Detection of the analyte by this process significantly reduces assay background caused by non-specific reporter conjugate binding.
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Citations
25 Claims
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1. A method for the detection of an analyte comprising:
(i) contacting at least one analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising;
a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte;
b) a nucleic acid label;
wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;
(ii) contacting said analyte dependent reporter complex with a enzyme composition wherein the nucleic acid labels on said reporter conjugates are joined to form an analyte specific amplicon;
(iii) contacting the analyte dependent amplicon with an replication composition wherein amplification products are produced; and
(iv) detecting said amplification products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20)
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12. A method for the detection of an analyte comprising:
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(i) immobilizing at least one analyte on a solid support, said analyte having at least two reporter conjugate binding sites;
(ii) contacting said analyte with at least one reporter conjugate pair, said reporter conjugate pair comprising a first reporter conjugate and a second reporter conjugate, each of said first and second reporter conjugates further comprising;
a) one member of a binding pair having an affinity for at least one reporter conjugate biding site on said analyte;
b) a nucleic acid label;
wherein said nucleic acid label of said first reporter conjugate comprises a 3′
hydroxyl group and wherein said nucleic acid label of said second reporter conjugate comprises a 5′
phosphoryl group and wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;
(iii) contacting said analyte dependent reporter complex with a DNA ligase,
wherein said first and second nucleic acid labels are ligated to form an analyte dependent amplicon;
(iv) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and
(v) detecting said amplification products.
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21. A method for the detection of an analyte comprising:
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(i) contacting at least one analyte with at least one reporter conjugate pair, said reporter conjugate pair comprising a first reporter conjugate and a second reporter conjugate, each of said first and second reported conjugates further comprising;
a) one member of a binding pair having an affinity for at least one reporter conjugate biding site on said analyte;
b) a nucleic acid label;
wherein said nucleic acid label of said first reporter conjugate comprises a 3′
hydroxyl group and wherein said nucleic acid label of said second reporter conjugate comprises a 5′
phosphoryl group and wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;
(ii) contacting said analyte dependent reporter complex with a DNA ligase,
wherein said first and second nucleic acid labels are ligated to form an analyte dependent amplicon;
(iii) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and
(iv) detecting said amplification products.
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22. A method for the detection of an analyte comprising:
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(i) contacting at least one analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising;
a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte;
b) a nucleic acid label;
wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;
(ii) contacting said analyte dependent reporter complex with;
a) an enzyme composition; and
b) a nucleic acid reporting label selected from the group consisting of fluorescent moieties, chemiluminescent moieties, particles, enzymes, radioactive tags, light emitting moieties and intercalating dyes;
wherein the nucleic acid labels on said reporter conjugates are joined to form an analyte specific amplicon and wherein said nucleic acid reporting label is incorporated into said amplicon; and
(iii) detecting said labeled amplicon. - View Dependent Claims (23, 24, 25)
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Specification