METHODS AND COMPOSITIONS FOR ENHANCING DETECTION IN DETERMINATIONS EMPLOYING CLEAVABLE ELECTROPHORETIC TAG REAGENTS
First Claim
1. A method for isolating signal produced by a detectable moiety of a released predetermined portion of a reagent from signal obtained from the intact reagent and from other fragments thereof, the method comprising:
- (a) combining (i) a first reagent comprising a releasable predetermined portion that includes a detectable moiety wherein the predetermined portion is attached to the first reagent by a cleavable moiety and wherein the first reagent comprises at least one interactive functionality adjacent the predetermined portion positioned in the first reagent such that the interactive functionality does not form part of the predetermined portion upon release thereof and (ii) a second reagent that potentiates the cleavage of the cleavable moiety, and (b) subjecting the combination to conditions under which the cleavable moiety is cleaved to release the predetermined portion, wherein the interactive functionality enables the isolation of the signal produced by the detectable moiety of the released predetermined portion from signal obtained from intact first reagent and from other fragments thereof.
6 Assignments
0 Petitions
Accused Products
Abstract
Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. The probes comprise interactive functionalities adjacent the cleaved portion positioned in the probes such that the interactive functionality does not form part of the e-tag reporters. Target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.
79 Citations
64 Claims
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1. A method for isolating signal produced by a detectable moiety of a released predetermined portion of a reagent from signal obtained from the intact reagent and from other fragments thereof, the method comprising:
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(a) combining (i) a first reagent comprising a releasable predetermined portion that includes a detectable moiety wherein the predetermined portion is attached to the first reagent by a cleavable moiety and wherein the first reagent comprises at least one interactive functionality adjacent the predetermined portion positioned in the first reagent such that the interactive functionality does not form part of the predetermined portion upon release thereof and (ii) a second reagent that potentiates the cleavage of the cleavable moiety, and (b) subjecting the combination to conditions under which the cleavable moiety is cleaved to release the predetermined portion, wherein the interactive functionality enables the isolation of the signal produced by the detectable moiety of the released predetermined portion from signal obtained from intact first reagent and from other fragments thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for isolating signal produced by a detectable moiety of a released predetermined portion of a reagent from signal obtained from the intact reagent and from other fragments thereof, the method comprising:
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(a) combining (i) an oligonucleotide reagent comprising a releasable predetermined portion that includes a detectable moiety and a mobility modifier wherein the predetermined portion is attached to a terminal nucleotide of the oligonucleotide reagent by a cleavable moiety and wherein the oligonucleotide reagent comprises at least one interactive functionality attached to the penultimate nucleotide adjacent the terminal nucleotide and (ii) a reagent that potentiates the cleavage of the cleavable moiety, and (b) subjecting the combination to conditions under which the cleavable moiety is cleaved to release the predetermined portion, wherein the interactive functionality enables the isolation of the signal produced by the detectable moiety of the released predetermined portion from signal obtained from intact oligonucleotide reagent and from other fragments thereof. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for separating a component of a mixture from other components thereof, the method comprising:
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(a) combining (i) a first reagent comprising a releasable predetermined portion that includes a detectable moiety wherein the predetermined portion is attached to the first reagent by a cleavable moiety and wherein the first reagent comprises at least one interactive functionality adjacent the predetermined portion positioned in the first reagent such that the interactive functionality does not form part of the predetermined portion upon release thereof and (ii) a second reagent that potentiates the cleavage of the cleavable moiety, (b) subjecting the combination to conditions under which the cleavable moiety is cleaved to release the predetermined portion thereby forming a mixture, (c) including in the mixture a third reagent that comprises a moiety that binds to the interactive functionality, and (d) separating the third reagent or the predetermined portion from the mixture. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35)
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36. A method for separating a component of a mixture from other components thereof, the method comprising:
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(a) combining (i) an oligonucleotide reagent comprising a releasable predetermined portion that includes a detectable moiety and a mobility modifier wherein the predetermined portion is attached to a terminal nucleotide of the oligonucleotide reagent by a cleavable moiety and wherein the oligonucleotide reagent comprises at least one interactive functionality attached to the penultimate nucleotide adjacent the terminal nucleotide and (ii) a reagent that potentiates the cleavage of the cleavable moiety, and (b) subjecting the combination to conditions under which the cleavable moiety is cleaved to release the predetermined portion thereby forming a mixture, (c) including in the mixture a third reagent that comprises a moiety that binds to the organic moiety comprising an interactive functionality, and (d) separating the third reagent or the predetermined portion from the mixture. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. A method for detecting the binding of or interaction between a first binding agent and each and any of a plurality of second binding agents, comprising:
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(a) subjecting a mixture comprising the first binding agent and the second binding agents to conditions for interaction therebetween, wherein the second binding agent comprises a cleavable reporter group and at least one interactive functionality adjacent the reporter group positioned in the second binding agent such that the interactive functionality does not form part of the reporter group upon release thereof, where the cleavable reporter group in each second binding agent includes;
(i) a cleavable moiety that is susceptible to cleavage, and (ii) at least one tag, wherein the at least one tag has a detectable moiety and a mobility unique to the second binding agent, (b) subjecting the mixture to conditions under which the cleavable moiety is cleaved to release the reporter groups, wherein the interactive functionality enables the isolation of the signal produced by the detectable moiety of the released reporter groups from signal obtained from intact second binding agent and from other fragments thereof, (c) separating the released reporter groups by their differences in mobility; and
(c) detecting the binding of or interaction between a first binding agent and each second binding agent based on the unique mobility of the corresponding reporter group. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55)
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56. A kit for use in detecting the presence and/or amount of each of a plurality of target molecules, comprising in packaged combination:
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(a) first binding agent capable of binding to a first binding site on said target molecules, (b) a plurality of second binding agents, each capable of target-specific binding to a second binding site on a selected target, and each having a unique cleavable reporter group and at least one interactive functionality adjacent the reporter group positioned in the second binding agent such that the interactive functionality does not form part of the reporter group upon release thereof, where the cleavable reporter group in each second binding agent includes (i) a cleavable moiety that is susceptible to cleavage, and (ii) an electrophoretic tag having a detectable reporter moiety and a unique electrophoretic mobility which allows the tag to be uniquely identified among all other tags associated with the plural binding agents, wherein the interactive functionality is effective in isolating the signal produced by released reporter groups from the signal produced by intact second binding agents and other fragments thereof. - View Dependent Claims (57, 58, 59, 60, 61, 62, 63, 64)
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Specification