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US 2023/0384295 A1Published application
Multiplexed Catalyzed Reporter Deposition
AssigneeUltivue, Inc.
InventorsMark Bobrow, Stephanie Rae Hennek, Mael Manesse
Life of this patent
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Abstract
A method for testing a sample for the presence of one or more targets comprises multiplexed catalyzed reporter deposition (CARD) is provided.
Claims18
A method for testing a sample for the presence of one or more targets comprising
- (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;
- (2) in one or two steps, contacting the sample with (a) an enzyme linked to a nucleic acid strand and (b) an intermediate moiety comprising a first domain capable of specifically binding to the nucleic acid strand linked to the target-specific binding partner;
- and a second domain capable of specifically binding to the nucleic acid strand linked to the enzyme;
- (3) contacting the sample from step (2) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a releasable linker;
- (4) optionally deactivating the bound enzyme;
- (5) optionally detect signals from the bound detectable labels;
- and
- (6) optionally repeating steps (1)-(5) or a subset thereof and optionally repeating steps (3)-(4) or any subset thereof prior to step (5) of imaging.
5 more elements
A method for testing a sample for the presence of one or more targets comprising
- (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;
- (2) optionally removing unbound target-specific binding partners;
- (3) performing (3-a) or (3-b):
- (3-a) contacting the sample from step (1) or optionally step (2) with an enzyme linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner;
- (3-b) contacting the sample from step (1) or optionally step (2) with a first member of a binding pair linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner;
- contacting the sample with a second member of the binding pair linked to an enzyme;
- and optionally removing unbound second member of the binding pair linked to the enzyme and/or optionally removing unbound second member of the binding pair linked to the enzyme;
- (4) optionally removing unbound enzyme linked to complimentary nucleic acid strands;
- (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a releasable linker;
- (6) optionally removing unbound substrate conjugate;
- (7) optionally deactivating the bound enzyme;
- (8) optionally imaging the sample to detect the bound detectable labels;
- and
- (9) optionally repeating steps (1)-(8) or any subset thereof and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to step (8) of imaging.
11 more elements
A method for testing a sample for the presence of one or more targets comprising
- (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity are linked to different nucleic acid strands;
- (2) optionally removing unbound target-specific binding partners;
- (3) performing one of the following (3-a) or (3-b):
- (3-a) contacting the sample from step (1) or optionally step (2) with an enzyme linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner, wherein the following (i) or (ii) or both is met:
- (i) the nucleic acid strand is linked to the target-specific binding partner with a first releasable linker;
- and
- (ii) the enzyme is linked to the complementary nucleic acid strand with a second releasable linker;
- (3-b) contacting the sample from step (1) with a first member of a binding pair linked to a nucleic acid strand complimentary to the nucleic acid strand linked to the target-specific binding partner, wherein:
- (i) the nucleic acid strand is linked to the target-specific binding partner with a first releasable linker;
- and
- (ii) the first member of the binding pair is linked to the complimentary nucleic acid strand with a second releasable linker;
- contacting the sample with a second member of the binding pair linked to an enzyme optionally with a third releasable linker;
- (4) optionally removing unbound enzyme linked to complementary nucleic acid strands;
- (5) contacting the sample from step (3) or optionally step (4) with a substrate conjugate comprised of a detectably labeled substrate, where the substrate and detectable label are linked optionally with a fourth releasable linker;
- (6) optionally removing unbound substrate conjugate;
- (7) optionally releasing the bound enzyme by cleaving the first, second, third, or fourth releasable linker;
- (8) optionally imaging the sample to detect the bound detectable labels;
- and
- (9) optionally repeating steps (1)-(8) or any subset thereof and optionally if more than one target-specific binding partner is used, repeating steps (1)-(7) or any subset thereof prior to step (8) of imaging.
16 more elements
Dependencies are inferred from the claim text.