Methods and systems for sensitive and multiplexed analysis of biological samples using cleavable fluorescent streptavidin and anti-hapten antibodies
AssigneeArizona Board of Regents on behalf of Arizona State University
InventorsJia Guo, Joshua LaBaer
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Abstract
Provided herein are methods and systems for sensitive and multiplexed in situ analysis of samples such as biological samples using cleavable hapten linked targeting agents and cleavable detectably-labeled hapten-binding agents. In particular, provided herein are methods for multiplexed single-cell in situ biomolecule profiling in samples, including fixed or fresh tissues, and also allows the investigation of the different cell compositions and their spatial organizations in intact tissues through consecutive cycles of probe hybridization, fluorescence imaging, and signal removal.
Claims21
A method of multiplexed in situ analysis of biomolecules in a sample, the method comprising the following steps:
- (a) performing a first contacting step comprising contacting a sample comprising a plurality of biomolecules with a first hapten-conjugated targeting agent, the first hapten-conjugated targeting agent comprising a first targeting agent conjugated to the hapten via a cleavable linker, wherein the first targeting agent is configured to specifically bind or hybridize to a first target biomolecule in the sample, and wherein the first contacting step occurs under conditions that promote binding or hybridization of the first targeting agent to the first target biomolecule in the sample;
- (b) performing a second contacting step comprising contacting the sample of step (a) with a hapten-binding composition comprising a hapten-binding agent linked to a detectable label via a cleavable linker, wherein the second contacting step occurs under conditions that promote conjugation of the hapten-binding composition to the hapten of the first hapten-conjugated targeting agent;
- (c) imaging the sample after step (b), whereby a detectable signal generated from an interaction of hapten-conjugated targeting agent with the hapten-binding composition is detected;
- (d) cleaving the cleavable linkers to remove the detectable label from the hapten-binding composition and unbound hapten from the first hapten-conjugated targeting agent;
- (e) blocking the remaining hapten-binding agent;
- and
- (f) repeating the first contacting step, second contacting step, and imaging step with a second hapen-conjugated targeting agent, wherein the first hapten-conjugated targeting agent and the second hapten-conjugated targeting agent are configured to specifically bind or hybridize to a different target biomolecule, whereby multiple target biomolecules are detected in situ in the same sample.
A method for in situ analysis of biomolecules in a sample, the method comprising the following steps:
- (a) performing a first contacting step comprising contacting a sample comprising a plurality of biomolecules with a first hapten-conjugated targeting agent, the first hapten-conjugated targeting agent comprising a first targeting agent conjugated to the hapten via a cleavable linker, wherein the first targeting agent is configured to specifically bind or hybridize to a first target biomolecule in the sample, and wherein the first contacting step occurs under conditions that promote binding or hybridization of the first targeting agent to the first target biomolecule in the sample;
- (b) performing a second contacting step comprising contacting the sample of step (a) with a hapten-binding composition comprising hapten-binding agent linked to a detectable label via a cleavable linker, wherein the second contacting step occurs under conditions that promote conjugation of the hapten-binding composition to the hapten of the first hapten-conjugated targeting agent;
- (c) performing a third contacting step comprising contacting the sample of step (b) with a second orthogonal hapten labeled antibody for signal amplification, wherein the third contacting step occurs under conditions that promote binding of the second orthogonal hapten labeled antibody to the hapten-binding composition;
- (d) performing a fourth contacting step comprising contacting the sample of step (c) with a second hapten-binding composition comprising a second hapten-binding agent linked to a detectable label via a cleavable linker under conditions that promote binding of the second hapten-binding agent to the second orthogonal hapten labeled antibody;
- and
- (e) imaging the sample after step (d), whereby a detectable signal generated from an interaction of hapten-conjugated targeting agents with the hapten-binding agent is detected.
Dependencies are inferred from the claim text.