Digital protein quantification

Patent metadata

Field	Value
Publication number	US-12467931-B2
Application number	US-202318354151-A
Country	US
Kind code	B2
Filing date	Jul 18, 2023
Priority date	Dec 30, 2015
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

Abstract

Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.

First claim

What is claimed is:

1 . A method comprising:
incubating a plurality of permeabilized cells with a plurality of structurally distinct binding elements, each binding element having a specific binding affinity for a target epitope of a cellular protein, and wherein each binding element is conjugated to an epitope-specific oligonucleotide (ESO) comprising a barcode sequence specific to the target epitope, thereby binding the binding elements to their corresponding epitopes to form binder-epitope complexes; washing away unbound binding elements; isolating single cells of the plurality of permeabilized cells into a partition of a plurality of partitions; providing to partitions of the plurality of partitions a plurality of partition-specific oligonucleotides (PSO) linked to a bead, each PSO comprising a partition-specific barcode sequence and a unique molecular identifier (UMI); and hybridizing, in one or more partitions, a PSO with an ESO and extending the hybridized PSO with a polymerase, thereby generating a nucleic acid comprising an ESO, a partition-specific barcode sequence, and a UMI.

2 . The method of claim 1 , wherein each partition-specific oligonucleotide further comprises a primer binding sequence.

3 . The method of claim 1 , wherein each binding element is an antibody.

4 . The method of claim 1 , wherein each binding element is an aptamer.

5 . The method of claim 1 , wherein the plurality of structurally distinct binding elements comprises a library of at least 10, and no more than about 10,000, structurally distinct antibodies conjugated to the epitope-specific oligonucleotides.

6 . The method of claim 1 , further comprising discarding partitions that do not contain a single cell or contain multiple cells.

7 . The method of claim 1 , further comprising discarding partitions that do not contain a single partition-specific barcode sequence or contain multiple partition-specific barcode sequences.

8 . The method of claim 1 , wherein the partition-specific oligonucleotides are covalently linked to a bead with a cleavable linker.

9 . The method of claim 1 , further comprising lysing the cells after the isolating step.

10 . The method of claim 1 , wherein the plurality of partitions comprise a primer that binds to a primer binding sequence.

11 . The method of claim 10 , wherein the primer binding sequence is within the epitope-specific oligonucleotides.

12 . The method of claim 1 , wherein the epitope-specific oligonucleotide comprises a unique molecular identifier.

13 . The method of claim 1 , wherein each binding element is conjugated to a unique molecular identifier (UMI).

14 . The method of claim 1 , wherein each epitope-specific oligonucleotide further comprises a universal primer binding site.

15 . The method of claim 1 , further comprising cleaving the epitope-specific oligonucleotide from each binding element to yield a cleaved antibody oligonucleotide and cleaving the partition-specific oligonucleotide linked to the bead to yield a cleaved partition-specific oligonucleotide.

16 . The method of claim 15 , further comprising amplifying the cleaved antibody oligonucleotide using a universal primer and the cleaved partition-specific oligonucleotide.

17 . The method of claim 1 , wherein the plurality of partitions comprise wells, microwells or nanowells.

18 . The method of claim 1 , wherein the plurality of partitions comprise droplets.

19 . The method of claim 10 , wherein the primer binding sequence is within the partition-specific oligonucleotides.

Assignees

• Bio Rad Laboratories Inc
• Bio Rad Europe Gmbh

Inventors

• Agresti Jeremy
• Lebofsky Ronald

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• B01J19/0046

  Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00) · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C40B70/00

  Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes · CPC title