Measurement of protein expression using reagents with barcoded oligonucleotide sequences

• US 10,338,066 B2
• Filed: 05/30/2018
• Issued: 07/02/2019
• Est. Priority Date: 09/26/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fan, Christina, Zoellner, Olaf
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/993,468
• Publication Number:
  US 20180267036A1
• Time in Patent Office: 398 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/00646 the compounds being bound to beads immobilised on the solid supports
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6857 : Antibody fragments
• CPC Class Codes: G16B 25/10 : Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 99/00 : Subject matter not provided for in other groups of this subclass
• CPC Class Codes: G16H 10/20 : for electronic clinical trials or questionnaires
• CPC Class Codes: G16H 20/13 : delivered from dispensers
• CPC Class Codes: G16Z 99/00 : Subject matter not provided for in other main groups of this subclass

Abstract

Some embodiments disclosed herein provide a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide. The oligonucleotide comprises a unique identifier for the protein binding reagent it is conjugated with, and the protein binding reagent is capable of specifically binding to a protein target. Further disclosed are methods and kits for quantitative analysis of a plurality of protein targets in a sample and for simultaneous quantitative analysis of protein and nucleic acid targets in a sample. Also disclosed herein are systems and methods for preparing a labeled biomolecule reagent, including a labeled biomolecule agent comprising a protein binding reagent conjugated with an oligonucleotide.

Claims

1. A method of measuring protein expression in cells, comprising:

(a) contacting a plurality of oligonucleotide-conjugated antibodies with a plurality of cells comprising a plurality of protein targets, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of protein targets;

(b) partitioning the plurality of cells associated with the plurality of oligonucleotide-conjugated antibodies to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the oligonucleotide-conjugated antibodies;

(c) in the partition comprising the single cell, contacting a barcoding particle with the antibody specific oligonucleotides, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from a diverse set of unique barcode sequences;

(d) extending the oligonucleotide probes hybridized to the antibody specific oligonucleotides via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the poly(T) regions of the oligonucleotide probes to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier, or a complementary sequence thereof, and a barcode sequence; and

(e) obtaining sequence information of the plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of protein targets in one or more of the plurality of cells.

2. The method of claim 1, comprising after step (a) removing the oligonucleotide-conjugated antibodies that are not associated with the plurality of cells.

3. The method of claim 1, comprising detaching the antibody specific oligonucleotides from the antibody.

4. The method of claim 1, wherein partitioning the plurality of cells comprises partitioning the plurality of cells associated with the plurality of oligonucleotide-conjugated antibodies and a plurality of barcoding particles comprising the barcoding particle to the plurality of partitions, wherein the partition of the plurality of partitions comprises the single cell from the plurality of cells associated with the oligonucleotide-conjugated antibody and the barcoding particle.

5. The method of claim 1, wherein step (e) obtaining sequencing information of the plurality of labeled nucleic acids or a portion thereof comprises subjecting the labeled nucleic acids to one or more reactions to generate a set of nucleic acids for nucleic acid sequencing.

6. The method of claim 1, wherein the antibody specific oligonucleotide is conjugated to the antibody through a chemical group selected from the group consisting of a UV photocleavable group, a streptavidin, a biotin, an amine, and a combination thereof.

7. The method of claim 1, wherein the plurality of protein targets comprises a cell-surface protein, an intracellular protein, a cell marker, a B-cell receptor, a T-cell receptor, an antibody, a major histocompatibility complex, a tumor antigen, a receptor, or a combination thereof.

8. The method of claim 1, wherein each of the oligonucleotide probes comprises a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof.

9. The method of claim 1, wherein the antibody specific oligonucleotide comprises a molecular label, a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof.

10. The method of claim 1, wherein the barcoding particle is a sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or a hydrogel bead.

11. The method of claim 1, wherein the partition is a well or a droplet.

12. The method of claim 1, wherein the plurality of cells comprises T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof.

13. A method for simultaneous measurement of protein and gene expressions in cells, comprising:

(a) contacting a plurality of oligonucleotide-conjugated antibodies with a plurality of cells comprising a plurality of protein targets and a plurality of mRNA target molecules, wherein each of the plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of protein targets;

(b) partitioning the plurality of cells associated with the oligonucleotide-conjugated antibodies to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the oligonucleotide-conjugated antibodies;

(c) in the partition comprising the single cell, contacting a barcoding particle with the antibody specific oligonucleotides, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence selected from a diverse set of unique barcode sequences;

(d) extending the oligonucleotide probes hybridized to the antibody oligonucleotides and the mRNA target molecules via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the mRNA target molecules, respectively, and the poly(T) regions of the oligonucleotide probes to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier or a sequence of an mRNA target molecule or a complementary sequence thereof, respectively, and a barcode sequence; and

(e) obtaining sequence information of the plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of protein targets and the quantity of one or more of the plurality of mRNA target molecules in one or more of the plurality of cells.

14. The method of claim 13, comprising lysing the single cell in the partition comprising the single cell.

15. The method of claim 13, wherein the partition is a well or a droplet.