MEASUREMENT OF PROTEIN EXPRESSION USING REAGENTS WITH BARCODED OLIGONUCLEOTIDE SEQUENCES

• US 20180088112A1
• Filed: 09/25/2017
• Published: 03/29/2018
• Est. Priority Date: 09/26/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co, Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fan, Christina, Chang, Christina, Shum, Eleen, Zoellner, Olaf, Bansal, Nidhanjali, Ghadiali, James, Lam, Gretchen Yinbon, Jensen, Eric, Rohrer, Jurg, Gaylord, Brent, Martin, Jody, Vidal, Jason G.
• Granted Patent: US 11,460,468 B2
• Time in Patent Office: Days
• 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 plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding reagent that it is conjugated therewith, and the protein binding reagent is capable of specifically binding to a protein target.

2. The plurality of compositions of claim 1, wherein the unique identifier comprises a nucleotide sequence of 25-45 nucleotides in length.

3. The plurality of compositions of claim 1, wherein the unique identifier is selected from a diverse set of unique identifiers.

4. The plurality of compositions of claim 3, wherein the diverse set of unique identifiers comprises at least 100 different unique identifiers.

7. The plurality of compositions of claim 1, comprising a plurality of antibodies, a plurality of aptamers, or a combination thereof.

8. The plurality of compositions of claim 1, wherein the oligonucleotide is conjugated to the protein binding reagent through a linker.

16. The plurality of compositions of claim 1, wherein the oligonucleotide comprises a barcode sequence, a poly(A) tail, or a combination thereof.

17. The plurality of compositions of claim 1, comprising at least 100 different protein binding reagents.

20. The plurality of compositions of claim 17, wherein each protein binding reagent is conjugated with one or more oligonucleotides comprising at least one barcode sequence selected from a set of at least 10 different barcode sequences.

23. The plurality of compositions of claim 1, wherein the plurality of compositions further comprises one or more second protein binding reagents which are not conjugated with the oligonucleotide.

24. The plurality of compositions of claim 23, where the protein binding reagent and the one or more second protein binding reagents are the same.

25. The plurality of compositions of claim 1, capable of specifically binding to a plurality of protein targets.

5. (canceled)

6. (canceled)

9-15. -15. (canceled)

18. (canceled)

19. (canceled)

21. (canceled)

22. (canceled)

26. (canceled)

27. (canceled)

28. A method of quantitative analysis of a plurality of protein targets in a sample comprising:

providing a sample comprising a plurality of protein targets;

providing a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding reagent that it is conjugated therewith, and the protein binding reagent is capable of specifically binding to at least one of the plurality of protein targets;

contacting the plurality of compositions with the sample for specific binding with the plurality of protein targets;

removing unbound compositions;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target binding region and a barcode sequence, wherein the barcode sequence is from a diverse set of unique barcode sequences;

contacting the plurality of oligonucleotide probes with the oligonucleotides of the plurality of compositions;

extending the oligonucleotide probes hybridized to the oligonucleotides to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier and a barcode sequence; and

determining the number of unique barcode sequences for each unique identifier,

whereby the quantity of each protein target in the sample is determined.

40. The method of claim 28, wherein the sample comprises a single cell.

41. The method of claim 40, wherein the plurality of protein targets is expressed on the surface of the single cell.

42. The method of claim 40, wherein the removing unbound compositions comprises washing the single cell with a washing buffer.

43. The method of claim 42, comprising lysing the single cell.

44. The method of claim 28, comprising detaching the oligonucleotides from the protein binding reagents.

45. The method of claim 44, wherein the oligonucleotides are detached from the protein binding reagent by UV photocleaving, chemical treatment (dithiothreitol), heating, enzyme treatment, or any combination thereof.

46. The method of claim 28, wherein each of the oligonucleotide probes comprises a cell label, a binding site for a universal primer, or any combination thereof.

48. The method of claim 28, wherein the plurality of oligonucleotide probes is immobilized on a solid support.

50. The method of claim 28, further comprising amplifying the plurality of labeled nucleic acids to produce a plurality of amplicons.

29-39. -39. (canceled)

47. (canceled)

49. (canceled)

51-58. -58. (canceled)

59. A method of simultaneous quantitative analysis of a plurality of protein targets and a plurality of nucleic acid target molecules in a sample comprising:

providing a sample comprising a plurality of protein targets and a plurality of nucleic acid target molecules;

providing a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding reagent that it is conjugated therewith, and the protein binding reagent is capable of specifically binding to at least one of the plurality of protein targets;

contacting the plurality of compositions with the sample for specific binding with the plurality of protein targets;

removing unbound compositions;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target binding region and a barcode sequence, wherein the barcode sequence is from a diverse set of unique barcode sequences;

contacting the plurality of oligonucleotide probes with the oligonucleotides of the compositions and the plurality of nucleic acid target molecules for hybridization;

extending the oligonucleotide probes hybridized to the oligonucleotides and nucleic acid target molecules to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier or a nucleic acid target molecule, and a barcode sequence; and

determining the number of unique barcode sequences for each unique identifier and each nucleic acid target molecule,

whereby the quantity of each protein target and each nucleic acid target molecule in the sample is determined.

60-89. -89. (canceled)

90. A kit for simultaneous quantitative analysis of a plurality of protein targets and a plurality of nucleic acid target molecules in a sample comprising a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding reagent that it is conjugated therewith, and the protein binding reagent is capable of specifically binding to a protein target, and a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target binding region and a barcode sequence, wherein the barcode sequence is from a diverse set of unique barcode sequences.

91-129. -129. (canceled)

130. A method of identifying a biomarker in a sample comprising:

providing a sample comprising a plurality of protein targets and a plurality of nucleic acid target molecules;

providing a plurality of compositions each comprising a protein binding reagent conjugated with an oligonucleotide, wherein the oligonucleotide comprises a unique identifier for the protein binding reagent that it is conjugated therewith, and the protein binding reagent is capable of specifically binding to at least one of the plurality of protein targets;

contacting the plurality of compositions with the sample for specific binding with the plurality of protein targets;

removing unbound compositions;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target binding region and a barcode sequence, wherein the barcode sequence is from a diverse set of unique barcode sequences;

contacting the plurality of oligonucleotide probes with the oligonucleotides of the compositions and the plurality of nucleic acid target molecules for hybridization;

extending the oligonucleotide probes hybridized to the oligonucleotides and nucleic acid target molecules to produce a plurality of labeled nucleic acids, wherein each of the labeled nucleic acid comprises a unique identifier or a nucleic acid target molecule, and a barcode sequence;

determining the number of unique barcode sequences for each unique identifier and each nucleic acid target molecule; and

identifying a biomarker using the quantity of a protein target or the quantity of a nucleic acid target molecule.

131. The method of claim 130, comprising determining the quantity of at least one protein target and at least one nucleic acid target molecule.

132. The method of claim 130, comprising comparing the quantity of at least one protein target and its corresponding nucleic acid target molecule.

133. The method of claim 132, comprising identifying a biomarker if the quantity of a protein target is greater than its corresponding nucleic acid target molecule.

135. The method of claim 132, comprising identifying a biomarker if the quantity of a protein target'"'"'s corresponding nucleic acid target molecule is less than 10.

134. (canceled)

136. (canceled)

137-1107. -1107. (canceled)