Protein detection via nanoreporters

• US 9,714,937 B2
• Filed: 10/13/2010
• Issued: 07/25/2017
• Est. Priority Date: 10/13/2009
• Status: Active Grant

• Current Assignee: Nanostring Technologies, Inc.
• Original Assignee: Nanostring Technologies, Inc.
• Inventors: Dunaway, Dwayne L.
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US12/904,078
• Publication Number:
  US 20110086774A1
• Time in Patent Office: 2,477 Days
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: G01N 2333/55 IL-2
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The invention provides methods, compositions, kits and devices for the detection of proteins. In some embodiments, the invention allows for multiplexed protein detection.

Claims

1. A method for determining the concentration of at least one protein in a sample comprising the steps:

(a) providing;

(i) at least one protein, and

(ii) a plurality of protein probe sets, wherein each protein probe set comprises

a first protein probe specific for a first region of said at least one protein, wherein said first protein probe is attached to a first capture region or a first matrix; and

a second protein probe specific for a second region of said at least one protein, wherein said second protein probe is directly attached to a partially double stranded nucleic acid;

wherein each protein probe set in the plurality of protein probe sets comprises a second protein probe comprising a different partially double stranded nucleic acid sequence;

(b) forming at least a first complex comprising said at least one protein, said first protein probe, said first capture region or said first matrix, and said second protein probe, wherein said at least one protein is bound to said first and second protein probes, and wherein when said first probe is attached to a first capture region said capture region is bound to a moiety in a second matrix capable of binding to said capture region;

(c) releasing one strand of said partially double stranded nucleic acid from said first complex thereby producing a unique signal oligo;

(d) forming a second complex comprising (1) at least said unique signal oligo and (2) at least one oligo probe comprising a region capable of hybridizing to said unique signal oligo and a region comprising a nanoreporter wherein said nanoreporter comprises a plurality of different detectable labels; and

(e) individually detecting said second complex or at least part of said second complex by a method comprising individually counting the presence of the plurality of different detectable labels of said nanoreporter, wherein the count of said plurality of different detectable labels of said nanoreporter is indicative of the concentration of said protein in said sample;

wherein steps (a) to (e) occur sequentially.

2. The method of claim 1 wherein said unique signal oligo is attached to a second capture region.

3. The method of claim 2 wherein said releasing of said unique signal oligo further comprises capturing directly or indirectly said signal molecule into a third matrix.

4. The method of claim 3 wherein said nanoreporter further comprising a constant region, wherein the constant region comprises a plurality of repeat nucleotide sequences.

5. The method of claim 4 further comprising binding said constant region to a second moiety in said third matrix, wherein said second moiety is capable of binding said constant region.

6. The method of claim 1 wherein said individually detecting further comprises detecting a digital signal.

7. The method of claim 1 wherein the first matrix is selected from the group consisting of a bead and an array.

8. The method of claim 1 wherein the second matrix is selected from the group consisting of a bead and an array.

9. The method of claim 1 further comprising determining the concentration of a plurality of target proteins by a method comprising forming a plurality of complexes, each complex comprising (i) at least one target protein (ii) a first protein probe specific for a first region of said at least one protein, wherein said first protein probe is attached to a capture region or a first matrix (iii) a second protein probe specific for a second region of said at least one protein, wherein said second protein probe comprises a signal molecule, wherein when said first probe is attached to a first capture region said capture probe is bound to said moiety in said second matrix, and wherein each second protein probe in each said plurality of complexes comprises a different signal oligo.

10. The method of claim 9 wherein the first matrix is a bead and the bead comprises a plurality of identical first protein probes.

11. The method of claim 9 wherein the concentration of two or more target proteins is determined.

12. The method of claim 9 wherein the concentration of 2, 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000 or more than 1000 different target proteins is determined.

13. The method of claim 9 wherein the concentration up to 2000 different target proteins is determined.

14. The method of claim 9 wherein the concentration up to 980 different target proteins is determined.

15. The method of claim 1 wherein said first protein probe and said second protein probe are independently selected from the group consisting of antibody, peptide, aptamer and peptoid.

16. The method of claim 1 wherein said nanoreporter comprises a single-stranded nucleic acid backbone, said backbone comprising a plurality of label attachment regions covalently attached together in a linear combination, wherein each label attachment region is hybridized to a complementary polynucleotide sequence having attached thereto the detectable label.

17. The method of claim 1, wherein said partially double stranded nucleic acid comprises one single-stranded nucleic acid to which is hybridized a single-stranded unique signal oligo and wherein said one single-stranded nucleic acid and said single-stranded unique signal oligo are partially complementary.