Spatially encoded biological assays

Patent metadata

Field	Value
Publication number	US-10983113-B2
Application number	US-202016837924-A
Country	US
Kind code	B2
Filing date	Apr 1, 2020
Priority date	Apr 5, 2010
Publication date	Apr 20, 2021
Grant date	Apr 20, 2021

Abstract

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

First claim

The invention claimed is:

1. A method of determining presence or abundance of a target protein at a region of interest in a tissue sample, comprising:
(a) delivering a plurality of probes to a tissue sample, wherein a probe of the plurality of probes comprises a capture agent that specifically binds a target protein of the tissue sample, wherein the capture agent is conjugated to an oligonucleotide;
(b) obtaining an image of the tissue sample to identify a region of interest in the tissue sample; and
(c) removing the oligonucleotide from the region of interest and determining all or a portion of a sequence of the oligonucleotide or a complement thereof of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target protein at the region of interest in the tissue sample, and using the sequence to determine the presence or abundance of the target protein at the region of interest in the tissue sample.

2. The method of claim 1 , wherein step (b) is performed using immunohistochemistry.

3. The method of claim 1 , wherein the method further comprises, between steps (a) and (c), separating the probes of the plurality of probes that are not specifically bound to the target protein at the region of interest in the tissue sample from the probe of the plurality of probes comprising the capture agent that is specifically bound to the target protein at the region of interest in the tissue sample.

4. The method of claim 3 , wherein the separating step comprises washing the tissue sample to separate the probes of the plurality of probes that are not specifically bound to the target protein at the region of interest in the tissue sample from the probe of the plurality of probes comprising the capture agent that is specifically bound to the target protein at the region of interest in the tissue sample.

5. The method of claim 1 , wherein step (c) comprises determining all or a portion of the sequence of the oligonucleotide or a complement thereof of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target protein at the region of interest in the tissue sample, via hybridization of a probe comprising a label.

6. The method of claim 5 , wherein step (c) comprises detecting the label.

7. The method of claim 6 , wherein the label is a fluorophore.

8. The method of claim 7 , wherein step (c) comprises detecting the fluorophore.

9. The method of claim 1 , wherein the capture agent is a protein.

10. The method of claim 9 , wherein the protein is an antibody.

11. The method of claim 1 , wherein the target protein is present in a subcellular target.

12. The method of claim 11 , wherein the subcellular target comprises a mitochondrion or a nucleus.

13. The method of claim 1 , wherein the determined sequence is used to determine the presence of the target protein at the region of interest in the tissue sample.

14. The method of claim 1 , wherein the determined sequence is used to determine the abundance of the target protein at the region of interest in the tissue sample.

15. The method of claim 1 , wherein the determined sequence is used to determine the presence and abundance of the target protein at the region of interest in the tissue sample.

16. The method of claim 1 , wherein step (a) is performed at substantially the same time as step (b).

17. The method of claim 1 , wherein step (a) is performed before step (b).

18. The method of claim 1 , wherein step (b) is performed before step (a).

19. The method of claim 1 , wherein two or more probes of the plurality of probes specifically bind to unique target proteins at the region of interest in the tissue sample.

20. The method of claim 19 , wherein the determined sequences are used to determine the presence or abundance or both, of the unique target proteins at the region of interest in the tissue sample in parallel.

21. The method of claim 19 , wherein the sequences of the oligonucleotides of the two or more probes are determined sequentially.

22. The method of claim 19 , wherein the determined sequences are used to determine the presence of greater than 75 unique target proteins at the region of interest in the tissue sample.

23. The method of claim 19 , wherein the determined sequences are used to determine the presence of greater than 100 unique target proteins at the region of interest in the tissue sample.

24. The method of claim 1 , wherein the plurality of probes is delivered to multiple regions of interest in the tissue sample.

25. The method of claim 1 , wherein the method further comprises determining the presence of the target protein at the region of interest in two or more serial sections of the tissue sample.

26. The method of claim 1 , wherein the tissue sample is a tissue section.

27. The method of claim 26 , wherein the tissue section is a fresh-frozen tissue section.

28. The method of claim 26 , wherein the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section.

29. The method of claim 1 , wherein the capture agent comprises an antibody and determining all or a portion of the sequence of the oligonucleotide or a complement thereof comprises hydridizing a probe comprising (i) a sequence that is complementary to the oligonucleotide or the complement thereof and (ii) a label.

30. The method of claim 1 , wherein the capture agent comprises an antibody and determining all or a portion of the sequence of the oligonucleotide or the complement thereof comprises sequencing.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S

Classifications

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title