Measurement of protein expression using reagents with barcoded oligonucleotide sequences
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.
1. A composition, comprising:
a plurality of oligonucleotide-associated antibodies each comprising an antibody associated with an antibody specific oligonucleotide, wherein
the antibody specific oligonucleotide comprises a binding site for a target sequence, a unique identifier sequence for the antibody that it is associated therewith, and a universal primer sequence, wherein the binding site for a target sequence is at the 3′ region of the antibody specific oligonucleotide;
the antibody is capable of specifically binding to at least one of a plurality of protein targets; and
at least 3 of the plurality of oligonucleotide-associated antibodies comprise different antibodies; and
a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target sequence and a molecular label sequence, wherein the molecular label sequence is from a diverse set of unique molecular label sequences, and wherein the target sequence comprises a poly(dT) region.
2. The composition of claim 1 , wherein the unique identifier sequence is located between the binding site for a target sequence and the universal primer sequence.
3. The composition of claim 1 , wherein the unique identifier sequence is 25 to 45 nucleotides in length.
4. The composition of claim 1 , wherein at least 10 of the plurality of oligonucleotide-associated antibodies comprise different antibodies.
5. The composition of claim 1 , wherein at least 10 of the plurality of oligonucleotide-associated antibodies comprise different unique identifier sequences.
6. The composition of claim 1 , wherein the oligonucleotide is associated with the antibody through a linker.
7. The composition of claim 1 , wherein the oligonucleotide is reversibly associated with the antibody.
8. The composition of claim 1 , wherein the antibody specific oligonucleotide is associated with 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.
9. The composition of claim 1 , wherein the antibody specific oligonucleotide comprises a molecular label sequence, a poly(A) sequence, or a combination thereof.
10. The composition of claim 9 , wherein the antibody specific oligonucleotides of at least 10 of the plurality of oligonucleotide-associated antibodies each comprises a different molecular label sequence.
11. The composition of claim 1 , wherein the antibody specific oligonucleotide comprises a molecular label sequence, a cell label sequence, an amplification adaptor, a sequencing adaptor, a random multimer sequence, or a combination thereof.
12. The composition of claim 1 , further comprising one or more second antibodies which are not associated with an oligonucleotide.
13. The composition of claim 12 , wherein one or more of the antibodies associated with an oligonucleotide in the plurality of oligonucleotide-associated antibodies are the same as one or more of the second antibodies.
14. The composition of claim 1 , wherein the plurality of protein targets comprises 10 to 400 different protein targets.
15. The composition of claim 1 , wherein the binding site for a target sequence comprises a poly(A) sequence.
16. The composition of claim 1 , wherein the protein target is 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 any combination thereof.
17. The composition of claim 1 , wherein the universal primer sequence comprises a sequence of a sequencing primer of a high-throughput sequencing platform.
18. The composition of claim 1 , wherein the antibody is a human antibody or a mouse antibody.
19. The composition of claim 1 , wherein the antibody is an antibody capable of specifically binding to a protein target selected from the group consisting of CD21, HLA-DR, CD8, CD34, ADAM10, CD156c, ANO6, ATP1B2, ATP1B3, BSG, CD147, CD109, CD230, CD29, CD298, ATP1 B3, CD44, CD45, CD47, CD51, CD59, CD63, CD97, CD98, SLC3A2, CLDND1, HLA-ABC, ICAM1, ITFG3, MPZL1, NA K ATPase alpha1, ATP1A1, NPTN, PMCA ATPase, ATP2B1, SLC1A5, SLC29A1, SLC2A1, and SLC44A2.
20. The composition of claim 1 , wherein the antibody specific oligonucleotide comprises a phosphorothioate backbone modification.
21. A kit, comprising:
a first oligonucleotide-associated antibody comprising a first antibody associated with an antibody specific oligonucleotide, wherein
the antibody specific oligonucleotide associated with the first antibody comprises a binding site for a target sequence, a unique identifier sequence for the first antibody, and a universal primer sequence, wherein the binding site for a target sequence is at the 3′ region of the antibody specific oligonucleotide; and
the first antibody is capable of specifically binding to a first protein target; and
a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target sequence and a molecular label sequence, wherein the molecular label sequence is from a diverse set of unique molecular label sequences, and wherein the target sequence comprises a poly(dT) region.
22. The kit of claim 21 , further comprising:
instructions for using the first oligonucleotide-associated antibody with one or more additional oligonucleotide-associated antibodies each comprising an antibody associated with an antibody specific oligonucleotide, wherein
the antibody specific oligonucleotide in each of the one or more additional oligonucleotide-associated antibodies comprises a binding site for a target sequence, a unique identifier sequence for the antibody that it is associated therewith, and a universal primer sequence; and
the antibody in each of the one or more additional oligonucleotide-associated antibodies is capable of specifically binding to a protein target.
23. The kit of claim 22 , wherein the instructions for
using the first oligonucleotide-associated antibody with one or more additional oligonucleotide-associated antibodies comprises instructions for using the first oligonucleotide-associated antibody with a second oligonucleotide-associated antibody, wherein the second oligonucleotide-associated antibody comprises a second antibody associated with an antibody specific oligonucleotide, wherein
the antibody specific oligonucleotide associated with the second antibody comprises a binding site for a target sequence, a unique identifier sequence for the second antibody, and a universal primer sequence; and
the second antibody is capable of specifically binding to a second protein target.
24. The kit of claim 23 , wherein the first protein target and the second protein target are different.
25. The kit of claim 23 , wherein the binding site for a target sequence in the antibody specific oligonucleotide associated with the second antibody is the same as the binding site for a target sequence in the antibody specific oligonucleotide associated with the first antibody.
26. The kit of claim 22 , wherein the antibody in each of the one or more additional oligonucleotide-associated antibodies is capable of specifically binding to a different protein target.
27. The kit of claim 21 , wherein the antibody specific oligonucleotide associated with the first antibody comprises a molecular label sequence, a poly(A) sequence, or a combination thereof.
28. The kit of claim 21 , wherein the first protein target is 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 any combination thereof.
29. The kit of claim 21 , wherein the antibody specific oligonucleotide comprises a phosphorothioate backbone modification.