Multi component detection

Patent metadata

Field	Value
Publication number	US-10577640-B2
Application number	US-201715420488-A
Country	US
Kind code	B2
Filing date	Jan 31, 2017
Priority date	Jul 28, 2011
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

Abstract

The disclosure provides methods for detecting the concurrent presence of at least two targets within a biological sample. The method includes contacting said biological sample with a first binding agent, said first binding agent operably linked to a first sortase molecule, wherein said first binding agent specifically binds to a first target; contacting said biological sample with a second binding agent, said second binding agent operably linked to a first sortase recognition sequence peptide, wherein said second binding agent specifically binds to a second target; adding a sortase substrate under conditions where a first sortase-mediated ligation of the sortase substrate to the first sortase recognition sequence will produce a ligation product, and detecting the ligation product wherein detection of said ligation product indicates the concurrent presence of the first target and the second target in the biological sample. Also disclosed are kits comprising reagents for performing the methods as claimed.

First claim

What is claimed is:

1. A method for detecting the concurrent presence of at least three targets within a biological sample, comprising:
(a) contacting said biological sample with a first binding agent, a second binding agent and a third binding agent,
wherein said first binding agent is operably linked to a first sortase and specifically binds to a first target,
wherein said second binding agent is operably linked to a first sortase recognition sequence and specifically binds to a second target,
and wherein said third binding agent is operably linked to a second sortase and specifically binds to a third target;
(b) adding an intermediate reagent comprising a first sortase substrate conjugated to a second sortase recognition sequence under conditions where a first sortase-mediated ligation of the intermediate reagent to the first sortase recognition sequence will produce an intermediate product,
(c) adding a second sortase substrate under conditions where a second sortase-mediated ligation of the second sortase substrate to the second sortase recognition sequence on the intermediate product will produce a ligation product, and
(d) detecting the ligation product, wherein detection of said ligation product indicates the concurrent presence of the first target, the second target, and the third target in the biological sample.

2. The method of claim 1 , wherein the first sortase is a Sortase A molecule.

3. The method of claim 2 , wherein the Sortase A molecule is from Staphylococcus aureus.

4. The method of claim 1 , wherein the first sortase recognition sequence comprises the amino acid sequence LPXTG, where X is any amino acid residue (SEQ ID NO: 1).

5. The method of claim 1 , wherein the second sortase is a Sortase B molecule.

6. The method of claim 1 , wherein the second sortase recognition sequence comprises the amino acid sequence NPQTN (SEQ ID NO: 2).

7. The method of claim 1 , wherein the first sortase substrate comprises the amino acid sequence GGG.

8. The method of claim 1 , wherein detecting the ligation product performed using a fourth binding agent that specifically binds to the ligation product.

9. The method of claim 8 , wherein the fourth binding agent is detectable.

10. The method of claim 1 , wherein the first sortase is directly attached to the first binding agent.

11. The method of claim 1 , wherein the first sortase is indirectly attached to the first binding agent.

12. The method of claim 1 , wherein the first sortase recognition sequence is directly attached to the second binding agent.

13. The method of claim 1 , wherein the first sortase recognition sequence is indirectly attached to the second binding agent.

14. The method of claim 1 , wherein the second sortase is directly attached to the third binding agent.

15. The method of claim 1 , wherein the second sortase is indirectly attached to the third binding agent.

16. The method of claim 1 , wherein the first target and the second target are on the same molecule.

17. The method of claim 1 , wherein the second sortase substrate is operably linked to a first member of a fourth binding member pair.

18. The method of claim 17 , wherein the ligation product is detected using a second member of the fourth binding member pair.

19. The method of claim 1 , wherein the first binding agent is an antibody.

20. The method of claim 1 , wherein the second binding agent is an antibody.

21. The method of claim 1 , wherein the third binding agent is an antibody.

22. The method of claim 8 , wherein the fourth binding agent is an antibody.

23. The method of claim 1 , wherein the biological sample is selected from the group consisting of a cell, a biopsy sample, a blood sample, a tissue sample, a saliva sample, a tear sample, a semen sample, cerebrospinal fluid sample, a bone marrow sample, a bone marrow sample, and a circulating tumor cell sample.

24. The method of claim 1 , wherein the biological sample is from a human.

25. A kit comprising
a first sortase recognition sequence directly attached to a first member of a first binding member pair,
a first sortase molecule directly attached to a first member of a second binding member pair,
a second sortase molecule directly attached to a first member of a third binding member pair,
a first sortase substrate conjugated to a second sortase recognition sequence,
a second sortase substrate;
and instructions for using the kit to detect the concurrent presence of at least three targets within a biological sample.

26. The kit of claim 25 , wherein the second sortase substrate is directly attached to a first member of a fourth binding member pair.

27. The kit of claim 26 , further comprising a detectable second member of the fourth binding member pair.

28. The kit of claim 25 , wherein the first binding member pair, the second binding member pair and the third binding member pair is each an antibody/antigen pair, where the antigen is an Fc portion of an antibody of a species and the antibody specifically binds to the Fc portion of the antibody of the species, and wherein the species of the first binding member pair, the species of the second binding member pair, and the species of the third binding member pair are different from each other.

29. The method of claim 1 , further comprising, after step (a) and prior to adding the sortase substrate in step (c), removing the first binding agent that is not bound to the first target and removing the second binding agent that is not bound to the second target and removing the third binding agent that is not bound to the third target.

Assignees

• Cell Signaling Technology Inc

Inventors

• Huynh Khanh Duc
• Cheung Wan Cheung
• Polakiewicz Roberto

Classifications

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• G01N33/5306

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N33/56966

  Animal cells · CPC title

• G01N33/50

  Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q) · CPC title