Use of antibody-surrogate antigen systems for detection of analytes

• US 9,575,069 B2
• Filed: 11/08/2013
• Issued: 02/21/2017
• Est. Priority Date: 03/15/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven, Lam, Joe Y., Ma, Zhaochun, Mullah, Khairuzzaman Bashar
• Primary Examiner(s): Lukton, David
• Application Number:
  US14/074,931
• Publication Number:
  US 20150198605A1
• Time in Patent Office: 1,201 Days
• Field of Search: 435/4
• US Class Current: 1/1
• CPC Class Codes: C07D 311/88 Nitrogen atoms
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Compositions, methods and kits for labeling proteins and uses in reporter systems for detecting, quantifying and/or characterizing analytes.

Claims

1. A labeling molecule useful for specifically labeling a protein comprising:

(a) a target moiety;

(b) a label moiety;

(c) a photo-activatable reactive moiety; and

(d) a cleavable tether linking the target moiety to the label moiety;

wherein the target moiety, label moiety, and reactive moiety are connected to one another such that the target moiety can bind the protein and the reactive moiety can crosslink the labeling molecule to the protein, and wherein the cleavable tether can be cleaved to allow detachment of the target moiety from the protein thereby resulting in a labeled moiety.

2. The labeling molecule of claim 1 in which the protein is an antibody and the target moiety is an antigen comprising an epitope recognized by said antibody.

3. The labeling molecule of claim 2 which has the structure:

TM-(L)_n-CT-(L)_n-LM-(L)_n-RM

wherein;

TM represents the target moiety;

CT represents the cleavable tether;

LM represents the label moiety;

RM represents the reactive moiety;

L represents an optional linker moiety; and

n is an integer from 0 to 5.

4. The labeling molecule of claim 3 in which the label moiety comprises a fluorescent moiety.

5. The labeling molecule of claim 4 in which the fluorescent moiety comprises a xanthene dye.

6. The labeling molecule of claim 5 in which the xanthene dye is selected from a fluorescein dye and a rhodamine dye.

7. The labeling molecule of claim 3 in which the label moiety comprises a quenching moiety.

8. The labeling molecule of claim 7 in which the quenching moiety is capable of quenching the fluorescence of a fluorescent moiety.

9. The labeling molecule of claim 3 in which the reactive moiety is specifically photo-activatable.

10. The labeling molecule of claim 1, wherein the reactive moiety comprises a benzophenone.

11. The labeling molecule of claim 3 in which the reactive moiety is linked to the label moiety via a linker moiety.

12. The labeling molecule of claim 11 in which the linker moiety comprises one or more nitrogen atoms.

13. The labeling molecule of claim 3 in which the cleavable tether comprises a cleavage moiety linked to one or more linker moieties, said cleavage moiety comprising a linkage that can be cleaved by a chemical cleaving agent.

14. The labeling molecule of claim 13 in which the chemical cleaving agent is fluoride.

15. The labeling molecule of claim 13 in which the cleavable tether has the structure:

(TE)_nCM(TE)_n

wherein;

CM represents the cleavage moiety;

TE represents an optional tether element; and

n is an integer from 0 to 5.

16. The labeling molecule of claim 15 in which tether elements are the same.

17. The labeling molecule of claim 15 in which tether elements are different.

18. The labeling molecule of claim 15 in which the tether elements comprise a substituted or unsubstituted, saturated or unsaturated hydrocarbon, having from 2 to 25 carbon atoms.

19. The labeling molecule of claim 15 in which the tether elements comprise a substituted or unsubstituted, saturated or unsaturated hydrocarbon, having from 2 to 10 carbon atoms.

20. The labeling molecule of claim 18 or 19 in which the tether elements are a linear, branched or cyclic, saturated or unsaturated alkyl.

21. The labeling molecule of claim 20 in which the tether elements is fully saturated n-alkyl.

22. The labeling molecule of claim 18 or 19 further comprising a substituted or unsubstituted heteroatom.

23. The labeling molecule of claim 15 in which the cleavable moiety comprises the structure:

24. The labeling molecule of claim 1 in which the label moiety, reactive moiety, and target moiety, are linked to one another via a multivalent linker.

25. The labeling molecule of claim 1, wherein the protein to be labeled comprises an enzyme having an active site, wherein said target moiety binds to the enzyme without interfering with the binding of a substrate to the active site of the enzyme.

26. The labeling molecule of claim 1, wherein the protein to be labeled comprises a receptor, wherein said target moiety binds to the receptor without interfering with the binding of a ligand to the receptor.

27. The labeling molecule of claim 1, wherein the protein to be labeled is a signal transduction protein, wherein said target moiety binds to signal transduction protein without interfering with the binding of a ligand to the signal transduction protein.