Enzyme-catalyzed metal deposition for the enhanced in situ detection of immunohistochemical epitopes and nucleic acid sequences

• US 20040265922A1
• Filed: 06/24/2004
• Published: 12/30/2004
• Est. Priority Date: 06/24/2003
• Status: Active Grant

• Current Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Original Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Inventors: Rodgers, Paula M., Wong, Jennifer, Bieniarz, Christopher, Kernag, Casey A., Kosmeder, Jerome W.
• Granted Patent: US 7,632,652 B2
• Time in Patent Office: Days
• US Class Current: 435/7.5
• CPC Class Codes: C07F 9/093 Polyol derivatives esterified at least twice by phosphoric acid groups
• CPC Class Codes: C07F 9/65517 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07H 15/20 Carbocyclic rings
• CPC Class Codes: C07H 15/24 Condensed ring systems having three or more rings
• CPC Class Codes: C07H 15/26 Acyclic or carbocyclic radicals, substituted by hetero rings
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The invention is directed to novel compositions of matter and methods of detecting in situ an immunohistochemical epitope or nucleic acid sequence of interest in a biological sample comprising binding an enzyme-labeled conjugate molecule to the epitope or sequence of interest in the presence of a redox-inactive reductive species and a soluble metal ion, thereby facilitating the reduction of the metal ion to a metal atom at or about the point where the enzyme is anchored. Novel phosphate derivatives of reducing agents are described that when exposed to a phosphatase are activated to their reducing form, thereby reducing metal ions to insoluble metal.

Claims

1. A method of detecting in situ an immunohistochemical epitope or nucleic acid sequence of interest in a biological sample comprising binding an enzyme-labeled conjugate molecule to the epitope or sequence of interest in the presence of a redox-inactive reductive species and a soluble metal ion, thereby facilitating the reduction of the metal ion to a metal atom at or about the point where the enzyme is anchored.

2. The method of claim 1 wherein said enzyme-labeled conjugate molecule comprises an antibody.

3. The method of claim 1 wherein said enzyme-labeled conjugate molecule comprises avidin or streptavidin.

4. The method of claim 1 wherein said enzyme-labeled conjugate molecule comprises a nucleotide sequence.

5. The method of claim 1 wherein said redox-inactive reductive species is a substrate for said enzyme-labeled conjugate molecule.

6. The method of claim 1 wherein said enzyme is selected from the group consisting of alkaline phospatase, acid phosphatase, alpha- and beta-galactosidases, alpha- and beta-glucosidases, esterases generally,.and beta-lactamases.

7. The method of claim 5 wherein said enzyme is alkaline phosphatase, and said substrate is ascorbic acid phosphate.

8. The method of claim 5 wherein said enzyme is alkaline phosphatase, and said substrate is a hydroquinone phosphate derivative.

9. The method of claim 1 wherein said redox-inactive reductive species is selected from the group consisting of hydroquinone mono- and di-phosphates, naphthohydroquinone mono- and di-phosphates, and anthrahydroquinone mono- and di-phosphates, or a derivative thereof.

10. The method of claim 1 wherein said redox-inactive reductive species is selected from the group consisting of sesamol phosphate, eugenol phosphate and alpha-tocopherol phosphate, or a derivative thereof.

11. The method of claim 1 wherein said metal is selected from the group consisting of silver and gold.

36. The method of claim 1 wherein the step of pre-treating the biological sample with gold is performed prior to the reduction of silver metal.

12. A compound having the general structure (IV) shown below:

13. The compound of claim 12 wherein both Zs are PO₃⁻

2.

14. A compound having the general structure (V):

33. The compound of claim 14 wherein said Z is phosphate.

15. A compound having the general structure (VI):

34. The compound of claim 15 wherein said Z is phosphate.

16. A compound having the general formula (VII):

17. The compound of claim 16 wherein R₁ is CH₂—

(CH₂—

CH₂—

CH(CH₃)—

CH₂)₃—

H.

35. The compound of claim 16 wherein said Z is phosphate and said R₁ is methyl.

18. A kit for detecting a biomarker of interest in a biological sample, comprising one or more containers, each container adapted to hold an anti-biomarker conjugate molecule, a redox-inactive reductive species, an enzyme for rendering said reductive species active, and a metal ion.

19. The kit of claim 18 wherein said anti-biomarker conjugate molecule is an antibody.

20. The kit of claim 19 wherein said antibody is labeled with a hapten.

21. The kit of claim 18 wherein said anti-biomarker conjugate molecule is an oligonucleotide sequence.

22. The kit of claim 21 wherein said oligonucleotide sequence is labeled with a hapten.

23. The kit of claim 18 wherein said enzyme label is a phosphatase.

24. The kit of claim 18 wherein said redox-inactive reductive species is ascorbic acid phosphate.

25. A method of in situ staining a biological sample having an epitope or nucleotide sequence of interest, comprising the steps of:

(a) contacting said tissue with a conjugate molecule having a hapten;

(b) contacting said hapten with a hapten-binding partner conjugated to a label enzyme; and

(c) contacting said biological sample with a redox-inactive reductive species that is a substrate for said label enzyme in the presence of a metal ion.

26. The method of claim 25 wherein said conjugate molecule is selected from the group consisting of antibodies, nucleotide sequences and affinity partners.

27. The method of claim 25 wherein said hapten is selected from the group consisting of fluoroscein, biotin, digoxigenin and dinitrophenol.

28. The method of claim 25 wherein said enzyme label is a phosphatase.

29. The method of claim 25 wherein said metal is selected from the group consisting of silver and gold.

30. The method of claim 25 wherein the step of pre-treating the biological sample with gold is performed prior to the reduction of silver metal.

31. The method of claim 25 wherein said redox-inactive reductive species is selected from the group consisting of hydroquinone mono- and/or di-phosphates, naphthohydroquinone mono- and/or di-phosphates, anthrahydroquinone mono- and/or di-phosphates, sesamol phosphate, eugenol phosphate and alpha-tocopherol phosphate, or a derivative thereof.

32. A method of in situ staining a biological sample having an epitope or nucleotide sequence of interest, comprising the steps of:

(a) contacting said tissue with a biotinylated primary antibody;

(b) contacting said biological sample having said biotinylated primary antibody bound to it with streptavidin-alkaline phosphatase; and

(c) contacting said biological sample of step (b) with ascorbic acid phosphate in the presence of silver ion at a pH greater than 7.