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.
- 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):
- 15. A compound having the general structure (VI):
- 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.
- 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.
- 33. The compound of claim 14 wherein said Z is phosphate.
- 34. The compound of claim 15 wherein said Z is phosphate.
- 35. The compound of claim 16 wherein said Z is phosphate and said R1 is methyl.
- 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.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application 60/482,596 filed Jun. 24, 2003, the contents of which are incorporated herein in their entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60482596 |
Jun 2003 |
US |