Enzyme-catalyzed metal deposition for the enhanced detection of analytes of interest

• US 20050100976A1
• Filed: 12/20/2004
• Published: 05/12/2005
• 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: Farrell, Michael, Bieniarz, Christopher
• Granted Patent: US 7,642,064 B2
• Time in Patent Office: Days
• US Class Current: 435/7.920
• 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 enhanced methods for detecting an analyte of interest in situ, by immunoassay, or by hybridization comprising binding an enzyme-labeled conjugate molecule to an analyte of interest in the presence of a redox-inactive reductive species and a soluble metal ion. The enzyme catalyzes the conversion of the inactive reductive species to an active reducing agent, which in turn reduces the metal ion to a metal atom thereby providing an enhanced means of detecting the analyte via metal deposition.

Claims

1. A method of detecting and quantifying an analyte of interest from a biological sample in an enzyme immunoassay (EIA) comprising the steps of:

(a) contacting a biological sample containing an analyte of interest with a primary antibody immobilized on a solid support, such that the presence of the analyte in the biological sample will result in the formation of a primary antibody-analyte complex;

(b) contacting said complex of step (a) with a secondary antibody conjugated to a label enzyme, such that the presence of analyte in the biological sample will result in a primary antibody-analyte-secondary antibody complex;

(c) contacting the complex formed in step (b) with a redox inactive reductive species in the presence of metal ion so that a metal precipitate will form on the solid support following reduction;

(d) enhancing said metal precipitate; and

(e) detecting and quantifying the amount of analyte in the sample by measuring the amount of metal precipitate formed on the solid support.

2. The method of claim 1, wherein said redox-inactive reductive species is a substrate for said secondary antibody conjugated to a label enzyme.

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

4. The method of claim 2 wherein said enzyme is alkaline phosphatase, and said substrate is ascorbic acid phosphate.

5. The method of claim 2 wherein said enzyme is alkaline phosphatase, and said substrate is a hydroquinone phosphate derivative.

6. The method of claim 5, where said substrate is hydroquinone diphosphate.

7. 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.

8. 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.

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

10. The method of claim 8 wherein said metal ion is silver.

11. The method of claim 10 wherein prior to the reduction of silver metal in step (c), the biological sample is pretreated with gold.

12. The method of claim 1 wherein said EIA is an enzyme-linked immunosorbent assay (ELISA).

13. A method of detecting a nucleic acid sequence of interest in a biological sample comprising the steps of:

(a) labeling DNA isolated from a biological sample with a first member of a specific binding pair;

(b) hybridizing said DNA of step (a) to a microarray of nucleic acid sequences immobilized on a solid support under appropriate hybridization conditions;

(c) incubating said microarray of step (b) with a label enzyme conjugated to a second member of said specific binding pair;

(d) incubating said microarray with a redox inactive reductive species in the presence of metal ion so that a metal precipitate will form on the microarray following reduction;

(e) enhancing said metal precipitate; and

(f) detecting nucleic acid sequences that are complementary to at least one of the microarray of nucleic acid sequences immobilized on the solid support by visualizing the metal precipitate formed on the solid support.

14. The method of claim 13, wherein said enzyme is selected from the group consisting of alkaline phosphatase, acid phosphatase, alpha- and beta-galactosidases, alpha- and beta-glucosidases, esterases generally, and beta-lactamases.

15. The method of claim 13 wherein said enzyme label is alkaline phosphatase, and said redox-inactive reductive species is ascorbic acid phosphate.

16. The method of claim 13 wherein said enzyme label is alkaline phosphatase, and said redox-inactive reductive species is a hydroquinone phosphate derivative.

17. The method of claim 13 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.

18. The method of claim 13 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.

19. The method of claim 13 wherein said metal is selected from the group consisting of silver and gold.

20. The method of claim 19 wherein said metal is silver.

21. The method of claim 20 wherein prior to the reduction of silver metal in step (d), the biological sample is pretreated with gold.

22. A kit for detecting an analyte of interest in a biological sample, said kit comprising one or more containers, each container adapted to hold a specific binding member for the analyte of interest, a redox-inactive reductive species, an enzyme label for rendering said reductive species active, a metal ion, and reagents for metal precipitate enhancement.

23. The kit of claim 22 wherein said specific binding member is immobilized on a solid support.

24. The kit of claim 23 wherein the metal ion is silver ion.

25. The kit of claim 22 wherein said specific binding member for the analyte of interest is an antibody.

26. The kit of claim 22 wherein said specific binding member for the analyte of interest is an oligonucleotide sequence.

27. The kit of claim 22 wherein said enzyme label is a phosphatase.

28. The kit of claim 27 wherein said phosphatase is alkaline phosphatase.

29. The kit of claim 28 wherein said redox-inactive reductive species is ascorbic acid phosphate.

30. The kit of claim 22 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.

31. The kit of claim 22 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.

32. The kit of claim 22 wherein said metal ion is selected from the group consisting of silver ion and gold ion.

33. The kit of claim 32 wherein said metal ion is silver ion.

34. The kit of claim 33 wherein said reagents for metal precipitate enhancement comprise reagents from Sigma'"'"'s Silver Enhancer Kit.