Claims for Patent: 7,691,598

Summary for Patent: 7,691,598

Title:	Method for detecting a target molecule by metal deposition
Abstract:	The present invention provides methods for the use of enzymes to selectively deposit metal to the vicinity of a target molecule. The invention also relates to applications of enzymatic metal deposition to sensitively and selectively detect target molecules such as biomarkers in various biological samples, such as chromogenic immunohistochemical (IHC) detection in situ by using bright field light microscope.
Inventor(s):	Hainfeld; James F. (Shoreham, NY), Liu; Wenqiu (Miller Place, NY)
Assignee:	Nanoprobes, Inc. (Yaphank, NY)
Application Number:	11/714,682
Patent Claims:	1. A method for detecting a target molecule in a test sample, comprising: binding an enzyme to the target molecule in the test sample; combining an enzyme with metal ions, an oxidizing agent and a reducing agent; incubating the enzyme with the metal ions in the presence of the oxidizing agent and the reducing agent, whereby the metal ions are reduced to elemental metal; depositing the elemental metal in the vicinity of the enzyme; and determining the presence, amount or level of the deposited metal in the vicinity of the enzyme by visualizing said deposited metal through a microscope thereby detecting said target molecule. 2. The method of claim 1, wherein the metal ions are selected from the group consisting of silver, gold, iron, mercury, nickel, copper, platinum, palladium, cobalt, iridium ions and a mixture thereof. 3. The method of claim 1, wherein the metal ions are silver ions. 4. The method of claim 1, wherein the enzyme is an oxido-reductase. 5. The method of claim 1, wherein the enzyme is peroxidase. 6. The method of claim 1, wherein the enzyme is horseradish peroxidase. 7. The method of claim 1, wherein the enzyme is conjugated to streptavidin. 8. The method of claim 1, wherein the enzyme is conjugated to an antibody. 9. The method of claim 1, wherein the oxidizing agent is an oxygen-containing oxidizing agent. 10. The method of claim 1, wherein the reducing agent is selected from the group consisting of hydroquinone, a hydroquinone derivative, n-propyl gallate, 4-methylaminophenol sulfate, 1,4 phenylenediamine, o-phenylenediamine, chloroquinone, bromoquinone, 2-methoxyhydroquinone, hydrazine, 1-phenyl-3-pyrazolidinone and dithionite salts. 11. The method of claim 1, wherein the metal ions act as a substrate for the enzyme. 12. The method of claim 1, wherein the enzyme is peroxidase and the metal ions are incubated with the peroxidase in the absence of an organic substrate of the peroxidase. 13. The method of claim 12, wherein the organic substrate is a colorless organic substrate that is converted by the peroxidase to a colored precipitate. 14. The method of claim 13, wherein the colorless organic substrate is 3,3'-diaminobenzidine or 5-bromo-4-chloro-3-indolyl phosphate. 15. The method of claim 1, wherein the step of binding includes binding the enzyme to the target molecule via a primary antibody that specifically binds to the target molecule, and a secondary antibody that is conjugated with the enzyme and binds to the primary antibody. 16. The method of claim 1, wherein the step of binding includes binding the enzyme to the target molecule via a nucleic acid probe that specifically hybridizes to the target molecule and is labeled with detectable marker, wherein the enzyme binds to the detectable marker via an antibody that specifically binds to the detectable marker. 17. The method of claim 16, wherein the detectable marker is biotin, dinitrophenyl, a radio-isotope or a fluorescent label. 18. The method of claim 17, wherein the fluorescent label is selected from the group consisting of fluorescein isothiocyanate (FTIC), Texas Red, rhodamine and Cy5. 19. The method of claim 16, wherein the enzyme binds to the detectable marker via a primary antibody that specifically binds to the detectable marker, and a secondary antibody that is conjugated with the enzyme and binds to the primary antibody. 20. The method of claim 1, wherein the target molecule is a gene, a gene product, or a genome. 21. The method of claim 20, wherein the gene is a gene encoding an angiogenic growth factor receptor selected from the group consisting of receptor for fibrin (VE-cadherin), receptors for VEGF (Flt1 and KDR), receptor for VEGF-C and VEGF-D (Flt4), receptor for VEGF-165 (NP-1 and NP-2), receptors for angiopoeitin-1, -2, -3, and -4 (Tie1 and Tie 2), receptors for FGF (FGF-R1, -R2, -R3 and -R4), receptor for PDGF (PDGF-R), receptor for ephrine A1-5 (Eph A 1-8), and receptor for ephrine B1-5 (Eph B1-8). 22. The method of claim 20, wherein the gene is a gene encoding a receptor tyrosine kinase selected from the group consisting of epidermal growth factor receptors (EGFR), platelet-derived growth factor receptors (PDGFR), vascular endothelial growth factor receptors (VEGFR), nerve growth factor receptors (NGFR), fibroblast growth factor receptors (FGFR), insulin receptors, ephrin receptors, Met, and Ror. 23. The method of claim 22, wherein the epidermal growth factor receptor is HER1, HER2/neu (or HER2/neu), HER3, or HER4. 24. The method of claim 20, wherein the gene is a gene encoding a non-receptor tyrosine kinase selected from the group consisting of Kit, Src, Fes, JAK, Fak, Btk, Syk/ZAP-70, and Ab1. 25. The method of claim 24, wherein the Kit is c-Kit. 26. The method of claim 1, further comprising: comparing the presence, amount or level of the deposited metal with that of a reference sample; and determining a disease status of a patient from whom the test sample is derived. 27. The method of claim 26, wherein the reference sample comprises a cell or tissue from a normal, healthy tissue. 28. The method of claim 26, wherein the disease status is disease determination or classification, prognosis, drug efficacy, patient responsiveness to therapy, whether adjuvant or combination therapy is recommended, or likelihood of recurrence of disease. 29. The method of claim 26, wherein the disease is selected from the group consisting of benign tumors, cancer, hematological disorders, autoimmune diseases, inflammatory diseases, cardiovascular diseases, nerve degenerative diseases and diabetes. 30. The method of claim 26, wherein the disease status is patient responsive to therapy. 31. The method of claim 30, wherein the disease is breast cancer; the therapy is trastruzumab or HERCEPTIN therapy; and the target molecule is HER2/neu gene or protein. 32. The method of claim 30, wherein the disease is gastrointestinal stromal tumor (GIST); the therapy is imatinib mesylate or GLEEVEC therapy; and the target molecule is c-Kit gene or protein.

Details for Patent 7,691,598

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	09/25/1998		2021-03-30
Genentech, Inc.	HERCEPTIN	trastuzumab	For Injection	103792	02/10/2017		2021-03-30