Methods for detecting targets

• US 20090305307A1
• Filed: 09/09/2005
• Published: 12/10/2009
• Est. Priority Date: 09/10/2004
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Sherwood, Steven W.
• Granted Patent: US 8,906,637 B2
• Time in Patent Office: Days
• US Class Current: 435/7.210
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

The present invention provides method and compositions for detecting target molecules present on cells and tissues. In particular, the methods involve adding primary antibodies such as scFv-targeted lactamase that are directed against a target of interest (e.g., cancer markers) to a tissue sample, followed by adding a lactam-containing compound and finally a lactamase reporter system. In some preferred embodiments, the lactamase reporters are fluorescent reporters that bind to the test tissue. In some particularly preferred embodiments, the test tissue contains at least once cancer cell and/or at least one cancer-associated marker.

Claims

1. A method for detecting at least one target on at least one cell, comprising the steps of:

a) providing at least one test cell having at least one target;

b) exposing said at least one test cell to a targeted agent that binds to said target, wherein said targeted agent is conjugated to an enzyme, to produce a bound target, and further wherein said enzyme is capable of cleaving a substrate to produce a detectable product;

c) exposing said bound target to said substrate under conditions such that said enzyme cleaves said substrate to produce a product; and

d) detecting said product.

2. The method of claim 1, wherein said at least one test cell is obtained from an animal.

3. The method of claim 2, wherein said at least one test cell is a human cell.

4. The method of claim 1, wherein said targeted agent is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and fragments of antibodies.

5. The method of claim 4, wherein said targeted agent is biotinylated.

6. The method of claim 1, wherein said substrate is biotinylated.

7. The method of claim 1, wherein said at least one target comprises at least one cancer marker.

8. The method of claim 1, wherein said at least one cell is in vitro, in vivo, ex vivo, or in situ.

9. A method for detecting at least one target on at least one cell, comprising the steps of:

a) providing at least one test cell having at least one target;

b) exposing said at least one test cell to a targeted agent that binds to said target, wherein said targeted agent is conjugated to a reporter molecule, to produce a bound target;

c) exposing said bound target to an enzyme to produce a targeted complex, wherein said enzyme is capable of cleaving a substrate to produce a detectable product;

d) exposing said targeted complex to said substrate under conditions such that said enzyme cleaves said substrate to produce a product; and

e) detecting said product.

10. The method of claim 9, wherein said at least one test cell is obtained from an animal.

11. The method of claim 10, wherein said at least one test cell is a human cell.

12. The method of claim 9, wherein said targeted agent is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and fragments of antibodies.

13. The method of claim 10, wherein said targeted agent is biotinylated.

14. The method of claim 9, wherein said substrate is biotinylated.

15. The method of claim 9, wherein said enzyme is beta-lactamase.

16. The method of claim 9, wherein said enzyme is labelled.

17. The method of claim 9, wherein said enzyme is biotinylated.

18. The method of claim 9, wherein said enzyme binds to said targeted complex due to binding between biotin and streptavidin/avidin.

19. The method of claim 9, wherein said at least one cell is in vitro, in vivo, ex vivo, or in situ.

20. The method of claim 9, wherein said targeted agent and said enzyme are coupled to produce a fusion protein.