Method for the diagnosis and prognosis of malignant diseases

• US 20030194754A1
• Filed: 04/08/2002
• Published: 10/16/2003
• Est. Priority Date: 04/08/2002
• Status: Active Grant

• Current Assignee: University of Louisville
• Original Assignee: University of Louisville
• Inventors: Bates, Paula J., Xu, Xiaohua, Trent, John O., Miller, Donald M.
• Granted Patent: US 7,541,150 B2
• Time in Patent Office: Days
• US Class Current: 435/7.23
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

Methods for the diagnosis of cancer by determining the neoplastic status of a cell by probing the cell plasma membrane for the presence of nucleolin are provided, as are kits to carry out such tests.

Claims

1. A method of determining a neoplastic state of a cell, comprising:

detecting the presence of plasma membrane nucleolin in the cell.

2. The method of claim 1, wherein the cell is mammalian.

3. The method of claim 2, wherein the cell is monkey, dog, cat, rabbit, cow, pig, goat, guinea pig, mouse, rat or sheep.

4. The method of claim 2, wherein the cell is human.

5. The method of claim 1, wherein the cell is lysed.

6. The method of claim 1 wherein the detecting comprises detecting nucleolin-anti-nucleolin agent complex.

7. The method of claim 6, wherein the anti-nucleolin agent is a monoclonal antibody that specifically binds nucleolin.

8. The method of claim 7, wherein the antibody is p7-1A4 mouse monoclonal antibody, sc-8031, sc-9893, sc-9892, 4E2 or 3G4B2.

9. The method of claim 6, wherein the anti-nucleolin agent is a nucleolin-binding oligonucleotide.

10. The method of claim 9, wherein the oligonucleotide is a guanosine-rich oligonucleotide.

11. The method of claim 10, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:

1-7;

9-17;

19-30 or 31.

12. The method of claim 6, wherein the complex is detected by detecting fluorescence, an enzyme, or radioactivity.

37. A method of diagnosing pre-malignant or malignant cells, comprising:

collecting a sample from a subject comprising lung cells;

sending the sample to a testing center;

determining the neoplastic state of the cells by the method of claim 1.

13. A method of determining a neoplastic state of a cell, comprising quantifying the amount of plasma membrane nucleolin in the cell.

14. The method of claim 13, wherein the cell is mammalian.

15. The method of claim 14, wherein the cell is monkey, dog, cat, rabbit, cow, pig, goat, guinea pig, mouse, rat or sheep.

16. The method of claim 15, wherein the cell is human.

17. The method of claim 13, wherein the cell is lysed.

18. The method of claim 13 wherein the quantifying comprises quantifying nucleolin-anti-nucleolin agent complex.

19. The method of claim 18, wherein the anti-nucleolin agent is a monoclonal antibody.

20. The method of claim 18, wherein the antibody is p7-1A4 mouse monoclonal antibody, sc-8031, sc-9893, sc-9892, 4E2 or 3G4B2.

21. The method of claim 18, wherein the anti-nucleolin agent is nucleolin-binding oligonucleotide.

22. The method of claim 21, wherein the oligonucleotide is a guanosine-rich oligonucleotide.

23. The method of claim 22, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:

1-7;

9-17;

19-30 or 31.

24. The method of claim 23, wherein the complex is quantified by quantifying fluorescence, an enzyme, or radioactivity.

25. A kit for determining a neoplastic state of a cell, comprising:

an anti-nucleolin agent and

a control sample.

26. The kit of claim 25, further comprising a second agent that detects a nucleolin-anti-nucleolin complex.

27. The kit of claim 26, wherein the anti-nucleolin agent is an anti-nucleolin antibody, and the second agent is a labeled secondary antibody.

28. The kit of claim 27, wherein the label is at least one enzyme, fluorophore, radioisotope or gold particle.

29. The kit of claim 28, wherein the anti-nucleolin agent comprises biotin, and the second agent comprises avidin or streptavidin.

30. The kit of claim 28, wherein the anti-nucleolin agent comprises digoxigenin, and the second agent comprises a labeled anti-digoxigenin antibody.

31. The kit of claim 25, further comprising at least one member selected from the group consisting of a fixative, a buffer, plasticware, serum, serum albumin, non-fat milk, membranes and instructions.

32. The kit of claim 25, wherein the anti-nucleolin agentt is labeled.

33. The kit of claim 32, wherein the label is at least one enzyme, fluorophore, radioisotope or gold particle.

34. A method of detecting small lung cell carcinoma in a subject, comprising:

collecting a sample from the subject containing lung cells; and

detecting the presence of nucleolin on the surface of the cells.

35. The method of claim 34, wherein the sample is sputum.

36. The method of claim 34, wherein the subject is human.

38. The method of claim 38, wherein the subject is human.

39. The method of claim 39, wherein the sample is sputum.

40. The method of claim 39, wherein the diagnosing is diagnosing lung small cell carcinoma.