MCM6 and MCM7 monoclonal antibodies and methods for their use in the detection of cervical disease

Abstract

Compositions and methods for diagnosing high-grade cervical disease in a patient sample are provided. The compositions include novel monoclonal antibodies, and variants and fragments thereof, that specifically bind to MCM6 or MCM7. Monoclonal antibodies having the binding characteristics of an MCM6 or MCM7 antibody of the invention are further provided. Hybridoma cell lines that produce an MCM6 or MCM7 monoclonal antibody of the invention are also disclosed herein. The compositions find use in practicing methods for diagnosing high-grade cervical disease comprising detecting overexpression of MCM6, MCM7, or both MCM6 and MCM7 in a cervical sample from a patient. Kits for practicing the methods of the invention are further provided. Polypeptides comprising the amino acid sequence for an MCM6 or an MCM7 epitope and methods of using these polypeptides in the production of antibodies are also encompassed by the present invention.

Description

Claims

1. A monoclonal antibody that is capable of specifically binding to MCM7, or a variant or fragment thereof, wherein the antibody is selected from the group consisting of: (a) the monoclonal antibody produced by the hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669;(b) a monoclonal antibody having the binding characteristics of the monoclonal antibody produced by the hybridoma cell line 2E6.2;(c) a monoclonal antibody that binds to an epitope capable of binding the monoclonal antibody produced by the hybridoma cell line 2E6.2;(d) a monoclonal antibody that binds to an epitope comprising the amino acid sequence set forth in SEQ ID NO:6;(e) a monoclonal antibody that competes in a competitive binding assay with the monoclonal antibody produced by the hybridoma cell line 2E6.2; and,(f) a monoclonal antibody that is an antigen binding fragment of a monoclonal antibody of (a)-(e), wherein the fragment retains the capability of specifically binding to MCM7, or a variant or fragment thereof.

2. The hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669.

3. A hybridoma cell line capable of producing a monoclonal antibody of claim 1.

4. A monoclonal antibody that is capable of specifically binding to MCM6, or a variant or fragment thereof, wherein the antibody is selected from the group consisting of: (a) the monoclonal antibody produced by the hybridoma cell line 9D4.3, deposited with the ATCC as Patent Deposit No. PTA-6911;(b) a monoclonal antibody having the binding characteristics of the monoclonal antibody produced by the hybridoma cell line 9D4.3;(c) a monoclonal antibody that binds to an epitope capable of binding the monoclonal antibody produced by the hybridoma cell line 9D4.3;(d) a monoclonal antibody that binds to an epitope comprising the amino acid sequence set forth in SEQ ID NO:5;(e) a monoclonal antibody that competes in a competitive binding assay with the monoclonal antibody produced by the hybridoma cell line 9D4.3; and,(f) a monoclonal antibody that is an antigen binding fragment of a monoclonal antibody of (a)-(e), wherein the fragment retains the capability of specifically binding to MCM6, or a variant or fragment thereof.

5. The hybridoma cell line 9D4.3, deposited with the ATCC as Patent Deposit No. PTA-6911.

6. A hybridoma cell line capable of producing a monoclonal antibody of claim 4.

7. A kit for diagnosing high-grade cervical disease comprising at least one monoclonal antibody according to claim 1.

8. The kit of claim 7, wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669.

9. A kit for diagnosing high-grade cervical disease comprising at least one monoclonal antibody according to claim 4.

10. The kit of claim 9, wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 9D4.3, deposited with the ATCC as Patent Deposit No. PTA-6911.

11. A kit comprising at least at least two monoclonal antibodies, wherein a first antibody is the MCM7 monoclonal antibody produced by the hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669, and a second antibody is the MCM6 monoclonal antibody produced by the hybridoma cell line 9D4.3, deposited with the ATCC as Patent Deposit No. PTA-6911.

12. The kit of claim 11, wherein each antibody is provided as a separate antibody reagent or as an antibody cocktail.

13. The kit according to claim 7, wherein said kit further comprises a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use.

14. The kit according to claim 7 further comprising reagents for Pap staining.

15. A method for diagnosing high-grade cervical disease in a patient, the method comprising: a) obtaining a cervical sample from the patient;b) contacting the sample with at least one monoclonal antibody according to claim 1 that specifically binds to MCM7; and,c) detecting binding of the antibody to MCM7.

16. The method of claim 15, wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669.

17. A method for diagnosing high-grade cervical disease in a patient, the method comprising: a) obtaining a cervical sample from the patient;b) contacting the sample with at least one monoclonal antibody according to claim 4 that specifically binds to MCM6; and,c) detecting binding of the antibody to MCM6.

18. The method of claim 17, wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 9D4.3, deposited with the ATCC as Patent Deposit No. PTA-6911.

19. The method of claim 17 further comprising contacting the sample with at least one monoclonal antibody that specifically binds to MCM7.

20. The method of claim 19, wherein the monoclonal antibody is the monoclonal antibody produced by the hybridoma cell line 2E6.2, deposited with the ATCC as Patent Deposit No. PTA-6669.

21. The method according to claim 19, wherein the antibodies are contacted with the sample sequentially as individual antibody reagents or as an antibody cocktail.

22. An isolated polypeptide comprising an epitope for binding an MCM7 monoclonal antibody, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6; and,(b) a polypeptide having at least 90% sequence identity to SEQ ID NO:6, wherein the polypeptide has antigenic activity.

23. An isolated polypeptide comprising an epitope for binding an MCM6 monoclonal antibody, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5; and,(b) a polypeptide having at least 90% sequence identity to SEQ ID NO:5, wherein the polypeptide has antigenic activity.

24. A method for producing an MCM7 antibody comprising immunizing an animal with a polypeptide according to claim 22.

25. A method for producing an MCM7 monoclonal antibody comprising: (a) immunizing an animal with a polypeptide according to claim 22 under conditions to elicit an immune response;(b) isolating antibody-producing cells from the animal;(c) fusing the antibody-producing cells with immortalized cells in culture to form monoclonal antibody-producing hybridoma cells;(d) culturing the hybridoma cells; and,(e) isolating monoclonal antibodies from culture.

26. A method for producing an MCM6 antibody comprising immunizing an animal with a polypeptide according to claim 2.

27. A method for producing an MCM6 monoclonal antibody comprising: (a) immunizing an animal with a polypeptide according to claim 23 under conditions to elicit an immune response;(b) isolating antibody-producing cells from the animal;(c) fusing the antibody-producing cells with immortalized cells in culture to form monoclonal antibody-producing hybridoma cells;(d) culturing the hybridoma cells; and,(e) isolating monoclonal antibodies from culture.