Assays for measurement of protein fragments in biological media

• US 6,030,792 A
• Filed: 11/02/1998
• Issued: 02/29/2000
• Est. Priority Date: 11/13/1997
• Status: Expired due to Fees

• Current Assignee: Pfizer Inc.
• Original Assignee: Pfizer Inc.
• Inventors: Downs, James T., Johnson, Kimberly S., Mezes, Peter S., Otterness, Ivan G.
• Primary Examiner(s): STUCKER, JEFFREY J
• Application Number:
  US09/184,658
• Time in Patent Office: 484 Days
• Field of Search: 435/7.1, 435/7.5, 435/7.7, 435/7.9, 435/7.92-7.95, 435/326, 435/331, 435/975, 435/328, 436/518, 436/531, 436/811, 530/356, 530/387.1, 530/387.9, 530/388.1, 530/388.85, 530/389.1, 530/391.1, 530/391.3, 530/866
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 2800/102 Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 530/866 involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain

Abstract

This invention provides novel antibodies and engineered versions thereof and methodology for monitoring biological media for protein fragments, especially collagen fragments resulting from collagenase cleavage of type II collagen.

Claims

1. A method for monitoring biological media for protein fragments which comprises;

contacting said biological media with a capture antibody;

said capture antibody being active against the sequences set forth in the Sequence Listing as SEQ ID NOS;

1 and 2; and

contacting said biological media with a detection antibody;

said capture antibody being active against the sequences set forth in the Sequence Listing as SEQ ID NOS;

3 and 4; and

detecting the amount of collagen fragments bound to said capture and detection antibodies;

or

contacting said biological media with a capture antibody;

said capture antibody being active against the sequences set forth in the Sequence Listing as SEQ ID NOS;

3 and 4; and

contacting said biological media with a detection antibody;

said detection antibody being active against the sequences set forth in the Sequence Listing as SEQ ID NOS;

1 and 2; and

detecting the amount of collagen fragments bound to said capture and detection antibodies.

2. A method of claim 1 wherein said protein fragments are collagen fragments generated by collagenase cleavage of articular cartilage.

3. A method of claim 2 wherein said protein fragments are generated from collagenase cleavage of type II collagen.

4. A method of claim 2 wherein said capture and detection antibodies are monoclonal antibodies.

5. A method of claim 1 wherein said capture and detection antibodies are monoclonal antibodies.

6. A method of claim 1 wherein said capture antibody is a genetically engineered antibody.

7. A method of claim 1 wherein said detection antibody is a genetically engineered antibody.

8. A method of claim 1 wherein said capture antibody, designated 9A4, has the V_H sequence as set forth in the Sequence Listing as SEQ ID NOS:

5 and 32 and the V_L sequence as set forth in the Sequence Listing as SEQ ID NOS;

6 and 33.

9. A method of claim 1 wherein said detection antibody, designated 5109, has the V_H sequence as set forth in the Sequence Listing as SEQ ID NOS:

10 and 48 and the V_L sequence as set forth in the Sequence Listing as SEQ ID NOS;

11 and 49.

10. An antibody of claim 1 which is labeled to facilitate detection.

11. An antibody of claim 10 wherein said label is radioactive, optical, enzymatic, fluorescent polarizing or fluorescent quenching.

12. An antibody of claim 1 which is labeled to facilitate capture of said antibody.

13. An antibody of claim 12 wherein said label is biotin or magnetic particles.

14. A method for monitoring biological media for protein fragments which comprises;

contacting said biological media with an antibody active against the sequences set forth in the Sequence Listing as SEQ ID NOS;

3 and 4; and

detecting the amount of protein fragments bound to said antibody.

15. A method of claim 14 wherein said protein fragments are collagen fragments.

16. A method of claim 14 wherein said antibody is a monoclonal antibody.

17. A method of claim 14 wherein said antibody is a genetically engineered antibody.

18. A method of claim 14 wherein said antibody, designated 5109, has the V_H sequence as set forth in the Sequence Listing as SEQ ID NOS:

10 and 48 and the V_L sequence as set forth in the Sequence Listing as SEQ ID NOS;

11 and 49.

19. The antibody, designated 9A4, having the V_H sequence as set forth in the Sequence Listing as SEQ ID NOS:

5 and 32 and the V_L sequence as set forth in the Sequence Listing as SEQ ID NOS;

6 and 33.

20. The antibody, designated 5109, having the V_H sequence as set forth in the Sequence Listing as SEQ ID NOS:

10 and 48 and the V_L sequence as set forth in the Sequence Listing as SEQ ID NOS;

11 and 49.

21. A cell line that produces a specific binding partner that binds to peptides consisting essentially of the structures as set forth in the Sequence Listing as SEQ ID NOS:

1 or 2 wherein the cell line is ATCC HB-12436.

22. A cell line that produces a specific binding partner that binds to peptides consisting essentially of the structures as set forth in the Sequence Listing as SEQ ID NOS:

3 or 4 wherein the cell line is ATCC HB-12435.

23. A genetically engineered form of antibody 9A4 such as that expressed by E. coli p9A4ICAT7-1 or p9A4IF-5 deposited as ATCC-98593 and ATCC-98592, respectively.

24. A genetically engineered form of antibody 5109 such as that derived from E. coli p5109CscFv7, which may be substituted for 5109 deposited as ATCC-98594.