Immunoassay for Collagen Type VI

• US 20190309055A1
• Filed: 06/05/2019
• Published: 10/10/2019
• Est. Priority Date: 04/01/2015
• Status: Active Grant

• Current Assignee: Nordic Bioscience A/S (C.C. Consulting A/S)
• Original Assignee: Nordic Bioscience A/S (C.C. Consulting A/S)
• Inventors: Nedergaard, Anders, Sand, Jannie Marie, Sun, Shu, Oersnes-Leeming, Diana Julie, Henriksen, Kim
• Granted Patent: US 11,634,479 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/32 specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention provides an immunological binding partner reactive with a C-terminal epitope of the C5 domain of the α3 chain of collagen Type 6, and a method of immunoassay using the immunological binding partner for detecting and quantifying the C-terminal epitope. The invention also provides a method of investigating the rate of formation of extracellular matrix and a method for identifying a subject suitable for treatment with an insulin sensitizer.

Claims

1. An immunological binding partner reactive with a C-terminal epitope of the C5 domain of the α

3 chain of collagen Type 6.

2. The immunological binding partner as claimed in claim 1, wherein said immunological binding partner specifically binds to a said C-terminal epitope comprised in a C-terminal amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1).

3. The immunological binding partner as claimed in claim 1, wherein said immunological binding partner is a monoclonal or polyclonal antibody.

4. The immunological binding partner as claimed in claim 1, wherein said immunological binding partner does not recognise or specifically bind an elongated version of said C-terminal amino acid sequence which is . . . KPGVISVMGTA-COOH (SEQ ID. NO:

2).

5. The immunological binding partner as claimed in claim 1, wherein the ratio of the affinity of said immunological binding partner for amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1) to the affinity of said immunological binding partner for elongated amino acid sequence . . . KPGVISVMGTA-COOH (SEQ ID. NO;

2) is greater than 10 to 1.

6. The immunological binding partner as claimed in claim 1, wherein said immunological binding partner does not recognise or specifically bind a truncated version of said C-terminal amino acid sequence which is . . . KPGVISVMG-COOH (SEQ ID. NO:

3).

7. The immunological binding partner as claimed in claim 1, wherein the ratio of the affinity of said immunological binding partner for amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1) to the affinity of said immunological binding partner for truncated amino acid sequence . . . KPGVISVMG-COOH (SEQ ID. NO;

3) is greater than 10 to 1.

8. A method of immunoassay for detecting in a sample a C-terminal epitope of the C5 domain of the α

3 chain of collagen type VI, wherein said method comprises contacting a sample comprising said C-terminal epitope of the α

3 chain of collagen type VI with an immunological binding partner as claimed in claim 1, and determining the amount of binding of said immunological binding partner.

9. The method as claimed in claim 8, wherein said C-terminal epitope is comprised in a C-terminal amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1).

10. The method as claimed in claim 8, wherein said method is used to quantify the amount of said C-terminal epitope of the α

3 chain of collagen type VI in a biofluid.

11. The method as claimed in claim 10, wherein said biofluid is serum, plasma, urine or amniotic fluid.

12. The method as claimed in claim 8, wherein said immunoassay is a competition assay or a sandwich assay.

13. The method as claimed in claim 12, wherein said immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

14. The method as claimed in claim 8, further comprising correlating the quantity of said C-terminal epitope of the α

3 chain of collagen type VI determined by said method with standard normal values of said C-terminal epitope of the α

3 chain of collagen type VI to evaluate a change thereof from normal levels.

15. A method of investigating the rate of formation of extracellular matrix comprising conducting an assay by a method as claimed in claim 10 to obtain a measure of the level in a biofluid sample of collagen type VI α

3 fragments comprising a C-terminal epitope comprised in a C-terminal amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO;

1).

16. The method as claimed in claim 15, further comprising forming an index comparing the said measured level of collagen type VI α

3 fragments with a measured level in the same sample of a biomarker of the degradation of collagen type VI.

17. A method for identifying a subject suitable for treatment with an insulin sensitizer, the method comprising the steps of:

i) quantifying the amount of a C-terminal epitope of the C5 domain of the α

3 chain of collagen type VI in a biofluid obtained from a subject as per the method of claim 10; and

ii) correlating an elevated value determined by step i) with a subject that is suitable for treatment with an insulin sensitizer.

18. The method as claimed in claim 17, wherein the insulin sensitizer is a thiazolidinedione.

19. The method as claimed in claim 17, wherein the C-terminal epitope is comprised in a C-terminal amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1).

20. The method as claimed in claim 17, wherein the elevated value of step ii) corresponds to a value falling within a second or third tertile.

21. The method as claimed in claim 17, wherein the elevated value of step ii) corresponds to 6.3 ng/mL or greater of a C-terminal epitope of the α

3 chain of collagen type VI.

22. An assay kit for determining the quantity of a C-terminal epitope of the C5 domain of the α

3 chain of collagen Type VI in a biological sample, comprising an immunological binding partner as claimed in claim 1 and at least one of;

a streptavidin coated 96 well plate;

a peptide which is reactive with said immunological binding partner, which may be a biotinylated peptide Biotin-L-KPGVISVMGT-COOH (SEQ ID. NO;

4), wherein L is an optional linker;

an optionally biotinylated secondary antibody for use in a sandwich immunoassay;

a calibrator peptide comprising the C-terminal sequence . . . KPGVISVMGT-COOH (SEQ ID. NO;

1);

an antibody HRP labeling kit;

an antibody radiolabeling kit; and

an assay visualization kit.

23. The assay kit as claimed in claim 22, wherein the C-terminal epitope is comprised in a C-terminal amino acid sequence . . . KPGVISVMGT-COOH (SEQ ID. NO:

1).