PCSK9 Function Assay

• US 20150037816A1
• Filed: 08/08/2014
• Published: 02/05/2015
• Est. Priority Date: 08/01/2013
• Status: Abandoned Application

• Current Assignee: Atherotech, Inc (Ningbo Medical System Biotechnology Co., Ltd.)
• Original Assignee: Atherotech, Inc (Ningbo Medical System Biotechnology Co., Ltd.)
• Inventors: YEH, Chen-Hsiung
• Application Number:
  US14/455,661
• Publication Number:
  US 20150037816A1
• Time in Patent Office: Days
• US Class Current: 435/7.4
• CPC Class Codes: G01N 2333/95 Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
• CPC Class Codes: G01N 2800/323 Arteriosclerosis, Stenosis
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Methods and apparatuses for measuring the concentration of functional proprotein convertase subtilisin/kexin type 9 (PCSK9). A method of measuring functional PCSK9 in a sample is provided, by contacting the sample with a PCSK9-binding agent capable of binding to the LDL-R-binding region of a PCSK9; and measuring the amount of functional PCSK9 from the sample bound to the binding agent. Diagnostic methods, kits, and reagents for using the method are also provided.

Claims

1. A method of selectively measuring functional proprotein convertase subtilisin-like/kexin type 9 (PCSK9) in a sample, the method comprising:

(a) contacting the sample with a PCSK9-binding agent, said binding agent comprising a first peptide sequence from the N-terminal region of the PCSK9 binding domain of a low-density lipoprotein receptor, for a period sufficient to allow substantially all of the PCSK9 in the sample to bind to the binding agent;

(b) contacting the binding agent with a signal compound, the signal compound comprising;

(i) a reporter, and (ii) a second peptide sequence from the catalytic domain of a PCSK9; and

(c) measuring the amount of signal compound bound to the binding agent.

2. The method of claim 1 comprising removing any unbound signal compound.

3. The method of claim 1 comprising centrifuging an aliquot of blood to remove substantially all of the LDL and to produce a supernatant, and wherein the supernatant is the sample.

4. The method of claim 1 wherein the sample is blood plasma.

5. The method of claim 1 wherein the sample is from a subject, further comprising measuring the total PCSK9 in the sample in addition to the functional PCSK9.

6. The method of claim 1 comprising removing free LDL from the sample.

7. The method of claim 1 wherein an excess of binding agent is present compared to the expected PCSK9 in the sample.

8. The method of claim 1 in which LDL has not been removed from the sample.

9. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-26 of SEQ ID NO:

26.

10. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-26 of SEQ ID NO:

10.

11. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-26 of SEQ ID NO:

9.

12. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-26 of SEQ ID NO:

25.

13. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 314-339 of at least one of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

15, and SEQ ID NO;

16.

14. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-40 of SEQ ID NO:

26.

15. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-40 of SEQ ID NO:

10.

16. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-40 of SEQ ID NO:

9.

17. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-40 of SEQ ID NO:

25.

18. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 314-353 of at least one of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

15, and SEQ ID NO;

16.

19. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-80 of SEQ ID NO:

26.

20. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-80 of SEQ ID NO:

10.

21. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-80 of SEQ ID NO:

9.

22. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 1-80 of SEQ ID NO:

25.

23. The method of claim 1, in which the first peptide sequence has at least 90% homology with positions 314-393 of at least one of:

SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

15, and SEQ ID NO;

16.

24. The method of claim 1, in which the second peptide sequence has at least 90% homology with SEQ ID NO:

23.

25. The method of claim 1, in which the second peptide sequence has at least 90% homology with SEQ ID NO:

14.

26. The method of claim 1, in which the second peptide sequence has at least 90% homology with SEQ ID NO:

13.

27. The method of claim 1, in which the first peptide sequence has at least 95% homology with SEQ ID NO:

26 and in which the second peptide sequence has at least 95% homology with SEQ ID NO;

23.

28. A diagnostic method of evaluating a subject'"'"'s risk of atherosclerotic disease, the method comprising:

performing the method of claim 1 on a plasma sample from the subject; and

determining the subject'"'"'s risk of atherosclerotic disease based on the amount of functional PCSK9 measured.

29. An apparatus for measuring functional PCSK9 in a sample, the apparatus comprising:

a substrate with low binding affinity to PCSK9; and

a PCSK9-binding agent associated with the substrate, said binding agent capable of binding to the LDL-R-binding region of a PCSK9.

30. A kit for fluorescence resonance energy transfer (FRET) detection of functional PCSK9, comprising:

a FRET reagent for the detection of functional PCSK9, comprising a PCSK9-binding agent conjugated to a first fluorophore, said binding agent comprising a first peptide sequence from the N-terminal region of the PCSK9 binding domain of a low-density lipoprotein receptor; and

a second FRET reagent comprising;

a second fluorophore that is a complimentary fluorophore to the first fluorophore, and a signal compound capable of binding to the binding agent, said binding agent comprising a second peptide sequence from the catalytic domain of a PCSK9.