PCSK9 Function Assay
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.
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.