JP6081714B2 - Method for detecting hypercholesterolemia and arteriosclerosis - Google Patents

Description

  The present invention relates to a detection method using the abundance of a specific component for hypercholesterolemia and arteriosclerosis as an index.

  The first cause of death in Japanese is malignant neoplasm (30.3%), the second is heart disease (15.8%), and the third is cerebrovascular disorder (11.5%). Heart diseases and cerebrovascular disorders are mainly caused by arteriosclerosis based on lifestyle-related diseases such as dyslipidemia and diabetes. Management of dyslipidemia is considered one of the most effective means of preventing and treating arteriosclerosis. Therefore, early diagnosis of dyslipidemia and appropriate treatment are considered to be a method for preventing heart disease and cerebrovascular disorder.

  Although there are various causes of dyslipidemia, the elucidation of the etiology and pathophysiology has been deepened. Among dyslipidemias, type II hyperlipidemia is a disease characterized by an increase in blood low density lipoprotein (LDL). Among them, familial hypercholesterolemia (FH) has been shown to be caused by genetic abnormalities of LDL receptors by Goldstein and Brown (Goldstein JL, Brown MS. Annu Rev Genet, 1979; 13 : 259-289). Later, the cause of type II hyperlipidemia caused by a single gene abnormality was established, and familial defective apolipoprotein B-100 (FDB) caused by an abnormality in apolipoprotein B-100 (familial defective apolipoprotein B-100: FDB) ( Schaefer JR, et al. Arterioscler Thromb Vasc Biol, 1997; 17: 348-353), ATP-binding cassette (ABC) transporter G5 / ABCG8 KE, et al. Science, 2000; 290: 1771-1775), autosomal recessive hypercholesterolemia (ARH) due to an abnormality in the ARH gene (Garcia CK, et al. Science, 2001; 292: 1394-1398), and proprotein convertase subtilin / Autosomal dominant hypercholesterolemia (Abifadel M, et al. Nat Genet, 2003; 34: 154-156) due to the kexin type 9 (PCSK9) gene has been demonstrated.

  Diagnosis of familial hypercholesterolemia (FH) is as follows: 1) LDL cholesterol level of 200 mg / dl or higher, 2) Juvenile coronary artery disease (male <55 years, female <65 years), and youngness of family up to second degree Diagnosed by history of coronary artery disease, 3) presence of tendon xanthoma or cutaneous nodular xanthoma, 4) presence of corneal ring. In addition to the diagnostic criteria of 1) to 4), if an LDL receptor gene abnormality is confirmed, a definitive diagnosis can be made regardless of the presence or absence of the diagnostic criteria of 1) to 4) Hyperlipidemia Research Group, http://www.nanbyou.or.jp/top.html).

  The frequency of FH expression due to an abnormality in the LDL receptor is about 1 in 500 people who inherited an abnormal gene from one parent (heterozygote), and 1 million people who inherited an abnormal gene from a parent (homozygote). One person. In the Japanese, there are about 240,000 heterozygotes and about 120 homozygotes. Recently, Mabuchi et al. Reported that the frequency of FH homozygotes in Japanese was 1 / 171,167 and the frequency of FH heterozygotes was 1/208 (Mabuchi H, et al. Atherosclerosis, 2011; 214: 404-407). Sitosterolemia, FDB, and ARH are extremely rare among Japanese, but the frequency of PCSK9 is estimated to be 10-20% of FH from the report of Mabuchi et al.

  PCSK9 (proprotein convertase subtilisin / kexin type 9) is also called NARC-1 (Neural apoptosis-regulated convertase 1) and is a subtilisin-like serine protease. It is the ninth enzyme in the proprotein convertase family belonging to kexin. PCSK9 is a secreted protein consisting of 692 amino acids expressed mainly in the liver, kidney, and small intestine (Non-patent Document 1: Seidah NG, et al. Proc Natl Acad Sci USA, 2003; 100: 928-933.) .

  The LDL receptor on the surface of the liver cell membrane binds to the ligand LDL and then is taken up into the cell (endocytosis). After endocytosis, the LDL receptor localizes on the cell membrane surface again after supplying LDL into the cell (recycling). This process is repeated to adjust the amount of cholesterol in the blood. However, the LDL receptor bound by PCSK9 is degraded after being transferred to lysosome after endocytosis, and the recycling mechanism of LDL receptor does not work. As a result, a quantitative abnormality of the LDL receptor expressed on the hepatocyte membrane occurs, and as a result, blood LDL uptake decreases and blood LDL cholesterol increases. That is, it has been clarified that PCSK9 is involved in LDL cholesterol metabolism by promoting degradation of the LDL receptor (Non-patent Document 2: Lagace TA, et al. J Clin Invest, 2006; 116: 2995-3005, non-patent document 2). Patent Document 3: Qian YW, et al., J Lipid Res, 2007; 48: 1488-1498, Non-Patent Document 4: Li J, et al. Biochem J, 2007; 406: 203-207, Non-Patent Document 5: Grefhorst A, et al. J Lipid Res, 2008; 49: 1303-1311).

  Human PCSK9 consists of a total length of 692 amino acids, amino acids 1-30 are signal sequence (secretion signal), 31-152 are pro domain (proprotein domain), 153-452 are catalytic domain (protease catalytic domain), and 453-692 are C. -Terminal domain (C-terminal domain) is formed.

  PCSK9 was synthesized as proPCSK9 (precursor PCSK9) of about 74 kDa, and then caused self-degradation between glutamine at amino acid sequence 152 and serine at amino acid sequence 153 by serine protease activity of PCSK9 itself in the endoplasmic reticulum. A pro segment (proprotein fragment) of about 14 kDa on the side and a mature segment (mature protein fragment) containing the protease catalytic domain on the C-terminal side of about 60 kDa. These two types of fragments form a complex (heterodimer structure) so that the pro segment closes the active center of the shape segment, then migrates to the Golgi body as the complex and is secreted outside the cell (Non-patent literature) 6: Seidah NG, et al. Proc Natl Acad Sci USA, 2003; 100: 928-933, Non-patent document 7: Naureckiene S, et al. Arch Biochem Biophys, 2003; 420: 55-67, Non-patent document 8: Benjannet S, et al. J Biol Chem, 2004; 279: 48865-48875).

  PCSK9 having a heterodimeric structure secreted in the blood binds to EGF-A of the LDL receptor and induces the receptor to degrade as described above. The promotion of degradation of LDL receptor by PCSK9 is a phenomenon independent of PCSK9's own protease activity (serine protease activity), and it inhibits recycling of LDL receptor by the molecular chaperone action of PCSK9, that is, induces to lysosome. This is considered, but no conclusion has been reached yet (Non-patent Document 9: McNutt MC, et al. J Biol Chem, 2007; 282: 20799-20803).

  In addition, it has been reported that part of PCSK9 is further degraded in the process of being secreted from the Golgi apparatus to the outside of the cell. That is, it is cleaved between 218th arginine and 219th glutamine by the action of proprotein convertase Furin and the like. By this action, the 60 kDa mature segment portion is decomposed into an N-terminal side of about 7 kDa and a C-terminal side of about 53 kDa (ΔN218 segment), and the hetero-dimer structure of the pro segment and the mature segment is destroyed. Fragments after degradation (7 kDa and 53 kDa) are also secreted outside the cell. PCSK9 (heterodimer type) that has not undergone degradation by Furin binds to the LDL receptor and inhibits the recycling described above, and has the function of leading to degradation of the LDL receptor, whereas it has undergone degradation by Furin. PCSK9 (non-heterodimer type) does not have such a function (Non-Patent Document 10: Benjannet S, et al. J Biol Chem, 2006; 281: 30561-30572).

  In 2003, Abifadel et al. Found that the PCSK9 gene was the third causative gene of autosomal dominant familial hypercholesterolemia (FH) (Non-Patent Document 11: Abifadel M, et al. Nat Genet, 2003; 34: 154-156).

  Over 100 mutations have been reported so far in the PCSK9 gene abnormality (http://www.ucl.ac.uk/ldlr/Current/), and the function of PCSK9 is enhanced by the gene abnormality, that is, LDL reception Two types of mutations have been found: gain-of-function that promotes degradation of the body and loss-of-function that loses function, that is, loss-of-function that cannot degrade the LDL receptor. It has been shown to exhibit cholesterolemia and hypocholesterolemia.

  It has been reported that PCSK9 is promoted by activation of transcription factor SREBPs (Sterol Regulatory Element-Binding Proteins) that regulate the expression of genes involved in cholesterol and triglyceride biosynthesis. (Non-Patent Document 12: Maxwell KN, et al. J Lipid Res, 2003; 44: 2109-2019).

  That is, a statin drug, which is a therapeutic drug for hypercholesterolemia, induces the expression of LDL receptor through the activation of SREBPs, but also induces the expression of PCSK9 through a similar pathway. From this, it is considered that PCSK9 acts antagonistically to the cholesterol-lowering action of statins (Non-patent Document 13: Dubuc G, et al. Arterioscler Thromb Vasc Biol, 2004; 24: 1454-1459). That is, PCSK9 is not only a cause of FH, but also has an effect of reducing the drug effect in the treatment of hypercholesterolemia. In addition, inhibitors have been developed as target molecules for therapeutic drugs for hypercholesterolemia by inhibiting the action of PCSK9 (Patent Documents 1 to 5).

Special table 2010-536384 gazette Special table 2010-523135 gazette Special table 2010-508817 Special table 2010-501952 gazette US Publication No. 2010/166768

Seidah NG, et al. Proc Natl Acad Sci U S A, 2003; 100: 928-933 Lagace TA, et al. J Clin Invest, 2006; 116: 2995-3005 Qian YW, et al., J Lipid Res, 2007; 48: 1488-1498 Li J, et al. Biochem J, 2007; 406: 203-207 Grefhorst A, et al. J Lipid Res, 2008; 49: 1303-1311 Seidah NG, et al. Proc Natl Acad Sci U S A, 2003; 100: 928-933 Naureckiene S, et al. Arch Biochem Biophys, 2003; 420: 55-67 Benjannet S, et al. J Biol Chem, 2004; 279: 48865-48875 McNutt MC, et al. J Biol Chem, 2007; 282; 20799-20803 Benjannet S, et al. J Biol Chem, 2006; 281: 30561-30572 Abifadel M, et al. Nat Genet, 2003; 34: 154-156 Maxwell KN, et al. J Lipid Res, 2003; 44: 2109-2019 Dubuc G, et al. Arterioscler Thromb Vasc Biol, 2004; 24: 1454-1459

  Based on the above, PCSK9 is an important factor involved in LDL cholesterol metabolism, and its expression level reflects the activity of SREBPs that control lipid synthesis in vivo, so measuring PCSK9 concentration in blood Is expected to be clinically useful.

  The present inventor has recently produced a monoclonal antibody that binds to PCSK9 at each stage, and quantifies PCSK9 in a blood sample using a PCSK9 detection system based on the monoclonal antibody, thereby reducing familial hypercholesterolemia. The present inventors have found that it is possible to detect hypercholesterolemia and arteriosclerosis.

  That is, the present invention quantifies PCSK9 in a blood sample using an antibody against the component, and detects hypercholesterolemia including familial hypercholesterolemia based on the quantified value. A method for detecting arteriosclerosis (hereinafter referred to as the present method), which provides a detection method (hereinafter also referred to as the present FH detection method), similarly quantifies PCSK9 in a blood sample, and detects arteriosclerosis using the quantitative value as an index. It is also an invention that provides an arteriosclerosis detection method. In addition, this FH detection method and this arteriosclerosis detection method are also called this detection method collectively. In addition, when “PCSK9” is described in this specification and the drawings, it means human PCSK9 unless otherwise specified.

  In this FH detection method, quantification of PCSK9 is carried out by quantification step (1) for both heterodimer and non-heterodimer, quantification step (2) for heterodimer PCSK9, and quantification step (3) for non-heterodimer PCSK9. ), One or two or more quantitative steps selected from the group consisting of:

Familial hypercholesterolemia detection, which is the subject of the present FH detection method, includes the following (A), (B), (C), (D), (E), (F), (G), and (H 1) or two or more indices selected from the group consisting of:
(A) An index for detecting a case where the quantitative value in the quantitative step (1) is significantly larger than that of a healthy person as hypercholesterolemia including familial hypercholesterolemia.
(B) An index for detecting a case where the quantitative value in the quantitative step (2) is significantly larger than that of a healthy person as hypercholesterolemia including familial hypercholesterolemia.
(C) An index for detecting a case where the quantitative value in the quantitative step (3) is significantly larger than that of a healthy person as hypercholesterolemia including familial hypercholesterolemia.
(D) Quantitative value in quantification step (1) / quantitative value in quantification step (2), where the quantification value is significantly larger than that of healthy individuals, and high cholesterol including familial hypercholesterolemia An indicator to detect as blood pressure.
(E) Quantitative value in quantification step (1) / quantitative value in quantification step (3), wherein the quantitative value is significantly smaller than that of a healthy person, and high cholesterol including familial hypercholesterolemia An indicator to detect as blood pressure.
(F) The sum of the quantitative values in the quantitative steps (2) and (3) / the quantitative value in the quantitative step (2), wherein the quantitative value is significantly larger than that of a healthy person, To detect as hypercholesterolemia including infectious disease.
(G) The sum of the quantitative values in the quantitative steps (2) and (3) / the quantitative value in the quantitative step (3), wherein the quantitative value is significantly smaller than that of a healthy person, To detect as hypercholesterolemia including infectious disease.
(H) Quantitative value in quantification step (2) / quantitative value in quantification step (3), where the quantification value is significantly smaller than that of a healthy person, high cholesterol including familial hypercholesterolemia An indicator to detect as blood pressure.

  Also in this arteriosclerosis detection method, quantification of PCSK9 is carried out by quantifying step (1) for both heterodimer and non-heterodimer, quantification step (2) for heterodimer PCSK9, and quantification step for non-heterodimer PCSK9. (3) It can be carried out by one or more quantitative steps selected from the group consisting of:

In addition, the detection of arteriosclerosis, which is the subject of the present arteriosclerosis detection method, is one or two selected from the group consisting of the following (A ′), (B ′), (C ′), and (D ′) This can be done with the above indicators.
(A ′) An index for detecting arteriosclerosis when the quantitative value in the quantitative step (1) is significantly larger than that of a healthy person.
(B ′) An index for detecting arteriosclerosis when the quantitative value in the quantitative step (2) is significantly larger than that of a healthy person.
(C ′) An index for detecting arteriosclerosis when the quantitative value in the quantitative step (3) is significantly larger than that of a healthy person.
(D ′) An index for detecting arteriosclerosis when the sum of the quantitative values of the quantitative steps (2) and (3) is significantly larger than that of a healthy person.

  In this detection method, examples of the blood sample include serum, plasma, or whole blood, but serum or plasma is preferable, and serum is particularly preferable.

  The present invention provides a method for detecting familial hypercholesterolemia and arteriosclerosis using PCSK9 in a blood sample as an index.

It is a structural schematic diagram of PCSK9. 1 is an SDS polyacrylamide electrophoresis image of purified recombinant PCSK9. It is drawing which shows the result of having examined the reactivity with respect to rhPCSK9 by Western blotting. It is drawing which shows the result of having examined the content of the cutting | disconnection by Furin process of rhPCSK9 by Western blotting. It is drawing which shows the result of having examined the reactivity with respect to Furin cutting | disconnection rhPCSK9 of each acquired monoclonal antibody by Western blotting. It is explanatory drawing of the Example which used this detection method as the PCSK9 molecular form specific protein mass measuring system by sandwich ELISA method. It is the figure which showed the analytical curve with respect to recombinant PCSK9 by the PCSK9 molecular form specific protein mass measuring system by the said sandwich ELISA method. It is the figure which showed the analytical curve with respect to the serum sample by the PCSK9 molecular form specific protein mass measuring system by the said sandwich ELISA method. It is drawing which showed the relationship between each measurement system of PCSK9 detection sandwich ELISA between healthy persons. It is drawing which showed the relationship between each measurement system of PCSK9 detection sandwich ELISA between FH patients. It is drawing which shows the result of having performed the immunoprecipitation of PCSK9 in human serum.

1. PCSK9
As described above, the base sequence and amino acid sequence (preproPCSK9, amino acids 1-692) of the gene of PCSK9 (human PCSK9) to be detected in this detection method are known (NCBI reference sequences: NM_174936 and NP_777596: SEQ ID NO: 1 and 2) The target of this detection method is all or part of the in vivo protein. As described above, PCSK9 is an in vivo protein that exists in vivo in several forms. FIG. 1 is a schematic diagram of the structure of PCSK9.

  As described above, PCSK9 is synthesized in vivo as proPCSK9 (precursor PCSK9) of about 74 kDa via preproPCSK9, and then the glutamine of amino acid sequence 152 and serine of 153 in the endoplasmic reticulum by the serine protease activity of PCSK9 itself. Between the N-terminal side of about 14 kDa pro segment (proprotein fragment) and the C-terminal side of about 60 kDa, which is converted into a mature segment containing the protease catalytic domain (mature protein fragment). After the pro segment forms a complex (heterodimer structure) so as to block the active center of the shape segment, it shifts to the Golgi body as the complex and is secreted outside the cell. PCSK9 pro segment functions as an inhibitor of protease activity, and the heterodimer structure is inactive as a proteolytic enzyme (serine protease). In addition, it has been reported that part of PCSK9 is further degraded in the process of being secreted extracellularly from the Golgi apparatus. That is, it is cleaved between 218th arginine and 219th glutamine by the action of proprotein convertase Furin and the like. By this action, the shape segment is decomposed into an N-terminal side of about 7 kDa and a C-terminal side of about 53 kDa (ΔN218 segment), the heterodimer structure of the pro segment and the mature segment is disrupted, and fragments after decomposition (7 kDa and 53 kDa) are also produced. Secreted extracellularly.

  The detection of PCSK9 is preferably performed using an antibody against PCSK9 and a detection means that uses an antigen-antibody reaction between PCSK9 and PCSK9 in a blood sample. Specifically, for example, an enzyme immunoassay (ELISA) method, a radioimmunoassay (RIA) method, an immunochromatography method, a latex agglutination turbidimetric method, an immunoprecipitation method, etc. The analysis method etc. which were performed can be illustrated. Among these detection means, it is preferable to select an ELISA method, particularly a sandwich ELISA method, because of its quantitativeness, detection sensitivity, safety, convenience, and accuracy. In recent years, an immunoassay (FEIA, CLIA, CLEIA, ECLIA) method using fluorescence or chemiluminescence for detection is also suitable.

2. Antibody used for detection As a premise for carrying out such detection means, an antibody that specifically binds to PCSK9 (monoclonal antibody and / or polyclonal antibody: hereinafter also referred to as an antibody against PCSK9) is required.

  A method for producing an antibody against PCSK9 can be performed according to a conventional method, but all or part of PCSK9 as an antigen is required. PCSK9 can be obtained by using a natural protein from a biomaterial or a protein that is naturally expressed in a cell line, but in practice, a recombinant (recombinant) protein of PCSK9 using genetic engineering techniques. Can be obtained by manufacturing. The recombinant protein can be obtained as a gene amplification product by preparing a primer for gene amplification with reference to the base sequence of the PCSK9 gene described above and performing RT-PCR using this primer. In order to produce an antibody against PCSK9 by purifying the PCSK9 recombinant protein produced from the forced expression cells obtained by introducing an expression vector incorporating the same into cells, etc., by the above-described commonly known purification methods. PCSK9 recombinant protein that can be used as an antigen of. In addition, a modified recombinant protein encoded by a partially modified PCSK9 gene obtained by partially modifying the above-mentioned PCSK9 gene base sequence can be used as long as it does not lose its properties as an antigen to PCSK9. Moreover, PCSK9 as an antigen used here does not necessarily need to be the whole of PCSK9, and may be a partial peptide. In particular, as described above, PCSK9 is known to take several characteristic forms, and selection of each form of PCSK9 as necessary is suitable for obtaining an antibody having a desired property. It is.

  PCSK9 partial fragment peptides include a PCSK9 fragment in which a partial fragment of the PCSK9 gene is expressed, a protease-treated product of PCSK9, a phosphite-triester method (Ikehara, M., et al., Proc. Natl. Acad. Sci. USA, 81, 5956 (1984)) and the like can be used as antigens as chemically synthesized peptides by a solid phase method or a liquid phase method.

  In PCSK9 obtained in this way, in particular, when PCSK9 used as an immunizing antigen is a partial peptide of a small molecule, a hapten can be bound to improve the immunogenicity of the antigen. As the hapten, it is possible to arbitrarily select a substance that can usually be used as a hapten. For example, umbrella hemocyanin (KLH), chicken egg albumin (OVA), bovine serum albumin (BSA), etc. are selected as haptens. can do.

  Irrespective of whether the desired antibody against PCSK9 is a polyclonal antibody or a monoclonal antibody, immunization is performed by a general method, for example, by immunizing the animal to be immunized intravenously, intradermally, subcutaneously, intramuscularly, It can be performed by administering intraperitoneally.

  Polyclonal antibodies against PCSK9 can be produced from immune sera derived from immunized animals using all or part of the PCSK9 molecule as an immunizing antigen.

  As a monoclonal antibody against PCSK9, a clone that produces an antibody that recognizes the PCSK9 molecule by producing a hybridoma between immune cells of the immunized animal and animal myeloma cells in the same manner as the above polyclonal antibody is selected. It can be produced by culturing this clone.

  The polyclonal and monoclonal antibodies described above can also be prepared by gene immunization with reference to known techniques such as Nature 1992 Mar12; 356, p152-154 and J. Immunol Methods Mar 1; 249, p147-154. Specifically, it can be produced by directly immunizing an animal with a vector expressing a gene encoding all or part of the PCSK9 molecule.

  The animal to be immunized is not particularly limited, and mice, rats, hamsters, rabbits, goats, sheep, chickens, etc. can be widely used. When producing monoclonal antibodies, they are used for cell fusion. It is desirable to select in consideration of compatibility with myeloma cells.

  More specifically, an immune serum for the production of polyclonal antibodies is administered several times by the above means every 7 to 21 days as a guide to an animal to be immunized in combination with an ordinary adjuvant if desired Alternatively, immune cells for monoclonal antibody production, for example, spleen cells after immunization can be obtained.

  When producing monoclonal antibodies, known monoclonal antibody production methods such as Tamie Ando, Takeshi Chiba, Co-author, “Introduction to Monoclonal Antibody Experimental Procedures” Kodansha (1991), EdHarlow and David Lane, “Antibodies: A Laboratory Manual ”, Cold Spring Harbor Laboratory, 1988.

  The thus obtained polyclonal antibody or monoclonal antibody is an antibody having specific reactivity with PCSK9. In the present invention, a commercially available antibody against PCSK9 can also be used.

  Furthermore, the antibody against PCSK9 can be subjected to a labeling treatment, that is, an enzyme labeling treatment, a fluorescence labeling treatment, an isotope labeling treatment, or the like according to a conventional method, if necessary.

  One preferred embodiment is that the antibody capable of capturing PCSK9 in a blood sample is used as an immobilized antibody immobilized on a solid phase.

  Examples of the solid phase include microplates and beads (agarose gel, sepharose gel, latex particles, magnetic particles, etc.) and the like.

  The immobilization method can be performed according to a conventional method in the antibody immobilization method corresponding to the type of solid phase.

  For example, an antibody can be immobilized on a microplate by performing a physical non-specific adsorption method according to a conventional method. Also, the beads can be immobilized according to a conventional method. For example, immobilization using a chemical cross-linking agent or affinity between other substances such as biotin-avidin is used to label an antibody with a compound in advance, and the compound is immobilized with affinity. The antibody can be immobilized by performing an immobilization method using a suitable protein, an immobilization method using an immobilized anti-immunoglobulin antibody, a protein having an affinity for an antibody such as protein A, or the like. it can.

3. This detection method As mentioned above, this detection method can be performed by using the antibody with respect to PCSK9 and using the element according to a detection means.

  When the detection means is an ELISA method, for example, a blood sample is brought into contact with an antibody against PCSK9 immobilized on a microplate, and soluble PCSK9 in the sample is bound to the immobilized antibody and bound to this solid phase. By detecting soluble PCSK9 using an enzyme-labeled antibody against another PCSK9, the determination method of the present invention can be carried out. It is also possible to detect by reacting the immobilized antibody, blood sample and enzyme-labeled antibody simultaneously.

  When immunoprecipitation is used as a detection means, for example, an antibody against PCSK9 immobilized on beads is contacted with a blood sample to bind PCSK9 to the immobilized antibody, and PCSK9 bound to this solid phase. This detection method can be carried out by detecting PCSK9 from the separated product. The detection of this PCSK9 by immunoprecipitation is performed, for example, by performing electrophoresis on the above-mentioned isolate, and allowing a labeled antibody against PCSK9 to act on the transcript of this electrophoresis pattern to detect a soluble PCSK9 band. And Western blotting.

  Further, the detection means may be a method mainly using Western blotting. For example, a cell-excluded sample obtained by removing cells from a blood sample is directly separated by electrophoresis, and a labeled antibody against PCSK9 is allowed to act on the transcript, thereby detecting PCSK9 in the blood sample. can do.

  Furthermore, it is also possible to use a latex agglutination turbidimetry as a detection means. For example, by contacting a blood sample with an antibody against PCSK9 bound to latex particles to form an aggregate of immune complex due to antigen-antibody reaction in the liquid phase, and measuring the change in turbidity, A detection method can be performed.

  As described above, the quantification of PCSK9 in the present detection method includes the quantification step (1) for both heterodimer and non-heterodimer, the quantification step (2) for heterodimer PCSK9, and the quantification step for non-heterodimer PCSK9. (3) It can be carried out by one or more quantitative steps selected from the group consisting of:

  When sandwich ELISA, which is one of the preferred detection means for carrying out this detection method, is used, the above-mentioned PCSK9 quantification step (1) for both heterodimer and non-heterodimer is performed as a solid phase antibody and a detection antibody. Both can be performed by sandwich ELISA using a monoclonal antibody that binds to both heterodimeric PCSK9 and non-heterodimeric PCSK9. In addition, the identification amount step (2) includes (a) a monoclonal antibody that binds to both heterodimeric human PCSK9 and non-heterodimeric human PCSK9, and (b) binds to heterodimeric human PCSK9. However, any one of the monoclonal antibodies that do not bind to non-heterodimeric human PCSK9 can be performed by sandwich ELISA using different solid phase antibodies and detection antibodies. In addition, the identity step (3) comprises (a) a monoclonal antibody that binds to both heterodimeric human PCSK9 and non-heterodimeric human PCSK9, and (b) non-heterodimeric human PCSK9 binding. However, any one of the monoclonal antibodies that do not bind to the heterodimeric human PCSK9 can be performed by a sandwich ELISA method using different solid phase antibodies and detection antibodies.

  As described above, the FH detection method is selected from the group consisting of (A), (B), (C), (D), (E), (F), (G), and (H). It is possible to carry out by one or more indicators. Moreover, in this arteriosclerosis detection method, it is possible to carry out with one or more indices selected from the group consisting of the above (A ′), (B ′), (C ′), and (D ′). is there. These will be specifically disclosed in Examples described later.

  Thus, by quantitatively detecting PCSK9 in the blood sample, cholesterolemia including familial hypercholesterolemia and arteriosclerosis in the sample provider can be detected using the quantitative value as an index. Examples of the arteriosclerotic diseases include coronary heart diseases such as myocardial infarction and angina pectoris, and cerebrovascular disorders such as cerebral infarction and cerebral thrombus. If the present FH detection method reveals hypercholesterolemia by the quantitative value of PCSK9 in the blood sample, it is possible to confirm the suspected arteriosclerosis by this arteriosclerosis detection method. That is, by performing this detection method, it is possible to check the possibility of arteriosclerotic diseases that are common in patients with hypercholesterolemia, particularly familial hypercholesterolemia.

  The present invention provides a kit for carrying out this detection method. That is, the present invention is also an invention that provides a kit (hereinafter also referred to as the present detection kit) including the elements for performing the above detection.

  This detection kit contains at least an antibody against PCSK9 as an element.

  For example, when the detection means is the ELISA method, it is different from the first antibody against PCSK9 immobilized on the solid phase or to be immobilized, and / or the epitope recognized by the immobilized first antibody. A second antibody for detection that recognizes a PCSK9 intramolecular epitope is preferably included as an element in the determination kit of the ELISA method. It is also preferable to include a detection reagent for detecting the second antibody, a blocking solution, a diluent, a PCSK9 standard product, and the like as elements of the ELISA method main determination kit.

  Further, when the detection means is combined with the Western blot method as the detection means in the analysis method using the immunoprecipitation method as described above, an antibody against PCSK9 for immobilizing PCSK9 in the blood sample, immobilization Included as an element of this detection kit for Western blotting is a detection antibody to PCSK9 for detection of soluble or isolated PCSK9 separated as an electrophoretic band and / or an antibody to PCSK9 to be immobilized or immobilized Is preferred. It is also preferable to include a reagent for detecting an antibody against PCSK9, a blocking solution, a diluent, a transfer membrane, and the like as elements of this detection kit for Western blotting.

  Further, in the analysis method in which the detection means utilizes latex agglutination, it is preferable that an antibody against PCSK9 bound to latex particles is included as an element in the detection kit.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the technical scope of this invention is not limited by this Example. The hybridomas producing the four types of monoclonal antibodies described below are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. That is, the hybridoma “Mouse-Mouse hybridoma PCSK9-1FB” that produces the monoclonal antibody “1FB” has the accession number FERM P-22109, and the hybridoma “Mouse-Mouse hybridoma PCSK9-B1G that produces the monoclonal antibody“ B1G ”has the accession number FERM P The hybridoma “Mouse-Mouse hybridoma PCSK9-B12E” producing the monoclonal antibody “B12E” is designated as accession number FERM P-22111 and the hybridoma “Mouse-Mouse hybridoma PCSK9-G12D” producing the monoclonal antibody “G12D”. Is received at the depository under the deposit number FERM P-22112.

1. Preparation of Recombinant PCSK9 (1) Preparation of Recombinant Human Full-Length PCSK9 Complementary strand DNA corresponding to human PCSK9 full-length (amino acid numbers 1 to 692) was amplified from total RNA derived from HepG2 cells by RT-PCR, and pEF321 vector (Kim DW , Gene. 1990 Jul 16; 91 (2): 217-23) to produce an expression vector pEF / PCSK9. In the PCR, a primer sequence was set so that the histidine 6 residue was fused to the C-terminus of the protein for metal affinity purification (described below). The thermal cycle of the PCR was 95 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 1 minute → 65 ° C for 2 minutes → 72 ° C for 5 minutes → 65 ° C for 1 minute. The reaction of minutes was performed.

As amplification primers,
“Gacgaattccagcgacgtcgaggcgctcatggttg” (forward primer: SEQ ID NO: 3),
“Gacgaattctcagtgatggtgatggtgatgctggagctcctgggaggcctgcgcc” (reverse primer: SEQ ID NO: 4) was used.

  The expression vector pEF / PCSK9 and the neomycin resistance gene expression vector pSV2Neo (Clontech) are co-introduced into the mammalian cell line CHO-K1 (RIKEN), and selection with Geneticin (Sigma) is repeated repeatedly for stability. Clone CHO-K1 / PCSK9 cells expressing recombinant human PCSK9 (rhPCSK9) were prepared.

The CHO-K1 / PCSK9 cells were cultured in serum-free and low protein medium CHO-S-SFM II (Invitrogen) at 37 ° C. under 5% CO ₂ and the culture supernatant was collected. Furthermore, 500 mL of the culture supernatant was mixed with 6 mL of TALON Resin (Clontech) and reacted at 4 ° C. overnight. The column is packed with TALON Resin and washed with 50 mL of 25 mM phosphate buffer (pH 8.0) containing 500 mM sodium chloride, followed by 10 mL of 20 mM phosphate buffer (pH 8.0) and containing 20 mL of 200 mM imidazole. Eluted with 20 mM phosphate buffer (pH 8.0). Next, this eluate was applied to a DEAE-Sepharose CL-6B (0.6 mL, GE Healthcare) column previously equilibrated with 20 mM phosphate buffer (pH 8.0), and 5 mL of 20 mM phosphate buffer. (pH 8.0) and then washed with 1.5 mL of 20 mM phosphate buffer (pH 8.0) containing 100 mM sodium chloride, and then with 3 mL of 20 mM phosphate buffer (pH 8.0) containing 200 mM sodium chloride. Eluted. This eluted fraction was designated as purified recombinant human full-length PCSK9 (rhPCSK9).

  Purified recombinant PCSK9 (rhPCSK9) was subjected to SDS polyacrylamide electrophoresis, the band was detected by silver staining of the gel, and the purity of the purification was confirmed (FIG. 2). As a result, rhPCSK9 exists in a pro segment away from the reduced or non-reduced conditions, and the mature segment formed by electrophoresis under reducing conditions is electrophoresed under non-reducing conditions. It was found that the molecular weight was slightly larger (approximately 60 kDa) than that of the mature segment.

(2) Preparation of Recombinant ΔN218 PCSK9 (53KDa) A complementary strand DNA corresponding to ΔN218 PCSK9 (amino acid numbers 219-692) was amplified by PCR using pEF / PCSK9 as a template, and pEF321 vector (Kim DW, Gene. 1990 Jul 16 ; 91 (2): 217-23) to produce the expression vector pEF / ΔN218 PCSK9. The thermal cycle of the PCR was 94 ° C for 30 seconds, followed by 23 cycles of 98 ° C for 10 seconds → 60 ° C for 5 seconds → 72 ° C for 1.5 minutes, and finally the reaction at 72 ° C for 2 minutes. It was.

As amplification primers,
“Agtcaagcttcaggccagcaagtgtgacagtcat” (forward primer: SEQ ID NO: 5),
“Agtagtcgacctggagctcctgggaggcctg” (reverse primer: SEQ ID NO: 6) was used.

  The expression vector pEF / ΔN218 PCSK9 was introduced into the mammalian cell line CHO-K1 in the same manner as described above to produce clone CHO-K1 / ΔN218 PCSK9 cells that stably express the recombinant ΔN218 PCSK9.

The cells were cultured in a 15 cmφ dish, the cells were collected by trypsin-EDTA (Gibco) treatment, and washed with PBS by centrifugation. After washing, the cells were suspended in 1x Complete mini EDTA-free (Roche) and 1% Nonidet P40-containing Tris buffer (TBS; 50 mM Tris-HCl containing 150 mM NaCl, pH 7.5) for 30 minutes in ice. Cooled down. Next, the cell suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes), and the supernatant was collected. This supernatant fraction was used as a cell lysate containing recombinant ΔN218 PCSK9 (rhΔN218 PCSK9).

2. Preparation of anti-human PCSK9 monoclonal antibody (1) Preparation of hybridoma producing anti-human PCSK9 monoclonal antibody Mice were immunized by protein immunization using purified rhPCSK9 as an antigen or gene immunization using expression vector pEF / PCSK9 as an immunogen.

  For protein immunization, 3 μg of purified rhPCSK9 was mixed with 12 μg of adjuvant Abisco-100 (ISCONOVA) and administered subcutaneously to the back of Balb / c mice (8 weeks old, female) (primary immunization). For booster immunization, the same operation as the first immunization was performed 3 times every 2 weeks.

  For gene immunization, 50 μg of expression vector pEF / PCSK9 dissolved in PBS per administration was administered subcutaneously to the Balb / c mouse (8 weeks old, female) tail (primary immunization). For booster immunization, the same operation as the first immunization was performed four times every two weeks.

Two weeks after the final administration of both protein immunization and gene immunization, 20 μg of purified rhPCSK9
Was administered intraperitoneally to mice. Three days after the intraperitoneal administration, the mouse spleen was removed and the spleen cells were collected. Spleen cells were fused with mouse myeloma cells (SP2 / 0-Ag14) with a polyethylene glycol 1500 solution (manufactured by Boehringer Mannheim). The fused cells (hybridoma) were selected with RPMI medium containing 3% hybridoma cloning factor (HCF, Air Brown), 1X HAT (Sigma) and 10% fetal bovine serum (FBS, Nichirei). Anti-PCSK9 monoclonal antibody-producing cells were selected by ELISA using a microplate on which purified rhPCSK9 was immobilized. Specifically, 25 ng of purified rhPCSK9 was immobilized on each well (4 ° C., overnight), and each well was blocked with PBS containing 1% bovine serum albumin (BSA, Sigma). After washing each well, 100 μL of the culture supernatant of each hybridoma was added and reacted at room temperature for 2 hours. After the reaction, each well was washed with a washing solution (PBS containing 0.1% Tween20), a peroxidase-labeled goat anti-mouse IgG antibody (BioSource) diluted 5000 times was added, and reacted at room temperature for 1 hour. After the reaction, the wells were washed 5 times with a washing solution, and 100 μL of a color developing solution [0.012% hydrogen peroxide, 0.4 mg / mL OPD (o-phenulenediamine dihydrochloride, Sigma-Aldrich) -containing citrate phosphate buffer solution] , PH 5.0] was added, and the mixture was reacted at room temperature for 30 minutes. After the reaction, 2N sulfuric acid was added to stop the reaction, and the absorbance at a wavelength of 492 nm was measured with a microplate reader. Wells having an absorbance of the hybridoma culture supernatant of 1.0 or more were selected as positive. The anti-PCSK9 monoclonal antibody-producing hybridoma was further cloned by limiting dilution to establish a cell line.

  Furthermore, positive hybridoma cells were selected by confirming the reactivity to rhPCSK9 by Western blotting or the reactivity to human serum PCSK9 by immunoprecipitation using the hybridoma cell culture supernatant. The resulting clones were finally selected.

  In Western blotting, 1 μg of purified rhPCSK9 per gel was subjected to SDS-PAGE without reduction and then transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with 0.1% Tween20-containing PBS (washing solution), each hybridoma culture supernatant was reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution, and then reacted with a peroxidase-labeled goat anti-mouse IgG antibody (BioSource) diluted 10,000 times at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution and then exposed to an X-ray film (manufactured by Kodak Co.) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected.

In the immunoprecipitation method, 200 μL of each hybridoma culture supernatant was added to 1.0 × 10 ⁷ Dynabeads M-280 anti-mouse IgG (manufactured by Veritas), and the antibody was bound to the beads with inversion mixing at room temperature for 2 hours. . The beads were washed three times with PBS containing 0.1% Tween 20, and then 100 μL of human serum was added and reacted at 4 ° C. for 16 hours with inversion mixing. After washing three times with PBS containing 0.1% Tween 20, the suspension was suspended in a non-reducing SDS sample solution, subjected to SDS-PAGE, and then transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with 0.1% Tween20-containing PBS (washing solution), 0.1 μg / mL peroxidase-labeled anti-PCSK9 antibody MAB38881 (manufactured by R & D) was reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution and then exposed to an X-ray film (manufactured by Kodak Co.) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected.

  Finally, a monoclonal antibody that reacts with human PCSK9 by selecting by ELISA using purified rhPCSK9 solid phase microplate, reactivity to purified rhPCSK9 by Western blotting, reactivity to human serum PCSK9 by immunoprecipitation Production hybridoma 25 clones were generated.

(2) Monoclonal antibody preparation from hybridoma Hybridoma cells 10 ⁶ to 10 ⁷ per Balb / c mouse (8 weeks old, male) previously administered intraperitoneally with pristen (0.5 ml / animal, Sigma) Pieces / 0.5 mL were injected intraperitoneally. Ten days after the injection, the mouse was opened, and ascites was collected. Cell components were removed from the obtained ascites by centrifugation, and an equal amount of ice-cooled saturated ammonium sulfate solution was added to the supernatant and mixed, ice-cooled and left for 2 hours. After centrifugation at 10000 × g for 10 minutes, the supernatant is discarded, and the precipitate is dissolved in a binding solution (1.5 M glycine solution containing 3 M sodium chloride, pH 8.9) and mixed with Protein A Sepharose (manufactured by GE Healthcare). And mixed by inversion at 4 ° C. overnight. The bound Protein A Sepharose was packed in a column, washed with 6 volumes of binding solution, and then eluted 1 mL at a time with an elution solution (0.1 M citric acid solution, pH 4.0). The eluted fraction was neutralized with 0.1 mL of 2M Tris solution (pH 10.0). The absorbance (280 nm) of each eluted fraction was measured, and the eluted fraction of the monoclonal antibody was collected. The collected monoclonal fraction was dialyzed against PBS (4 ° C., overnight) to obtain a purified monoclonal antibody.

  The isotype of the obtained monoclonal antibody was measured using a mouse monoclonal antibody isotype determination kit (manufactured by Boehringer) according to the operation procedure attached to the kit. The monoclonals against human PCSK9 were IgG1 (17 clones), IgG2a (2 clones), and IgG2b (6 clones). Among these, the monoclonal antibodies derived from the hybridomas 1FB, B12E, G12D, and B1G [antibody 1FB (IgG1), B12E (IgG1), G12D (IgG1), B1G (IgG1)] have the following antibody specificities: confirmed.

(3) Confirmation of Monoclonal Antibody Specificity The specificity of the monoclonal antibody was confirmed by examining reactivity to rhPCSK9 by Western blotting, Furin cleavage by immunoprecipitation, and reactivity to rhPCSK9.

(4) Preparation of peroxidase-labeled monoclonal antibody Antibodies 1FB, B12E, G12D, B1G, and commercially available anti-human PCSK9 antibody MAB38881 (R & D) and anti-histidine tag antibody (Tetra-His Antibody, Qiagen) are Peroxidase Labeling Kit-SH ( Peroxidase-labeled antibodies for each antibody were prepared using Dojindo Laboratories). Antibody labeling was performed according to the kit instructions.

(5) Specificity of antibody against rhPCSK9 by Western blotting 1 μg of purified rhPCSK9 per gel was subjected to SDS-PAGE under non-reducing and reducing conditions, and then transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with a washing solution (PBS containing 0.1% Tween20), 10 ng / mL of each peroxidase-labeled antibody was reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution, and then exposed to an X-ray film (manufactured by Kodak) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected (FIG. 3).

  The results of reactivity to rhPCSK9 by Western blotting revealed that antibodies 1FB, B12E, and B1G reacted with PCSK9 mature segment, and this reactivity was lost by antigen reduction treatment. The antibody G12D reacted only with the PCSK9pro segment, and it became clear that this reactivity was not affected by the reduction treatment of the antigen.

(6) Cleavage of rhPCSK9 by Furin treatment 1 μg of purified rhPCSK9 and recombinant human Furin (R & D) were mixed in 25 mM Tris-HCl (pH 9.0) solution containing ₂ mM CaCl ₂ and 0.5% Triton X-100. The reaction was carried out at 6 ° C. for 6 hours. Thereafter, 1 μL of 0.5 M EDTA was added to stop the reaction. This reaction solution was named Furin-cut rhPCSK9 solution.

  The cleavage of rhPCSK9 by Furin treatment was confirmed by SDS-PAGE and Western blotting as follows. Furin-untreated rhPCSK9 and Furin-treated rhPCSK9 (20 ng) were each subjected to SDS-PAGE under reducing conditions, and then transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with a washing solution (PBS containing 0.1% Tween 20), 10 ng / mL peroxidase-labeled anti-human PCSK9 antibody MAB38881 (manufactured by R & D) was reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution, and then exposed to an X-ray film (manufactured by Kodak) using a chemiluminescence reagent (manufactured by Perkin Elmer), and a specific band was detected (FIG. 4).

  As a result, the Furin-untreated rhPCSK9 detected a mature segment band with a molecular weight of about 60 kDa (corresponding to the mature segment in the reduction treatment in FIG. 2), whereas the Furin-treated rhPCSK9 was mostly a band with a molecular weight of 53 kDa. Was detected.

(7) Reactivity of monoclonal antibody against Furin-cleaved rhPCSK9 For anti-PCSK9 antibodies 1FB, B12E, B1G, G12D and control antibody 16G5, Dynabeads M-280 Tosyl washed with 0.1 M borate buffer (pH 9.5) -activated (manufactured by Veritas) was suspended in 0.1M borate buffer solution (pH 9.5) containing 3M ammonium sulfate, and 10μg of antibody per 1.0 × 10 ⁸ beads was added and tumbled at 4 ° C. for 20 hours. Each antibody was bound to the beads while mixing. After the reaction, the supernatant was discarded, 0.5% bovine serum albumin (BSA) -containing PBS (pH 7.4) was added, and the mixture was reacted at room temperature for 2 hours with gentle stirring to inactivate the beads. Washed with PBS containing BSA (pH 7.4) to prepare antibody-bound beads. 20 μg of rhPCSK9 or 20 μg of Furin-cleaved rhPCSK9 was added to 1.0 × 10 ⁷ antibody-bound beads and reacted at 4 ° C. for 16 hours while mixing by inversion. After washing with PBS containing 0.1% Tween20 three times, it was suspended in a non-reducing SDS sample solution, subjected to SDS-PAGE, and then transferred to a PVDF membrane (Millipore). The transferred film was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with a washing solution (PBS containing 0.1% Tween 20), 0.1 μg / mL peroxidase-labeled anti-human PCSK9 antibody MAB38881 (R & D) was reacted at room temperature for 2 hours. After the reaction, the plate was washed 5 times with a washing solution, and then exposed to an X-ray film (manufactured by Kodak Co.) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected (FIG. 5).

  As described above, G12D, which was confirmed to be an antibody against Pro segment by Western blotting, immunoprecipitates mature segment (60 kDa) in Furin-untreated rhPCSK9, whereas mature segment (53 kDa) in Furin-cleaved rhPCSK9. Does not immunoprecipitate. From this, it was confirmed that Furin-untreated rhPCSK9 formed a heterodimer, whereas Furin-cleaved rhPCSK9 did not form a heterodimer (non-heterodimer).

  Antibodies 1FB and B12E against mature segment immunoprecipitates both Furin-untreated rhPCSK9 (heterodimer) and Furin-cleaved rhPCSK9 (non-heterodimer), and thus reacts with both 60kDa and 53kDa cleaved mature segments of PCSK9. Confirmed to do. The antibody B1G does not immunoprecipitate a 60 kDa mature segment in Furin-untreated rhPCSK9 (heterodimer), whereas it immunoprecipitates a 53 kDa cleaved mature segment in Furin-cleaved rhPCSK9 (non-heterodimer). Reaction was confirmed.

3. Construction of PCSK9 molecular form-specific protein mass measurement system by sandwich ELISA method (1) Combination of antibodies Using the monoclonal antibody prepared as described above, a combination of antibodies capable of sandwich ELISA was examined. Total PCSK9 measurement system (A system) by combination of antibody 1FB and detection antibody peroxidase-labeled B12E, heterodimeric PCSK9 measurement system (B system) by combination of solid-phase antibody B12E and detection antibody peroxidase-labeled G12D, solid-phase antibody B1G and detection antibody A non-heterodimer PCSK9 measurement system (C system) was combined with a combination of peroxidase-labeled B12E (FIG. 6).

(2) Construction of sandwich ELISA 100 μl of each solid phase antibody (A system: 1FB 1 μg / mL, B system: B12E 0.3 μg / mL, C system: B1G 1 μg / mL) diluted with PBS on an immunoplate (Nunc) The solution was added at / well and allowed to stand at 4 ° C. overnight.
After washing twice with 0.1% Tween20-containing PBS (washing solution), 1% BSA and 10% Sucrose-containing PBS were added at 200 μl / well and blocked at room temperature for 2 hours. After removing the liquid from each well, a standard solution diluted with PBS containing 0.3% BSA and 0.1% Tween20 (A system and B system rhPCSK9, C system rhΔN218 PCSK9), or human serum at 100 μl / well Added and allowed to stand at room temperature for 2 hours (Table 1).

  After the reaction, each detection antibody was washed 4 times with a washing solution and diluted with PBS containing 10% StabilZyme (SurModics), 0.3% BSA and 0.1% Tween20 (A system: peroxidase-labeled B12E 10 ng / mL, B system: Peroxidase-labeled G12D 25 ng / mL, C system: peroxidase-labeled B12E 35 ng / mL) was added at 100 μl / well and allowed to stand at room temperature for 1 hour. After the reaction, the plate was washed four times with a washing solution, 100 μl / well of enzyme substrate solution TMB + (Dako) was added, color was developed for 30 minutes at room temperature, 100 μl / well of 0.5 M sulfuric acid was added to stop the enzyme reaction, Multiskan The absorbance of each well was measured at a wavelength of 450 nm (background 560 nm) with Ascent (manufactured by Thermo Labsystems).

  The calibration curve in each ELISA measurement system is as shown in FIG. A and B systems showed good calibration curves in the range of 0 to 20 ng / ml and C system in the range of 0 to 4 ng / ml.

  FIG. 8 shows the results of a dilution linearity test of serum samples. The linearity was shown about any system and the sample, and the accuracy of each ELISA system was confirmed.

4). Measurement of PCSK9 in human serum (1) Quantification of PCSK9 in healthy subjects and patients with familial hypercholesterolemia (FH) Measure PCSK9 concentrations in sera of 108 healthy subjects and 194 patients with familial hypercholesterolemia (FH) did. As a result, the serum PCSK9 concentration in healthy subjects was 262.7 ± 80.2 (mean ± SD) ng / mL for the A system measurement system, 252.5 ± 79.1 ng / mL for the B system measurement system, and the C system measurement. The system was 28.9 ± 10.6 ng / mL. The serum PCSK9 concentration in FH patients is 382.9 ± 132.6 ng / mL in the A system measurement system, 310.3 ± 146.6 ng / mL in the B system measurement system, and 89.7 in the C system measurement system. It was ± 90.6 ng / mL. In any system, FH patients had significantly higher values than healthy subjects (A system and C system measurement system, p <0.0001; B system measurement system, p = 0.016).

(2) Relationship between measurement systems of PCSK9 detection sandwich ELISA In healthy subjects, the correlation between the A system measurement system and the B system measurement system is y = 0.957x + 1.114, R ² = 0.940, and the A system measurement system. The correlation with the C system measurement system is y = 0.115x-1.449, R ² = 0.768, and the correlation between the A system measurement system and (B system measurement system + C system measurement system) is y = 1.072x-0.335, R ² = 0.961 Met. From this result, it was shown that the PCSK9 A system measurement system almost coincides with (PCSK9 B system measurement system + PCSK9 C system measurement system). That is, it was shown that most of PCSK9 in serum exists in a heterodimer type, and some 10% exist as a non-heterodimer type (FIG. 9).

In FH patients, the correlation between the A system measurement system and the B system measurement system is y = 0.768x + 20.598, R ² = 0.515, and the correlation between the A system measurement system and the C system measurement system is y = 0.283x-20.745, R ² = 0.207, the correlation between the A-system measurement system and (B-system measurement system + C-system measurement system) was y = 1.051x−0.147, and R ² = 0.942. In FH patients, the correlation between the A system measurement system and the (B system measurement system + C system measurement system) is the same as that of healthy subjects, but the correlation between the A system measurement system and the B system measurement system or the C system measurement system is Unlike healthy individuals, it was shown that the correlation was poor. In other words, in FH patients, the total amount of serum PCSK9 is higher than that in healthy subjects, and is largely due to the fact that the ratio of the B-system measurement system or C-system measurement system is greatly different. It was shown to be due to the increase in terror dimer type.

  In addition, although the abundance ratio of heterodimer and non-heterodimer varies, A-system and B-system + C-system show good positive correlation, so B-system and C-system correctly correct heterodimer and non-heterodimer in serum respectively It was shown that it was quantified (FIG. 10).

  Furthermore, the ratio of the A system measurement system to the B system measurement system (A system / B system ratio) is 1.05 ± 0.08 for healthy subjects and 1.88 ± 3.88 for FH patients, as well (B system measurement system + C system measurement system). The ratio of the B system to the B system ((B system + C system) / B system ratio) is 1.12 ± 0.02 for healthy subjects and 1.84 ± 3.39 for FH patients, both of which are significantly higher than those of healthy subjects (P <0.0001) It was shown to be high. The ratio of the A system measurement system to the C system measurement system (A system / C system ratio) is 9.39 ± 1.55 for healthy subjects and 6.73 ± 3.21 for FH patients, as well (B system measurement system + C system measurement system). The ratio of the measurement system to the C system ((B system + C system) / C system ratio) is 10.08 ± 1.93 for healthy subjects and 7.18 ± 3.62 for FH patients, both of which are significantly higher than those of healthy subjects (P <0.0001) It was shown to be low. The B / C ratio was 9.08 ± 1.93 for healthy subjects and 6.18 ± 3.65 for FH patients, indicating that FH patients were significantly lower (P <0.0001) than healthy subjects (Table). 2).

  These results indicate that in FH patients, the amount of PCSK9 (heterodimer type PCSK9) by the B system measurement system is higher than that of healthy subjects, the amount of PCSK9 (non-heterodimer type PCSK9) by the C system measurement system is high, and the heterodimer type PCSK9. It was shown that FH can be distinguished by these ratios.

(3) Immunoprecipitation of PCSK9 in human serum Serum PCSK9 molecules were examined by immunoprecipitation using a non-heterodimeric PCSK9-specific anti-PCSK9 antibody B1G. As described above, 100 μL of FH patient serum was reacted with anti-PCSK9 antibody B1G, and immunoprecipitation was performed.

  As a result, a 53 kDa band was detected in a serum sample (sample numbers 1 to 4) having a high amount of non-heterodimer type PCSK9, similar to Furin-cleaved rhPCSK9.

5. Association between PCSK9 and arteriosclerosis Serum lipid parameters [total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDLC), LDL] for 148 FH patients Cholesterol (LDLC), Remnant cholesterol (RLP-C), Lp (a), Apolipoprotein AI (Apo AI), Apolipoprotein B (Apo B), Apolipoprotein E (Apo E)], Fasting blood glucose (FBS), fasting insulin, hemoglobin A1c (HbA1c), high sensitivity CRP (hsCRP), carotid artery thickness (IMT) max-IMTR · max-IMTL · mean-IMTR · mean-IMTL, Achilles tendon xanthoma (Ach R, Ach L), liver / kidney function (ALT, AST, -GTP, CPK, Cr), diastolic blood pressure (SBP), systolic blood pressure (DBP), and were examined the correlation between the pulse rate.

  As a result, the PCSK9 A system measurement system (total of heterodimer type PCSK9 and non-heterodimer type PCSK9) is TG (r = 0.201, p = 0.0332), Lp (a) (r = 0.253, p = 0.0087), max-IMTR (r = 0.234, p = 0.0339), max-IMTL (r = 0.327, p = 0.0029), mean-IMTR (r = 0.325, p = 0.0033), Achilles tendon xanthoma R (r = 0.300, p = 0.0070), Achilles tendon xanthoma L (r = 0.250, p = 0.0242), ALT (r = 0.286, p = 0.0025), AST (r = 0.214, p = 0.0235) were significantly positively correlated with PCSK9 (A system measurement) System + B system measurement system) (total of heterodimer type PCSK9 and non-heterodimer type PCSK9) is TG (r = 0.200, p = 0.0341), max-IMTL (r = 0.305, p = 0.0055), mean-IMTR (r = 0.316, p = 0.0042), Achilles tendon xanthoma R (r = 0.282, p = 0.0113), Achilles tendon xanthoma L (r = 0.236, p = 0.0340), ALT (r = 0.308, p = 0.0011), AST (r = 0.239, p = 0.0116) significantly positive correlation, PCSK9 B system measurement system (heterodimer type PCSK9 amount) is mean-IMTR (r = 0.268, p = 0.0161), Achilles tendon Significantly positive correlation with xanthoma R (r = 0.300, p = 0.0070), Achilles tendon xanthoma L (r = 0.250, p = 0.0242), ALT (r = 0.212, p = 0.0246) Non-heterodimer type PCSK9 amount) was significantly positively correlated with max-IMTL (r = 0.255, p = 0.0200), mean-IMTR (r = 0.421, p = 0.0001), CPK (r = 0.200, p = 0.0340) Was significantly inversely correlated with HDLC (r = −213, p = 0.0242). That is, by measuring the amount of PCSK9, it was revealed that not only the diagnosis of FH but also the relationship with arteriosclerosis was shown (Table 3).

  From the above, it was revealed that this measurement system (PCSK9 molecular form-specific protein mass measurement system by sandwich ELISA method) can quantify the total amount of PCSK9, the amount of heterodimer, and the amount of non-heterodimer in human blood. In particular, non-heterodimer is about 10% of all PCSK9 in healthy subjects, whereas the proportion of non-heterodimer is high in FH patients, and high-value non-heterodimer is derived from the 53kDa segment It was revealed that this measurement system is useful for diagnosis of FH and arteriosclerosis.

Claims (10)

A method for detecting hypercholesterolemia, wherein human PCSK9 in a blood sample is quantified using an antibody against the component, and hypercholesterolemia is detected based on the quantified value, wherein the quantification of human PCSK9 is a heterodimer performed by the quantification step of human PCSK9 non heterodimer of human PCSK9 formed by Furin process, the quantification step, bind to both human PCSK9 of (a) heterodimer of human PCSK9 and the non heterodimer monoclonal antibodies, and, (b) the bind against non heterodimer human PCSK9, monoclonal antibody does not bind to human PCSK9 heterodimer, one of mutually different solid-phase antibody and detection Performed by sandwich ELISA used as an antibody Detection method of hypercholesterolemia. The detection of hypercholesterolemia is performed by one or more indicators selected from the group consisting of the following (A), (B), (C), (D), and (E). 2. The method for detecting hypercholesterolemia according to 1 .
( A ) An index for detecting hypercholesterolemia when the quantitative value in the quantitative step according to claim 1 is significantly larger than that of a healthy person.
( B ) Quantitative value of human PCSK9 of both heterodimer and non-heterodimer obtained by Furin treatment of heterodimer human PCSK9 / quantitative value in the quantification step according to claim 1 , wherein the quantified value is a healthy person This is an index for detecting cases significantly lower than that as hypercholesterolemia.
( C ) The sum of the quantitative value of heterodimer human PCSK9 and the quantitative value in the quantitative step according to claim 1 / the quantitative value of heterodimer human PCSK9 , wherein the quantitative value is significantly greater than that of a healthy person It is an index for detecting cases as hypercholesterolemia.
( D ) The sum of the quantitative value of heterodimer human PCSK9 and the quantitative value in the quantitative step according to claim 1 / the quantitative value in the quantitative step according to claim 1 , wherein the quantitative value is higher than that of a healthy person This is an index for detecting a significantly small case as hypercholesterolemia.
( E ) Quantitative value of heterodimer human PCSK9 / quantitative value in the quantification step according to claim 1 , wherein the quantitative value is significantly smaller than that of a healthy person as an index for detecting hypercholesterolemia is there. Quantification step of quantitative values of the heterodimer and formed by Furin treated human PCSK9 heterodimer non heterodimer both human PCSK9 as detection antibody and solid phase antibody, both heterodimeric human PCSK9 and the said non heterodimer The method for detecting hypercholesterolemia according to claim 2, which is carried out by sandwich ELISA using a monoclonal antibody that binds to both human PCSK9. The quantification step of the quantitative value of the heterodimeric human PCSK9 is as follows: (a) a monoclonal antibody that binds to both the heterodimeric human PCSK9 and the non-heterodimeric human PCSK9 obtained by Furin treatment of the heterodimeric human PCSK9. , and, as binding to relative (b) heterodimer of human PCSK9, but the non-monoclonal antibody that does not bind against heterodimer of human PCSK9, either one, mutually different solid-phase antibody and detection antibody The method for detecting hypercholesterolemia according to claim 2 or 3, which is carried out by a sandwich ELISA method to be used. The method for detecting hypercholesterolemia according to any one of claims 1 to 4 , wherein the hypercholesterolemia to be detected is familial hypercholesterolemia. The method for detecting hypercholesterolemia according to any one of claims 1 to 5 , wherein the blood sample is serum. A method for detecting arteriosclerosis in which human PCSK9 in a blood sample is quantified using an antibody against the component, and arteriosclerosis is detected based on the quantified value . Human PCSK9 is quantified by quantifying the heterodimeric human PCSK9. performed by quantification step of human PCSK9 of Furin treated to become non-heterodimer, the quantification step, a monoclonal antibody that binds to both the (a) human human PCSK9 and the non heterodimers heterodimers PCSK9, and , (b) to bind to the non heterodimer human PCSK9, but the sandwich used monoclonal antibodies that do not bind to human PCSK9 heterodimer, either one of, as a solid phase and detecting antibodies different from each other A method for detecting arteriosclerosis, which is performed by ELISA. The method for detecting arteriosclerosis according to claim 7 , wherein the detection of arteriosclerosis is performed according to the following index (A) or (B) .
( A ) An index for detecting arteriosclerosis when the quantitative value in the quantitative step according to claim 1 is significantly larger than that of a healthy person.
( B ) An index for detecting arteriosclerosis when the sum of the quantitative value of human PCSK9 as a heterodimer and the quantitative value in the quantitative step according to claim 1 is significantly larger than that of a healthy person. The quantification step of the quantitative value of the heterodimeric human PCSK9 is as follows: (a) a monoclonal antibody that binds to both the heterodimeric human PCSK9 and the non-heterodimeric human PCSK9 obtained by Furin treatment of the heterodimeric human PCSK9. , and, as binding to relative (b) heterodimer of human PCSK9, but the non-monoclonal antibody that does not bind against heterodimer of human PCSK9, either one, mutually different solid-phase antibody and detection antibody The method for detecting arteriosclerosis according to claim 8, which is performed by a sandwich ELISA method to be used. The method for detecting arteriosclerosis according to any one of claims 7 to 9 , wherein the blood sample is serum.