JP2015006174A - Standard substance for pcsk9 measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a standard substance in a detection system of a human PCSK9 60 kDa segment (un-heterodimer) used for e.g. screening of lipid metabolism improvement agents, or to provide a monoclonal antibody specific to the human PCSK9 segment concerned, and thereby to support implementation of the detection system concerned.SOLUTION: The invention relates to obtaining a human PCSK9 60 kDa segment (un-heterodimer) as a recombinant peptide whose host is an animal cell; and further to obtaining, using this as the above standard substance, a hybridoma producing a monoclonal antibody corresponding to a conformation of the human PCSK9 which reacts with the PCSK9 segment concerned, and does not crossreact with the human PCSK9 heterodimer and a human 53 kDa segment.

Description

  The present invention relates to a measurement standard substance used in a method for measuring a specific physiologically active substance useful for screening a new drug, confirming the effect of the drug, and the like.

  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.

  In such a background, PCSK9 (proprotein convertase subtilisin / kexin type 9) has attracted attention. PCSK9, also called NARC-1 (Neural apoptosis-regulated convertase 1), is the ninth enzyme of the proprotein convertase family belonging to the subtilisin-like serine protease kexin. Human 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 (signal peptide), 31-152 is pro domain (prodomain), 153-452 is catalytic domain (protease catalytic domain), and 453-692 is C-. It forms a terminal domain (C-terminal domain).

  Human PCSK9 is an approximately 74 kDa proPCSK9 (PCSK9 precursor) from which the above signal peptide has been removed, and autolysis between the glutamine at amino acid sequence 152 and the serine at 153 amino acid by the serine protease activity of PCSK9 itself in the endoplasmic reticulum. And is converted into a mature segment that contains a protease catalytic domain at the N-terminal side of about 14 kDa pro segment (prosegment) and the C-terminal side at about 60 kDa. These two fragments form a complex (PCSK9 heterodimer) so that the prosegment closes the active center of the mature segment, and then move to the Golgi body as the complex and are secreted extracellularly (Non-patent Document) 1: Seidah NG, et al. Proc Natl AcadSci USA, 2003; 100: 928-933, Non-patent document 6: Naureckiene S, et al. Arch Biochem Biophys, 2003; 420: 55-67, Non-patent document 7: Benjannet S, et al. J Biol Chem, 2004; 279: 48865-48875).

  Heterodimeric PCSK9 secreted into the blood binds to the LDL receptor EGF-A and induces the receptor to degrade as described above. The promotion of degradation of the LDL receptor by PCSK9 is a phenomenon independent of PCSK9's own protease activity (serine protease activity), and it inhibits recycling of the LDL receptor by the molecular chaperone action of PCSK9, that is, induction to lysosomes. It has been considered, but no conclusion has yet been reached (Non-patent Document 8: 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. For example, in human PCSK9, cleavage of PCSK9 between the amino acid sequence 218th arginine and the 219th glutamine is recognized by the action of proprotein convertase Furin and the like. By this action, the mature segment domain of the PCSK9 heterodimer is decomposed into a segment of about 7 kDa on the N-terminal side (7 kDa segment) and a ΔN218 segment (53 kDa segment) of about 53 kDa on the C-terminal side. The structure collapses. Fragments after degradation (7 kDa segment and 53 kDa segment) are also secreted outside the cell. Heterodimer PCSK9 that has not undergone degradation by Furin has a function of binding to the LDL receptor and inhibiting the above-mentioned recycling and leading to degradation of the LDL receptor, whereas non-heterodimer having undergone degradation by Furin. Dimer-type PCSK9 does not have such a function (Non-patent Document 9: 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 10: 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 gain-of-function and loss-of-function that can not promote LDL receptor degradation are found It has been clarified that they exhibit hypercholesterolemia and hypocholesterolemia, respectively.

  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 11: 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 12: 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).

  Under such a background, the present applicant quantifies PCSK9 in a blood sample using an antibody against the component, and detects hypercholesterolemia based on the quantified value. (Patent document 6) was filed.

Special table 2010-536384 gazette Special table 2010-523135 gazette Special table 2010-508817 Special table 2010-501952 gazette US Publication No. 2010/166768 JP 2012-237752 A

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 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

  FIG. 1 is a schematic diagram of PCSK9 (human) also disclosed in Patent Document 6, and simply represents recognition of conventional PCSK9. Although it overlaps with the above, it is an important premise and will be described again. In the description of FIG. 1, the two terms “domain” and “segment” may be used with the same sign, but “domain” is an expression that emphasizes the presence state that is potentially recognized in PCSK9. “Segment” is an expression that emphasizes the state in which the relevant part is actually separated from other PCSK9 molecules.

  Human PCSK9 is a peptide (1) consisting of a total length of 692 amino acids, wherein amino acids 1-30 are a signal peptide (11), 31-152 is a prodomain (12), 153-452 is a protease catalytic domain (13), 453-692 Forms the C-terminal domain (14). FIG. 1 (a) shows this peptide (1), which is “preproPCSK9” (pre-PCSK9 precursor), the first form of human PCSK9 in vivo. The pre-PCSK9 precursor has a known nucleotide sequence and amino acid sequence (preproPCSK9, amino acids 1-692) (NCBI reference sequences: NM_174936 and NP_777596: SEQ ID NOs: 1 and 2).

  Next, FIG. 1 (b) shows the formation of a PCSK9 precursor (2) of about 74 kDa from which the signal peptide (11) is removed, which acts on the extracellular secretion of the prePCSK9 precursor (1).

  The PCSK9 precursor (2) synthesized in vivo in this manner causes autolysis between the glutamine at amino acid sequence 152 and the serine at 153 amino acid sequence by the serine protease activity of PCSK9 itself in the endoplasmic reticulum. It is converted to a pro-segment (12) about 14 kDa on the side and a mature segment (13.14) containing the protease catalytic domain (13) on the C-terminal side about 60 kDa. These two regions [(12) and (13 · 14)] have a heterodimeric structure in which the prosegment (12) is non-covalently bonded so as to block the active center of the mature segment (13 · 14). After forming the PCSK9 heterodimer (3), the heterodimer (3) is transferred to the Golgi apparatus and secreted extracellularly. FIG. 1 (c) shows the formation of this PCSK9 heterodimer (3). The prodomain of PCSK9 functions as an inhibitor of protease activity, and although the PCSK9 heterodimer (3) is inactive as a proteolytic enzyme (serine protease), LDL-degrading activity is observed. This PCSK9 heterodimer (3) secreted into the blood binds to the LDL receptor EGF-A and induces the receptor to degrade. Thus, the promotion of degradation of the LDL receptor by PCSK9 is a phenomenon independent of the protease activity (serine protease activity) of PCSK9 itself. This phenomenon is a factor causing an increase in blood LDL cholesterol level.

  On the other hand, it is reported that some PCSK9 is further decomposed in the process of being secreted from the Golgi apparatus outside the cell. That is, it is cleaved between 218th arginine and 219th glutamine by the action of proprotein converting enzyme Furin and the like. By this action, the mature segment (13, 14) is decomposed into a 7 kDa segment (15) of about 7 kDa on the N-terminal side and a 53 kDa segment (ΔN218 segment) (4) of about 53 kDa on the C-terminal side, and the pro-segment (12) The heterodimeric structure with the mature segment (13, 14) is disrupted, and the fragments (7 kDa segment (15) and 53 kDa segment (4)) after degradation are also secreted outside the cell. FIG. 1 (d) illustrates this last stage. The 53 kDa segment (4) has no LDL-degrading activity like PCSK9 heterodimer.

  Thus, PCSK9 actually present in the blood in the living body is the PCSK9 heterodimer (3) of FIG. 1 (c) having the activity of promoting the degradation of the LDL receptor, or the heterozygous in which the degradation promoting activity has been lost. Only the 53 kDa segment (4), 7 kDa segment (15), and pro-segment (12) after degradation of the dimer were recognized. That is, the “60 kDa segment (non-heterodimer)” in which only the prodomain (12) is removed from the PCSK9 heterodimer in FIG. 1 (c) is obtained by in vitro treatment in the laboratory by acid treatment of the PCSK9 heterodimer (3). It was recognized that it was only present in, and at least not in human blood.

  It was the first challenge that led to the idea of the present invention to further examine the behavior of PCSK9 in vivo, to approach its essence and provide a way to contribute to the advancement of the diagnosis and medical fields. At that time, the contents of the present invention described later could not be predicted at all.

  The present inventor has disclosed three types of human PCSK9 detection systems disclosed in Patent Document 6, that is, “all human PCSK9 detection system”, “heterodimer-specific detection system”, and “non-heterodimer-specific detection system”. As a result of measurement of serum samples, a sample with “low heterodimer and non-heterodimer high” was found in part. According to the technical common sense at that time, the non-heterodimer component was considered to be the 53 kDa segment (4). In order to confirm this, immunoprecipitation was performed on the above serum sample using a non-heterodimer specific antibody [B1G: reacts with both 53 kDa segment (4) and 60 kDa segment (non-heterodimer)], When the molecular weight of the non-heterodimer component was confirmed, it was confirmed that it was a 60 kDa segment (non-heterodimer), which was completely different from the prediction based on the common general technical knowledge. As described above, PCSK9 is known to have an important role in human lipid metabolism, and the presence of this “60 kDa segment (non-heterodimer) in blood” also plays an important role in human lipid metabolism. It is thought to have.

  From this surprising result, in view of the critical role expected in lipid metabolism of the newly recognized “60 kDa segment in blood (non-heterodimer)”, the present inventor A novel antibody that reacts and does not cross-react with human PCSK9 heterodimer and 53 kDa human PCSK9 (hereinafter also referred to as the antibody of the present invention) ”was produced. Details of this will be described later.

  When this monoclonal antibody was used to directly reexamine the existence of a 60 kDa segment (non-heterodimer) in a human blood sample, it was confirmed that a “60 kDa segment (non-heterodimer)” exists in human blood. Was found.

  As will also be described later, the “60 kDa segment (non-heterodimer)”, like “human PCSK9 (53k) subjected to degradation by Furin”, has a reduced ability to degrade the LDL receptor. Confirmed by the inventor. Therefore, if the value of the “60 kDa segment (non-heterodimer)” in this blood is high and the amount of heterodimeric human PCSK9 is small, the LDL receptor involved in the first step of degrading LDL is difficult to be degraded. As a result, the LDL blood cholesterol level is expected to decrease. In addition, as described above, heterodimeric human PCSK9 tends to increase with the administration of statin drugs, which are therapeutic agents for hypercholesterolemia, and this is a resistance to the therapeutic agent treatment. On the other hand, if a drug capable of dissociating a prosegment from heterodimeric human PCSK9 is provided, it can be used not only as a therapeutic agent for hypercholesterolemia but also as a statin drug enhancer. Is expected to be possible.

  Therefore, a method for measuring human PCSK9 is provided that makes it possible to select candidate substances having the dissociation action in the course of developing therapeutic agents that dissociate prosegments as described above. In addition, providing a method for measuring human PCSK9 that makes it possible to monitor the effects of such therapeutic agents in a clinical setting becomes a general issue.

  And the subject in this invention is contributing to appropriate implementation of this measuring method by providing human PCSK9 used as a reference material in the said measuring method.

  The present inventor has conceived that the above problem can be solved by providing a PCSK9 measurement standard substance (hereinafter also referred to as a standard substance of the present invention) comprising human PCSK9 60 kDa segment (non-heterodimer). .

  The measurement of human PCSK9 in which the standard substance of the present invention is used is as follows: “In a test system for a test drug that decomposes human PCSK9 heterodimer into a prosegment and a 60 kDa segment (non-heterodimer), 60 kDa before and after use of the drug. "Measurement method for determining the ratio of segment (non-heterodimer) to human PCSK9 heterodimer or total human PCSK9" (hereinafter also referred to as the measurement method of the present invention). This measurement method of the present invention is novel as described later. It is possible to carry out using the antibody specific for a 60-kDa segment (non-heterodimer) of this, and it is suitable that the antibody used for this is a monoclonal antibody.

  When the measurement method of the present invention is used in a clinical field or an in vivo test field, that is, “whether or not human PCSK9 heterodimer has been broken down into a prosegment and a 60 kDa segment (non-heterodimer). "Is preferably performed using a blood sample. The blood sample includes whole blood, serum, plasma, etc., preferably serum or plasma is used, and serum is particularly preferred. Furthermore, when conducting drug screening in a basic drug discovery experiment, that is, when verifying whether or not “human PCSK9 heterodimer has a function of decomposing pro-segment and 60 kDa segment (non-heterodimer)”. Can use a solvent system to which human PCSK9 heterodimer is added.

  Based on the significance of the existence of the 60 kDa segment (non-heterodimer) as described above, the determination that the test drug is effective is based on the ratio of the 60 kDa segment (non-heterodimer) to the human PCSK9 heterodimer or the total human PCSK9. It is made with the rise after use.

  The measurement using the standard substance of the present invention may be a measurement for analyzing the function of a 60 kDa segment (non-heterodimer). That is, the 60 kDa segment (non-heterodimer) is a newly found form of PCSK9 in the living body newly discovered recently, and analyzing the detailed function of this existing form is a more useful method of using PCSK9. It is an important measurement in finding out. The standard substance of the present invention can be used for the measurement. The measurement method is not particularly limited, and may be, for example, in vitro, in vivo, or in situ. For example, when analyzing the LDL receptor degradation promoting action and the like in a 60 kDa segment (non-heterodimer), the standard substance of the present invention can be suitably used, but is not limited thereto.

  “Human PCSK9 60 kDa segment (non-heterodimer)”, which is an essential component of the standard substance of the present invention, is produced by using the host as an animal cell based on the base sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2. It is a human PCSK9 60 kDa segment (non-heterodimer) obtained by dissociating the prosegment in the human PCSK9 heterodimer and removing the prosegment and the mixed heterodimer.

  Examples of the above-mentioned “method for dissociating prosegments in human PCSK9 heterodimer” include acid treatment, heat treatment, protein denaturation treatment, surfactant treatment, and the like. Among these, acid treatment is preferred. . Specifically, the pH of the acid treatment is 5 or less, preferably 4 or less.

Furthermore, the above-mentioned “removal of prosegment and mixed heterodimer dissociated from human PCSK9 heterodimer”
(1) capturing a prosegment with an antibody specific to the prosegment, preferably a monoclonal antibody, and removing the captured prosegment and the mixed heterodimer, or
(2) An antibody specific to a human PCSK9 60 kDa segment (non-heterodimer), preferably a monoclonal antibody, is allowed to capture the human PCSK9 60 kDa segment (non-heterodimer) and the captured 60 kDa segment (non-heterodimer) ) Selectively,
Can be performed.

  In addition to the above, the present invention is based on the nucleotide sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2, and the prosegment is dissociated in human PCSK9 heterodimer produced using the host as an animal cell. A human PCSK9 60 kDa segment (non-heterodimer) production method (hereinafter also referred to as the production method of the present invention) is obtained by removing a segment and mixed heterodimer to obtain a human PCSK9 60 kDa segment (non-heterodimer). .

  Regarding the “method for dissociating prosegments in human PCSK9 heterodimer” and the “removal of prosegments and mixed heterodimers dissociated from human PCSK9 heterodimer” in the production method of the present invention, the above-mentioned standard substances of the present invention According to

  Furthermore, the present invention relates to the use of human PCSK9 60 kDa segment (non-heterodimer) as a standard substance for measuring PCSK9, that is, a method of using human PCSK9 60 kDa segment (non-heterodimer) as a standard substance for measuring PCSK9 (hereinafter, Also referred to as use of the present invention).

  “Measurement of human PCSK9” in the use of the present invention is the same as the above-mentioned standard substance of the present invention. In the test system of a test drug that decomposes human PCSK9 heterodimer into a prosegment and a 60 kDa segment (non-heterodimer), the drug Is a measurement for determining the ratio of the 60 kDa segment (non-heterodimer) before and after use to human PCSK9 heterodimer or total human PCSK9. In addition, as described above, it may be a measurement for functional analysis of a 60 kDa segment (non-heterodimer).

  Furthermore, the present invention relates to a hybridoma that produces a monoclonal antibody that reacts with the human PCSK9 60 kDa segment (non-heterodimer) and does not cross-react with the human PCSK9 heterodimer and the human 53 kDa segment (hereinafter referred to as the hybridoma of the present invention), Monoclonal antibodies produced by hybridomas (hereinafter also referred to as monoclonal antibodies of the present invention) are provided. Specific examples of the hybridoma and the monoclonal antibody of the present invention include a hybridoma deposited under the accession number NITE P-01582 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, and a monoclonal antibody produced therefrom.

  According to the present invention, (1) when selecting a drug having the action of degrading human PCSK9 heterodimer into a prosegment and a 60 kDa segment (non-heterodimer) at the development stage, (2) monitoring the clinical effect of the agonist Or (3) a standard substance composed of a 60 kDa segment (non-heterodimer) used for functional analysis of the 60 kDa segment (non-heterodimer) in vivo, and a 60 kDa segment (non-non-standard) as the standard substance Heterodimers) or methods of use are provided. Furthermore, a method for producing a human PCSK9 60 kDa segment (non-heterodimer), which is an essential component of the standard substance, is provided, and a monoclonal antibody specific for the human PCSK9 60 kDa segment (non-heterodimer) is provided.

It is the schematic which shows the conventional recognition content about human PCSK9. It is an electrophoretic drawing which shows the result of refinement | purification of a rh60 kDa segment (non-heterodimer). It is drawing which shows the result of confirmation of the purity by SDS polyacrylamide electrophoresis of a refined rh60 kDa segment (non-heterodimer). It is an electrophoretic drawing which shows the result of having examined the contents of cleavage by Furin processing of rhPCSK9 by Western blotting. It is an electrophoretic drawing which shows the result of having examined the reactivity with respect to rhPCSK9 by Western blotting. It is an electrophoretic drawing which shows the result of having examined the reactivity with respect to Furin cutting rhPCSK9 of each acquired monoclonal antibody by the immunoprecipitation method. It is an electrophoretic drawing by Western blotting showing the reactivity of a monoclonal antibody (8H9) specific for a 60 kDa segment (non-heterodimer). It is the electrophoresis drawing by a Western blotting which shows the result of having confirmed the specificity by a Furin process about the monoclonal antibody (8H9) specific for a 60 kDa segment (non-heterodimer). It is an electrophoretic drawing which shows the result of having confirmed the specificity by the immunoprecipitation method about the monoclonal antibody (8H9) specific to a 60 kDa segment (non-heterodimer). It is explanatory drawing of the human PCSK9 molecular form specific protein mass measuring system (A system, B system, C system) by sandwich ELISA method. It is an electrophoretic drawing which shows the result of having performed the immunoprecipitation about human PCSK9 in the serum sample (1-11) used in the Example. It is drawing which showed the calibration curve in the human PCSK9 molecular form specific protein mass measuring system (A system, B system, C system) by sandwich ELISA method. It is the figure which showed the dilution linearity with respect to the serum sample by the human PCSK9 molecular form specific protein mass measuring system (A system, B system, C system) by sandwich ELISA method. It is the figure which showed the analytical curve in the human PCSK9 molecular form specific protein mass measuring system (D system) by sandwich ELISA method. It is drawing which showed the dilution linearity with respect to the serum sample by the human PCSK9 molecular form specific protein mass measuring system (D system) by sandwich ELISA method. It is an electrophoresis drawing based on the immunoprecipitation method using monoclonal antibody 8H9, showing the presence of a 60 kDa segment (non-heterodimer) in human serum. It is drawing which shows the quantity of various PCSK9 contained in each fraction of the refinement | purification process of a 60 kDa segment (non-heterodimer). It is an electrophoretic drawing which shows the result of examination of the binding property of the monoclonal antibody specific for human PCSK9 to rhΔ152PCSK9 expressed in an insect cell expression system. It is a figure which shows the result of having compared the degradation activity of the LDL receptor in HepG2 cell in a human PCSK9 heterodimer and a 60 kDa segment (non-heterodimer). It is drawing which shows the result of having examined the usefulness in the case of using the measuring method of this invention for the screening of the chemical | medical agent which decomposes | disassembles human PCSK9 heterodimer.

1. Standard substance of the present invention The essential component of the standard substance of the present invention is “human PCSK9 60 kDa segment (non-heterodimer)” (sometimes simply referred to as “60 kDa segment (non-heterodimer)”). As described above, the 60 kDa segment (non-heterodimer) is a prosegment in a human PCSK9 heterodimer produced using the host as an animal cell based on the base sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2. It can be produced by dissociating to remove the prosegment and the mixed heterodimer.

  As an example of the base sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2, the base sequence shown in SEQ ID NO: 1 is preferably exemplified. Typically, the base sequence is amplified by using a gene amplification method such as RT-PCR using an appropriate amplification primer, the amplified product is transfected into an animal cell as a host, and the recombinant Obtain human PCSK9 (heterodimer) produced by the body. And it can produce by dissociating this prosegment and removing the said prosegment and the heterodimer mixed from here. The signal peptide originally present in the recombinant is cleaved when human PCSK9 is secreted from the host animal cell. Actually obtained human PCSK9 is a heterodimer in which 1 to 30 of SEQ ID NO: 2 are cleaved and the prosegment is non-covalently bound to the mature segment.

  The animal cell that is the host is not particularly limited, and for example, CHO cells, HEK293 cells, COS7 cells, NS0 cells and the like can be used.

  The desired human PCSK9 60 kDa segment (non-heterodimer) can be obtained by dissociating the human PCSK9 heterodimer produced in the recombinant animal cell and removing the prosegment and the mixed heterodimer.

  The outline of the heterodimer dissociation step and the prosegment and mixed heterodimer removal step are as described above, and specific examples will be described later in Examples.

  Known purification steps performed on recombinant peptides, ie, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, on the crude product of human PCSK9 60 kDa segment (non-heterodimer) obtained through these steps , Affinity chromatography, dialysis and the like can be performed.

  In this way, a human PCSK9 60 kDa segment (non-heterodimer) that is an essential component of the standard substance of the present invention can be obtained. And this can be used as a standard substance when performing the measuring method of the present invention.

2. Measurement method of the present invention The measurement method in which the standard substance of the present invention can be used is a method whose gist is to measure the 60 kDa segment (non-heterodimer) in the test system as described above, but it is roughly divided into three types. There is a purpose.

  The first object of the measurement method of the present invention is drug screening in drug discovery. In this case, an artificial human PCSK9 heterodimer is prepared in advance by a protein engineering method, and this is added to an appropriate solvent system, so that the human PCSK9 heterodimer and the 60 kDa segment (non-heterodimer) before addition of the test drug are added. Measure the ratio. Next, the test drug is added to the test system, and the ratio after incubation is measured. If the ratio of the 60 kDa segment (non-heterodimer) after the addition of the test drug is significantly increased at this ratio, the test drug has the action of dissociating the prosegment from the human PCSK9 heterodimer, and the desired lipid metabolism It can be listed as a candidate for an improving agent.

  When drug screening is performed by the measurement method of the present invention in a form using this solvent, the solvent environment is (1) the first solvent environment that “reacts the human PCSK9 heterodimer with the candidate drug”, and (2) “Reaction for performing quantification after reaction with candidate drug (antigen-antibody reaction etc.)” second solvent environment is used. In the first solvent environment, a solvent environment in which human PCSK9 heterodimer dissociates or denatures without a candidate drug is not preferred. In the second solvent environment, a solvent environment that inhibits a reaction that is a basis of a quantitative signal such as an antigen-antibody reaction is not preferable. In addition, it is preferable that the first solvent environment and the second solvent environment are similar to each other as much as possible. When the first solvent environment and the second solvent environment are different, for example, the concentration of human PCSK9 heterodimer in the first solvent environment is set high, and this can be performed by diluting in the second solvent environment. . This dilution ratio is preferably about 10 to 100 times.

  The solvent and buffer used through the first solvent environment and the second solvent environment are not particularly limited as long as this condition is satisfied. Specific examples include sodium phosphate, potassium phosphate, Tris-HCl, HEPES and the like.

  The pH of the solvent environment needs to be 5 or higher. This is the “condition for the human PCSK9 heterodimer not to dissociate” in the first solvent environment. Since the drug to be screened is a medicine, a pH of about 6.5 to 7.5 close to physiological conditions is preferable. Furthermore, the temperature of the solvent environment is 4 ° C. or higher which does not freeze, and the upper limit is a range where the human PCSK9 heterodimer is not thermally denatured or a candidate drug is not deactivated, but about 30 to 40 ° C. close to physiological conditions is preferable. .

  The concentration of human PCSK9 heterodimer in the first solvent environment is a concentration detectable by the quantification method used. For example, in the case of sandwich ELISA, the lower limit is about 1 ng / mL. However, in consideration of dilution in the second solvent environment, this concentration of about 10 times is necessary. A suitable concentration is about 100 to 500 ng / mL, which is the concentration of PCSK9 in human serum.

  The second object of the measurement method of the present invention is to check the effectiveness of the administered drug. In this case, the human PCSK9 heterodimer and the 60 kDa segment (non-heterogeneous) in the blood sample of the subject before and after administration of the lipid metabolism improving agent having the action of dissociating the prosegment from the human PCSK9 heterodimer developed by the above screening or the like. Measure the ratio of dimer). When the ratio of the 60 kDa segment (non-heterodimer) in the ratio after administration is significantly higher than that before administration of the drug, this is an index indicating that the drug is effective.

  The third object of the measurement method of the present invention is the functional analysis of “60 kDa segment (non-heterodimer)” newly discovered as an embodiment of PCSK9 that is present in the living body as described above.

  Thus, the measurement method of the present invention that can measure a 60 kDa segment (non-heterodimer) in a solvent system or blood has very important significance.

  The measurement of the 60 kDa segment (non-heterodimer) is preferably performed using the antibody of the present invention and a detection means that utilizes the antigen-antibody reaction between the antibody and the 60 kDa segment (non-heterodimer) in the test system. Specific examples include analysis methods using enzyme immunoassay (ELISA) method, radioimmunoassay (RIA) method, immunochromatography method, latex agglutination turbidimetric method, immunoprecipitation method, and the like. 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 addition, an immunoassay (FEIA, CLIA, CLEIA, ECLIA) method using fluorescence or chemiluminescence for detection is also suitable. In these measurements, the standard substance of the present invention is used as a standard substance for quantifying the 60 kDa segment (non-heterodimer).

  When the detection means is an ELISA method, for example, a blood sample or a test solvent (hereinafter may be collectively referred to as a test system solvent) is brought into contact with the antibody of the present invention immobilized on a microplate, and the test system is used. A soluble 60 kDa segment (non-heterodimer) in a solvent is bound to an immobilized antibody, and the soluble 60 kDa segment (non-heterodimer) bound to the solid phase is detected using another enzyme-labeled antibody against human PCSK9 or the like. Thus, the 60 kDa segment (non-heterodimer) in the test system constituting the measurement method of the present invention can be quantified. It is also possible to detect by reacting the immobilized antibody, the test system solvent and the enzyme-labeled antibody simultaneously.

  When immunoprecipitation is used as a detection means, for example, a 60 kDa segment (non-heterodimer) is bound to an immobilized antibody by contacting a test solvent with the antibody of the present invention immobilized on beads. By separating the 60 kDa segment (non-heterodimer) bound to this solid phase and detecting the 60 kDa segment (non-heterodimer) from this isolate, the quantification of the 60 kDa segment (non-heterodimer) in the test system solvent can be performed. It can be carried out. The means for detecting the 60 kDa segment (non-heterodimer) by immunoprecipitation is, for example, performing electrophoresis on the above-mentioned separation, and labeling the transcript of the electrophoresis pattern on the 60 kDa segment (non-heterodimer). There may be mentioned Western blotting in which an antibody is allowed to act to detect a band of a soluble 60 kDa segment (non-heterodimer).

  It is also possible to use a latex agglutination turbidimetry as a quantitative means. For example, an antibody against a 60 kDa segment (non-heterodimer) bound to latex particles is brought into contact with a test system solvent to form an aggregate of immune complexes due to an antigen-antibody reaction in a liquid phase, and the turbidity changes. By measuring the quantification of the 60 kDa segment (non-heterodimer) can be performed.

  In addition, human PCSK9 heterodimer is quantified in place of the antibody of the present invention by reacting with an antibody specific to human PCSK9 heterodimer, ie, human PCSK9 heterodimer, but with a 60 kDa segment (non-heterodimer). And an antibody that does not cross-react with the 53 kDa segment.

  As described above, the quantification of human PCSK9 in the measurement method of the present invention is performed by the quantification step (1) of the 60 kDa segment (non-heterodimer) and the quantification step (2) of the human PCSK9 heterodimer. These quantification steps (1) and (2) are performed in a common analyte or solvent system, but it is preferable that each quantification step itself is performed separately. For example, these quantification steps (1) and (2) in the case of drug screening are performed separately in a second solvent environment, preferably under the same conditions, after contact with the candidate drug in the common first solvent environment described above. It is preferred that this is done.

  When sandwich ELISA, which is one of the preferred quantification means for carrying out the measurement method of the present invention, is used, the quantification step (1) of the 60 kDa segment (non-heterodimer) is performed as a solid phase antibody as described above. Using the antibody of the present invention, the detection can be performed by sandwich ELISA using an antibody that binds to a 60 kDa segment (non-heterodimer) or a 53 kDa segment. Conversely, an antibody that binds to a 60 kDa segment (non-heterodimer) or a 53 kDa segment can be used as a solid phase antibody, and the antibody of the present invention can be used as a detection antibody. In the identification amount step (2), a monoclonal antibody that binds to human PCSK9 heterodimer but does not bind to non-heterodimer human PCSK9 is used as a solid phase antibody, and a 60 kDa segment (nonheterogeneous) is detected as a detection antibody. Dimer) and a 53 kDa segment can be performed by a sandwich ELISA method using an antibody. Conversely, an antibody that binds to both a 60 kDa segment (non-heterodimer) and a 53 kDa segment is used as a solid phase antibody and binds to human PCSK9 heterodimer as a detection antibody, but binds to non-heterodimer human PCSK9. It is also possible to carry out by sandwich ELISA using a monoclonal antibody that does not.

  An example of a suitable functional analysis when performing the measurement method of the present invention is an analysis of LDL receptor degradation promoting action. This is, for example, after culturing by adding a PCSK9 heterodimer purified product or a 60 kDa segment (non-heterodimer) purified product to cells expressing the LDL receptor, and then quantifying the cell LDL receptor by a method such as ELISA, Western blot, or FACS. By doing so, the LDL receptor degradation promoting function of the 60 kDa segment (non-heterodimer) can be analyzed.

  In this way, by measuring the ratio of the 60 kDa segment (non-heterodimer) in the test system solvent to human PCSK9 heterodimer or all human PCSK9, using the measured value as an index, (1) Screening, (2) It is possible to judge the effectiveness of a drug that converts human PCSK9 heterodimer into a 60 kDa segment (non-heterodimer). In addition, (3) functional analysis of a 60 kDa segment (non-heterodimer) can be performed by measuring the interaction between a specific biological substance and a 60 kDa segment (non-heterodimer).

3. As described above, the antibody of the present invention is “an antibody that reacts with a 60 kDa segment (non-heterodimer) and does not cross-react with the human PCSK9 heterodimer and the 53 kDa segment” (the antibody of the present invention). . The antibody of the present invention is a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody and is practical. This is because a monoclonal antibody has only a specific antigenic determinant, and the antigenic determinant of an antibody having a desired property is limited.

  As described above, the antibody of the present invention is suitably used in the “step of removing heterodimers mixed with prosegments” for producing the 60 kDa segment (non-heterodimer) that is an essential component of the standard substance of the present invention. It is done. In the measurement method of the present invention, it is necessary to quantify the 60 kDa segment (non-heterodimer) in the test system. In this case, direct measurement by using an antibody specific for the 60 kDa segment (non-heterodimer) among human PCSK9 is efficient.

  The antibody production method of the present invention can be carried out according to a conventional method, but requires all or a part of human PCSK9 as an antigen. Human 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, recombination of human PCSK9 (recombinant) using protein engineering techniques. Obtained by producing protein. The recombinant protein can be produced and obtained according to the above-described production method of recombinant human PCSK9 heterodimer or 60 kDa segment (non-heterodimer). A modified recombinant protein encoded by a partially modified gene of human PCSK9, in which the nucleotide sequence of the original human PCSK9 gene is partially modified, is also possible as long as it does not lose its properties as an antigen for human PCSK9. Human PCSK9 as an antigen used here does not necessarily need to be the whole of human PCSK9, and may be a partial peptide fragment thereof. Since the antigenic determinant of an antibody having particularly desirable properties is in the N-terminal side about 7 kDa segment (15) (SEQ ID NO: 2 153 to 218) in FIG. 1 (d), at least the about 7 kDa segment is immunized. It is reasonable to choose as the original. By using such a pre-screened immunogen, the antibody of the present invention can be produced more efficiently. A partial peptide of human PCSK9 includes a human PCSK9 fragment in which a partial fragment of the human PCSK9 gene is expressed, a protease-treated product of human 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, such as chemically synthesized peptides by a solid phase method or a liquid phase method.

  In human PCSK9 obtained in this manner, a hapten can be bound to improve the immunogenicity of the antigen, particularly when human PCSK9 used as an immunizing antigen is a partial peptide of a small molecule. 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.

  Immunization can be performed by a general method, for example, by administering the immunizing antigen to an animal to be immunized intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, or the like.

  Polyclonal antibodies can be produced from immune sera derived from immunized animals using all or part of the 7 kDa segment of human PCSK9 molecule as an immunizing antigen.

  The monoclonal antibody of the present invention produces a hybridoma of an animal immune cell immunized with all or part of the human PCSK9 molecule and an animal myeloma cell, thereby selecting a clone that produces an antibody having a desired property. 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 human PCSK9 molecule.

  Also, the animal to be immunized is not particularly limited, and mice, rats, hamsters, rabbits, goats, sheep, chickens, etc. can be widely used, but 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 production of monoclonal antibodies, such as spleen cells and ascites 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 “desired property” to be provided for the antibody of the present invention is “reacts with a 60 kDa segment (non-heterodimer) and does not cross-react with human PCSK9 heterodimer and 53 kDa segment” as described above. Because of the presence of the antigenic determinant in the 7 kDa segment, there is no antigenic determinant in the 53 kDa segment, so no cross-reaction occurs. Heterodimer). In addition to this, human PCSK9 heterodimer has a prosegment that blocks the serine protease active center composed of aspartic acid at amino acid sequence 186 (within the 7 kDa segment region), histidine at 226, and serine at 386. Therefore, it is difficult for an antibody having an antigenic determinant in the 7 kDa segment region and a portion that is three-dimensionally covered by the prosegment to bind to the human PCSK9 heterodimer.

After clones producing monoclonal antibodies are obtained by the above-described operation, it is necessary to screen clones producing monoclonal antibodies having desired properties from these clones. Examples of the screening for this antibody include conventional methods, that is, immunoprecipitation, ELISA, Western blotting and the like. In any of these methods, the reactivity of various forms of human PCSK9 produced by protein engineering techniques with the monoclonal antibodies obtained from each clone is examined, and whether the monoclonal antibodies have the desired properties. This is a method for determining whether or not. Specifically, a monoclonal antibody production clone having the following properties (1) to (3) can be selected as the monoclonal antibody production clone of the present invention. In addition, a polyclonal antibody having the following properties (1) to (3) can be used as the antibody of the present invention.
(1) Although it binds to the 7 kDa segment, it does not bind to the separated 53 kDa segment.
(2) It does not bind to human PCSK9 heterodimer to which a prosegment is bound.
(3) It binds to a 60 kDa segment (non-heterodimer) that is not bound to a prosegment.

  The monoclonal antibody or polyclonal antibody produced from the thus selected clone can be used as the antibody of the present invention having specific reactivity with the 60 kDa segment (non-heterodimer). As described above, the antibody of the present invention selectively captures the 60 kDa segment (non-heterodimer) when producing the 60 kDa segment (non-heterodimer) that is an essential component of the standard substance of the present invention, Can be used to remove heterodimers mixed with prosegments found in

  Furthermore, the antibody of the present invention can be used, for example, in the measurement method of the present invention. The antibody is subjected to labeling treatment, that is, enzyme labeling treatment, fluorescence labeling treatment, isotope labeling treatment, etc. Can be done according to

  As will be described later, an antibody capable of capturing a 60 kDa segment (non-heterodimer) in the test system is used as an immobilized antibody immobilized on a solid phase, which is one of preferred embodiments.

  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.

  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 five types of monoclonal antibodies described below are deposited at the Patent Microorganism Depositary Center for Product Evaluation 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” as -22110 is the accession number FERM P-22111, and the hybridoma “Mouse-Mouse hybridoma PCSK9-G12D” producing the monoclonal antibody “G12D” is The depository number FERM P-22112 and the hybridoma “Mouse-Mouse hybridoma PCSK9-8H9” producing the monoclonal antibody “8H9” have been received at the depository organization as the accession number NITE P-01582.

  In the description of the present specification, “PCSK9” means “human PCSK9” unless otherwise specified.

1. Production of essential components of the standard substance of the present invention After preparing the basic recombinant human full-length PCSK9 and confirming that its form is a heterodimer, the human PCSK9 60 kDa segment which is an essential component of the standard substance of the present invention (Non-heterodimer) was produced in two ways.

(1) Preparation of Recombinant Human Full-Length PCSK9 A complementary strand DNA corresponding to human PCSK9 full-length (amino acid number 1-692) 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 prepare 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 and stably. Clone CHO-K1 / PCSK9 cells expressing recombinant human PCSK9 (rhPCSK9) were prepared.

The CHO-K1 / PCSK9 cells were cultured in a serum-free 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 (manufactured by Clontech) and reacted at 4 ° C. overnight. The column is packed with TALON Resin, 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 20 mL of 20 mM containing 200 mM imidazole. Elute with phosphate buffer (pH 8.0). Then, 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 ( After washing with 1.5 mL of 20 mM phosphate buffer solution (pH 8.0) containing 100 mM sodium chloride, elution was performed with 3 mL of 20 mM phosphate buffer solution (pH 8.0) containing 200 mM sodium chloride. This eluted fraction was designated as purified rhPCSK9.

  As mentioned later in “2 (6) Reactivity of monoclonal antibody to Furin-cleaved rhPCSK9”, when immunoprecipitation using an antibody against a pro-segment (G12D) is performed, a mature segment (60 kDa) is found in Furin-untreated rhPCSK9. In contrast to immunoprecipitation, the mature segment (53 kDa) does not immunoprecipitate with Furin-cleaved rhPCSK9. From this, it is clear that Furin-untreated rhPCSK9, that is, the purified rhPCSK9 described above, forms a heterodimer.

  In the example disclosed in Patent Document 6 (paragraph [0067] of Patent Document 6), it was disclosed that when the purified rhPCSK9 was subjected to SDS polyacrylamide electrophoresis, the pro-segment was present in an off form. However, since SDS is a protein denaturant, the non-covalent bond connecting the pro-segment and the 60 kDa segment (non-heterodimer) is considered to be a result of removal.

(2) Method for producing essential components of standard substance of the present invention using monoclonal antibody specific to prosegment <Preparation of culture supernatant containing rhPCSK9 heterodimer>
The above-described CHO-K1 / PCSK9 cells were cultured in serum-free, low protein medium CHO-S-SFM II (manufactured by Invitrogen) at 37 ° C. and 5% CO ₂ , and rhPCSK9 heterodimer-containing culture supernatant was collected.

<Preparation of anti-prosegment antibody G12D-binding sepharose (G12D-seph)>
The column was filled with 0.5 mL of NHS-activated Sepharose (manufactured by GE Healthcare) and washed with 10 mL of 1 mM HCl. Subsequently, it mixed with 6 mL of G12D (manufacturing example mentioned later) solution melt | dissolved in the density | concentration of 1 mg / mL in 100 mM sodium hydrogencarbonate buffer (pH 8.3) containing 500 mM sodium chloride, and was made to react at room temperature for 2 hours. Sepharose after the reaction was packed in a column and washed with 100 mM sodium bicarbonate buffer (pH 8.3) containing 5 mM 500 mM sodium chloride. Next, the cells were washed with 5 mL of 100 mM Tris-HCl buffer (pH 8.5), suspended in 1 mL of 100 mM Tris-HCl buffer (pH 8.5), and reacted at 4 ° C. overnight. After the reaction, Sepharose was packed into a column, washed with 1.5 mL of 100 mM sodium acetate buffer (pH 4.0) containing 500 mM sodium chloride, and then washed with 1.5 mL of 100 mM Tris-HCl buffer (pH 8.5). . Washing with these two buffer solutions was repeated 6 times, followed by washing with 5 mL of PBS to obtain G12D-seph.

<Purification of rh60 kDa segment (non-heterodimer)>
18 mL of 1M citrate buffer (pH 3.0) was added to 300 mL of the above culture supernatant, the pH of the culture supernatant was adjusted to 4.0, and then left at room temperature for 1 hour to dissociate the heterodimer. 27 mL of 2M Tris was added, the culture supernatant was adjusted to pH 8.0, mixed with 7 mL of TALON Resin (Clontech), and reacted at 4 ° C. overnight. This TALON Resin is packed in a column, washed with 35 mL of 25 mM phosphate buffer (pH 8.0) containing 500 mM sodium chloride, followed by 10 mL of 20 mM HEPES buffer (pH 7.5), and 20 mL of 20 mM HEPES containing 200 mM imidazole. Elute with buffer (pH 7.5). Next, this eluate was applied to a column packed with 0.6 mL of DEAE-Sepharose CL-6B (GE Healthcare) previously equilibrated with 20 mM HEPES buffer (pH 7.5), and 5 mL of 20 mM HEPES buffer ( After washing with 2 mL of 20 mM HEPES buffer (pH 7.5) containing 100 mM sodium chloride, elution was performed with 2 mL of 20 mM HEPES buffer (pH 7.5) containing 150 mM sodium chloride. Next, this eluate was applied to a column packed with 0.1 mL of G12D-seph previously equilibrated with 20 mM HEPES buffer (pH 7.5) containing 150 mM sodium chloride, and the flow-through fraction was purified to a purified rh60 kDa segment (non-heterodimer). It was.

  The purified rh60 kDa segment (non-heterodimer) was subjected to SDS polyacrylamide electrophoresis, the band was detected by silver staining of the gel, and the purified purity was confirmed (FIG. 2).

(3) Method for producing essential components of standard substance of the present invention using monoclonal antibody specific to 60 kDa segment (non-heterodimer) <Preparation of culture supernatant containing rhPCSK9 heterodimer>
The above-described CHO-K1 / PCSK9 cells were cultured in serum-free, low protein medium CHO-S-SFM II (manufactured by Invitrogen) at 37 ° C. and 5% CO ₂ , and rhPCSK9 heterodimer-containing culture supernatant was collected.

<Preparation of 60 kDa segment (non-heterodimer) antibody 8H9-binding sepharose (8H9-seph)>
The column was filled with 0.5 mL of NHS-activated Sepharose (manufactured by GE Healthcare) and washed with 10 mL of 1 mM HCl. Next, 6 mL of 8H9 solution dissolved at a concentration of 1 mg / mL in 100 mM sodium hydrogen carbonate buffer (pH 8.3) containing 500 mM sodium chloride was mixed and reacted at room temperature for 2 hours. Sepharose after the reaction was packed in a column and washed with 100 mM sodium bicarbonate buffer (pH 8.3) containing 5 mM 500 mM sodium chloride. Next, the cells were washed with 5 mL of 100 mM Tris-HCl buffer (pH 8.5), suspended in 1 mL of 100 mM Tris-HCl buffer (pH 8.5), and reacted at 4 ° C. overnight. After the reaction, Sepharose was packed into a column, washed with 1.5 mL of 100 mM sodium acetate buffer (pH 4.0) containing 500 mM sodium chloride, and then washed with 1.5 mL of 100 mM Tris-HCl buffer (pH 8.5). . The washing with these two buffer solutions was repeated 6 times, and then washed with 5 mL of PBS to obtain 8H9-seph.

<Purification of rh60 kDa segment (non-heterodimer)>
18 mL of 1M citrate buffer (pH 3.0) was added to 300 mL of the above culture supernatant, the pH of the culture supernatant was adjusted to 4.0, and then left at room temperature for 1 hour to dissociate the heterodimer. 27 mL of 2M Tris was added, the culture supernatant was adjusted to pH 8.0, mixed with 7 mL of TALON Resin (Clontech), and reacted at 4 ° C. overnight. This TALON Resin is packed in a column, washed with 35 mL of 25 mM phosphate buffer (pH 8.0) containing 500 mM sodium chloride, followed by 10 mL of 20 mM HEPES buffer (pH 7.5), and 20 mL of 20 mM HEPES containing 200 mM imidazole. Elute with buffer (pH 7.5).

  To this eluate, 0.5 mL of 8H9-seph was mixed, and gently mixed by inversion for 1 hour at room temperature. Next, 8H9-seph was packed in the column, washed with 2 mL of PBS, and eluted with 2.7 mL of 100 mM sodium citrate buffer (pH 3.0). Next, the eluate was neutralized by adding 0.3 mL of 2M Tris solution. At this stage, the rh60 kDa segment (non-heterodimer) is concentrated and purified utilizing the specificity of the monoclonal antibody 8H9, but polymer aggregates are mixed.

  In two NAP-25 columns (GE Healthcare) equilibrated with 20 mM HEPES buffer (pH 7.5) in advance, 1.5 mL was applied to each column, and 1 mL of 20 mM HEPES buffer was further added. Elution was carried out at pH 7.5, and the eluates for two columns were combined into one, and the buffer was exchanged for use in the next DEAE-Sepharose.

  This eluate was applied to a column packed with DEAE-Sepharose CL-6B 0.6 mL (GE Healthcare) equilibrated in advance with 20 mM HEPES buffer (pH 7.5), and 5 mL of 20 mM HEPES buffer (pH 7. After washing in 5), elution was performed with 2 mL of 20 mM HEPES buffer (pH 7.5) containing 150 mM sodium chloride. Thereby, the polymer aggregate was removed. This eluate was used as a purified rh60 kDa segment (non-heterodimer).

  The purified rh60 kDa segment (non-heterodimer) was subjected to SDS polyacrylamide electrophoresis for each fraction in the purification process, and the band was detected by Coomassie brilliant blue staining to confirm the purity of each gel (FIG. 3). In FIG. 3, lane 1 is a culture supernatant before addition of citrate buffer, 2 is a culture supernatant after addition of a Tris solution, 3 is a TALON Resin non-adsorbed fraction, 4 is a TALON Resin washed fraction, and 5 is TALON Resin. Elution fraction, 6 is an 8H9-seph non-binding fraction, 7 is an 8H9-seph washed fraction, 8 is an 8H9-seph elution fraction, 9 is a NAP-25 elution fraction, 10 is a DEAE non-adsorbed fraction, 11 Is the DEAE wash fraction and 12 is the DEAE elution fraction. It can be seen that the 60 kDa band appears more specifically as the purification step proceeds.

2. Preparation of other recombinant PCSK9 used in the measurement method of the present invention (1) Preparation of recombinant human ΔN218PCSK9 (53 kDa segment) Complementary strand DNA corresponding to ΔN218PCSK9 (amino acid numbers 219-692) was obtained by PCR using pEF / PCSK9 as a template. Amplified and incorporated into the pEF321 vector (Kim DW, Gene. 1990 Jul 16; 91 (2): 217-23) to produce the expression vector pEF / ΔN218PCSK9. The thermal cycle of the PCR was performed at 94 ° C. for 30 seconds, followed by 23 cycles of “98 ° C./10 seconds → 60 ° C./5 seconds → 72 ° C./1.5 minutes”. -The reaction was performed for 2 minutes.

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

  In the same manner as described above, the expression vector pEF / ΔN218PCSK9 was introduced into the mammalian cell line CHO-K1, and a clone CHO-K1 / ΔN218PCSK9 cell stably expressing the recombinant ΔN218PCSK9 was produced.

  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 1 × Complete mini EDTA-free (Roche) and 1% Nonidet P40-containing Tris buffer (TBS; 150 mM NaCl-containing 50 mM Tris-HCl, pH 7.5), and 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 ΔN218PCSK9 (rhΔN218PCSK9).

(2) Preparation of Recombinant Human ΔN152 PCSK9 In order to confirm the specificity of the monoclonal antibody 8H9 described later by immunoprecipitation, a segment consisting of the amino acid sequence of human PCSK9 (SEQ ID NO: 153-1692) was prepared.

  Specifically, a complementary strand DNA corresponding to ΔN152PCSK9 (SEQ ID NO: 2: 153-692) was amplified by PCR using the complementary strand DNA of full-length PCSK9 as a template, and the pEF321 vector (Kim DW, Gene. 1990 Jul 16; 91 (2): 217-23) to produce the expression vector pEF / ΔN152PCSK9. The thermal cycle of the PCR was performed at 94 ° C. for 30 seconds, followed by 23 cycles of “98 ° C./10 seconds → 60 ° C./5 seconds → 72 ° C./1.5 minutes”. -The reaction was performed for 2 minutes.

As amplification primers,
“Agtcaagcttagcatcccgtggaacctggagcgga” (forward primer: SEQ ID NO: 7)
“Agtagtcgacctggagctcctgggaggcctg” (reverse primer: SEQ ID NO: 6)
Was used.

  In the same manner as described above, the expression vector pEF / ΔN152PCSK9 was introduced into the mammalian cell line CHO-K1, and a clone CHO-K1 / ΔN152PCSK9 cell stably expressing the recombinant ΔN152PCSK9 was prepared.

  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 1 × Complete mini EDTA-free (Roche) and 1% Nonidet P40-containing Tris buffer (TBS; 150 mM NaCl-containing 50 mM Tris-HCl, pH 7.5), and 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 ΔN152PCSK9 (rhΔN152PCSK9).

(3) Preparation of Furin-cleaved rhPCSK9 (substantially corresponding to a 53 kDa segment) In 25 mM Tris-HCl (pH 9.0) solution containing ₂ mM CaCl ₂ and 0.5% Triton X-100, 1 μg of the purified rhPCSK9 described above was used. Recombinant human Furin (R & D) was mixed and reacted at 37 ° 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, a Furin-untreated rhPCSK9 detected a band with a molecular weight of about 60 kDa (corresponding to a 60 kDa segment (non-heterodimer)), whereas a Furin-treated rhPCSK9 was mostly a band with a molecular weight of about 53 kDa (corresponding to a 53 kDa segment). Detected). Specifically, it is a mixture of a 53 kDa segment, a 7 kDa segment, a pro segment, and a residual PCSK9 heterodimer, but 90% or more are 53 kDa segments.

  Therefore, the above Furin-cut rhPCSK9 solution was used as a 53 kDa segment-containing solution.

3. Preparation of anti-human PCSK9 monoclonal antibody (1) (reference example)
(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) per time 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 tail of Balb / c mice (8 weeks old, female) (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 for 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 in RPMI medium containing 3% hybridoma cloning factor (HCF, Air Brown), 1 × 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% Tween 20), 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 coloring 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 allowed to react 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 while mixing by inversion for 2 hours at room temperature. . 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, 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 PBS containing 0.1% Tween 20 (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) Preparation of Monoclonal Antibody from Hybridoma Hybridoma cells 10 ⁶ to 10 ⁷ per Balb / c mouse (8 weeks old, male) previously administered intraperitoneally with pristene (0.5 mL / animal, manufactured by 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. Subsequently, after centrifuging at 10,000 × 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 Protein A Sepharose (manufactured by GE Healthcare) and Mix and invert at 4 ° C overnight. The bound protein A sepharose was packed in the column, washed with 6 times the amount of the binding solution, and then eluted with 1 mL each of the 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 recovered. 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 the reactivity to rhPCSK9 by Western blotting and the reactivity to Furin-cleaved rhPCSK9 by immunoprecipitation.

(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 available from 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% Tween 20), each peroxidase-labeled antibody was allowed to react 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).

  The results of reactivity to rhPCSK9 by Western blotting revealed that antibodies 1FB, B12E, and B1G reacted with a mature segment of about 60 kDa, and this reactivity was lost by antigen reduction treatment. The antibody G12D reacted only with the prosegment, and it became clear that this reactivity was not affected by the reduction treatment of the antigen.

(6) Reactivity of the monoclonal antibody against Furin-cleaved rhPCSK9 For the anti-PCSK9 antibodies 1FB, B12E, B1G, G12D and the control antibody 16G5, Dynabeads M-280 Tosyl washed with 0.1 M borate buffer (pH 9.5) -activated (manufactured by Veritas) is suspended in 0.1 M borate buffer solution (pH 9.5) containing 3 M ammonium sulfate, and 10 μg of antibody per 1.0 × 10 ⁸ beads is added and mixed by inversion at 4 ° C. for 20 hours. Each antibody was then bound to the beads. 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. It was washed with PBS (pH 7.4) containing% BSA 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 3 times with PBS containing 0.1% Tween 20, 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) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected (FIG. 6).

  As described above, G12D, which was confirmed to be an antibody to a prosegment by Western blotting, immunoprecipitated a mature segment (60 kDa) in Furin-untreated rhPCSK9, whereas a 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 the mature segment immunoprecipitates both Furin-untreated rhPCSK9 (heterodimer) and Furin-cleaved rhPCSK9 (non-heterodimer: 53 kDa segment), and thus react with both PCSK9 heterodimer and 53 kDa segment Confirmed to do. Antibody B1G does not immunoprecipitate Furin-untreated rhPCSK9 (heterodimer), whereas it immunoprecipitates Furin-cleaved rhPCSK9 (non-heterodimer: 53 kDa segment), so that the non-heterodimer mature segment (53 kDa segment) specifically It was confirmed to react.

4). Preparation of anti-human PCSK9 monoclonal antibody (2) (Example)
An attempt was made to prepare a monoclonal antibody of the present invention, that is, a “monoclonal antibody that reacts with a 60 kDa segment (non-heterodimer) and does not cross-react with PCSK9 heterodimer and 53 kDa PCSK9”. This is an example with the detection of the 60 kDa segment (non-heterodimer) in mind, in light of the results in familial hypercholesterolemia (FH) serum described below.

(1) Preparation of hybridoma producing anti-human PCSK9 monoclonal antibody Mice were immunized with purified rhPCSK9 heterodimer as an antigen.

  Specifically, 3 μg of purified rhPCSK9 per one time was mixed with 25 μg of adjuvant Abisio-100 (ISCONNOVA) and administered subcutaneously to the back of Balb / c mice (8 weeks old, female) (primary immunization). Booster immunization was performed in the same manner as the initial immunization 3 times every 2 weeks.

  Two weeks after the final administration, 20 μg of purified rhPCSK9 was administered intraperitoneally to mice. Three days after the intraperitoneal administration, the spleen of the mouse was taken out 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 on HAT medium (Sigma) supplemented with 10% hybridoma cloning factor (HCF, Air Brown). Cells producing anti-PCSK9 monoclonal antibody 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 after washing, each well was blocked with PBS containing 5% skim milk. After washing each well, 100 μL of the culture supernatant of each hybridoma was added, and the reaction was performed at room temperature for 2 hours. After the reaction, each well was washed with a washing solution (PBS containing 0.1% Tween 20), a peroxidase-labeled goat anti-mouse IgG antibody (BioSource) diluted 5000 times was added, and the reaction was performed at room temperature for 1 hour. After the reaction, the wells were washed 5 times with a washing solution, and 100 μL of a coloring solution [0.012% hydrogen peroxide, 0.4 mg / mL OPD (o-phenylenediamine dihydrochloride, Sigma-Aldrich) -containing citrate phosphate buffer solution] , PH 5.0] was added and 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. The hybridoma culture supernatant was selected as a well positive showing an absorbance of 1.0 or more. The anti-PCSK9 monoclonal antibody-producing hybridoma was further cloned by limiting dilution to establish a cell line.

(2) Selection of hybridoma 8H9 Hybridoma 8H9 was selected from the above-mentioned positive hybridoma strains on the basis of reactivity to rhPCSK9 by Western blotting using a hybridoma culture supernatant.

  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 membrane was allowed to stand overnight at 4 ° C. in PBS containing 5% skim milk. After washing twice with PBS containing 0.1% Tween 20 (washing solution), the culture supernatant of each hybridoma 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) using a chemiluminescent reagent (manufactured by Perkin Elmer), and a specific band was detected (FIG. 7).

  In rhPCSK9, His × 6 tag is added to the C-terminus. In the anti-histidine tag antibody that recognizes His × 6 tag, a small amount of a band of about 50 kDa is detected in addition to a band around 60 kDa. Since the anti-histidine tag antibody recognizes the C-terminus of the mature segment, this approximately 50 kD band is a molecule produced by degradation of the N-terminal side of the mature segment, which is considered to be a 53 kDa segment. On the other hand, with the monoclonal antibody 8H9, although the 60 kDa segment was detected, the band of about 50 kDa was not detected. Instead, a band of less than 19.8 kDa that was not detected with the anti His-tag antibody was detected. From this, hybridoma 8H9 was considered to be a characteristic clone producing a monoclonal antibody that recognizes the N-terminus of the mature segment, and was selected as a clone worthy of preparing monoclonal antibodies from ascites.

(3) Preparation of monoclonal antibody from hybridoma Balb / c mice (female) were injected with 10 ⁶ to 10 ⁷ hybridoma cells per mouse intraperitoneally. Ten days after the injection, the mouse was opened, and ascites was collected. Cell components were removed from the obtained ascites using centrifugation, and an equal amount of ice-cooled saturated ammonium sulfate solution was added to the supernatant, mixed, ice-cooled, and left for 2 hours. After centrifugation at 10,000 × 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 recovered. The collected monoclonal fraction was dialyzed overnight at 4 ° C. with PBS 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. As a result, the isotype of monoclonal antibody 8H9 was IgG1.

(4) Confirmation of specificity of monoclonal antibody 8H9 <Western blot against Furin-cleaved rhPCSK9>
20 ng each of rhPCSK9 before and after cleavage with Furin was subjected to SDS-PAGE under reducing conditions, and then transferred to a PVDF membrane (Millipore). The transferred membrane 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 (R & D: anti-human PCSK9 heterodimer antibody) or peroxidase-labeled monoclonal antibody 8H9 was added at room temperature. Reacted for hours. After the reaction, the plate was washed 5 times with a washing solution and then exposed to an X-ray film (Kodak) using a chemiluminescence reagent (Perkin Elmer), and a specific band was detected (FIG. 8).

  Prior to Furin cleavage, both antibodies detected a mature segment of 60 kDa. After Furin cleavage, MAB38881 detected a 53 kDa C-terminal molecule in addition to the remaining 60 kDa mature segment, whereas monoclonal antibody 8H9 detected a 7 kDa N-terminal molecule. From the above, it was revealed that the monoclonal antibody 8H9 is a monoclonal antibody having an epitope in the 7 KDa region on the N-terminal side of the PCSK9 mature segment.

<Immunoprecipitation>
Immunoprecipitation was performed using rhPCSK9 heterodimer, 53 kDa segment, 60 kDa segment (non-heterodimer) as an immunoprecipitation sample, 8H9 and B12E as immunoprecipitation antibodies, and peroxidase-labeled B12E as detection antibodies.

For each immunoprecipitated antibody, Dynabeads M-280 anti-mouse IgG (manufactured by Veritas) washed with 0.1 M borate buffer was suspended in 0.1 M borate buffer (pH 9.5) containing 3 M ammonium sulfate. 0.0 × 10 ⁸ μg of antibody per ⁸ beads was added, and the antibody was bound to the beads by inversion mixing at 4 ° C. for 20 hours. 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. The antibody-bound beads were prepared by washing with PBS (pH 7.4) containing 1% BSA. 100 μL of serum specimen was added to 1.0 × 10 ⁷ antibody-bound beads, and reacted at 4 ° C. for 16 hours while mixing by inversion. After washing three times with PBS containing 0.1% Tween 20, the sample was suspended in a non-reducing SDS sample solution, subjected to SDS-PAGE, and then transferred to a PVDF membrane (Millipore). The transferred membrane 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 detection 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. 9).

  B12E immunoprecipitated all PCSK9, whereas 8H9 immunoprecipitated only the 60 kDa segment (non-heterodimer). From this, it was revealed that the monoclonal antibody 8H9 is an antibody specific for the 60 kDa segment (non-heterodimer).

5. PCSK9 molecular form-specific protein mass measurement system by sandwich ELISA method (reference example)
Using the monoclonal antibodies (excluding 8H9) prepared as described above, a combination of antibodies capable of sandwich ELISA was examined. Finally, a “total PCSK9 measurement system using a combination of solid phase antibody 1FB and detection antibody peroxidase-labeled B12E” (A system), “heterodimer PCSK9 measurement system” (B system) by combination of solid phase antibody B12E and detection antibody peroxidase labeled G12D, “non-heterodimer PCSK9 measurement by combination of solid phase antibody B1G and detection antibody peroxidase label B12E” A "system" (C system) was constructed (FIG. 10).

  The sandwich ELISA system of these measurement systems (A system, B system, and C system) is as shown in Patent Document 6. If the B system value >> C system value, it indicates that the non-heterodimer type PCSK9 value is small, and conversely, if the B system value << C system value, the non-heterodimer type PCSK9 value is large.

  The PCSK9 concentration in the serum of 108 healthy subjects and 194 patients with familial hypercholesterolemia (FH) was measured using these measurement systems. (A) Non-heterodimeric PCSK9 with B system value >> C system value A serum sample (sample numbers 1 to 4) having a small value and a serum sample (sample numbers 5 to 11) having a large non-heterodimeric PCSK9 value of (b) B system value << C system value were picked up. Table 1 shows the A system value, the B system value, and the C system value of these samples 1 to 11.

6). Detection of 60 kDa segment (non-heterodimer) in serum sample Next, serum samples 1 to 11 were examined by immunoprecipitation of serum PCSK9 molecule using non-heterodimeric PCSK9-specific anti-PCSK9 antibody B1G. . Peroxidase labeling of each antibody performed in this immunoprecipitation was performed using Peroxidase Labeling Kit-SH (manufactured by Dojindo Laboratories). The labeling method was performed according to the instructions attached to the kit.

In the immunoprecipitation method, Dynabeads M-280 anti-mouse IgG (manufactured by Veritas) washed with 0.1M borate buffer (pH 9.5) was washed with 0.1M borate buffer containing 3M ammonium sulfate. The suspension was suspended in (pH 9.5), 10 μg of antibody per 1.0 × 10 ⁸ beads was added, and the antibody was bound to the beads by inversion mixing at 4 ° C. for 20 hours. 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. The antibody-bound beads were prepared by washing with PBS (pH 7.4) containing 1% BSA. 100 μL of serum specimen was added to 1.0 × 10 ⁷ antibody-bound beads, and reacted at 4 ° C. for 16 hours while mixing by inversion. After washing three times with PBS containing 0.1% Tween 20, the sample 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-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.

  The results are shown in FIG.

  As a result, in a serum sample (sample numbers 1 to 4) having a small non-heterodimer type PCSK9 value, a 53 kDa band was detected similarly to Furin-cleaved rhPCSK9, and the intensity of the band signal was high in the non-heterodimer type PCSK9 value. There was a tendency for serum samples (sample numbers 5 to 11) to be dense, that is, high in concentration.

  And in the serum sample (sample numbers 5-11) with a high non-heterodimeric PCSK9 value, in addition to the thin 53 kDa band, a 60 kDa band was unexpectedly detected deeply. The molecular weight of this 60 kDa band is the size corresponding to the 60 kDa mature domain, which was found to be a 60 kDa segment (non-heterodimer). To date, the mature domain in human serum has only been reported to have a PCSK9 heterodimer and a 53 kDa segment (Piper DE et al., Structure, 2007: 15; 545-552, Dubuc G et al , J Lipid Res, 2010: 51; 140-149), it was first discovered that a 60 kDa segment (non-heterodimer) exists in human serum.

7). Construction of sandwich ELISA 100 μL / well of each solid phase antibody (A system: 1 FB 1 μg / mL, B system: B12E 0.3 μg / mL, C system: B1G 1 μg / mL) diluted with PBS on an immunoplate (Nunc) And left at 4 ° C. overnight.

  After washing twice with PBS containing 0.1% Tween 20 (washing solution), PBS containing 1% BSA and 10% sucrose was added at 200 μL / well and blocked at room temperature for 2 hours. After removing the solution from each well, a standard solution diluted with PBS containing 0.3% BSA and 0.1% Tween 20 (A and B systems are rhPCSK9 heterodimer, C system is rhΔN218 PCSK9 (53 kDa segment)), or human Serum was added at 100 μL / well and allowed to stand at room temperature for 2 hours (Table 2).

  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, and 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. 13 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.

8). Construction of PCSK9 non-heterodimer 60 kDa specific protein mass measurement system by sandwich ELISA method (Example)
A 60 kDa segment (non-heterodimer) specific antibody 8H9 and an anti-PCSK9 mature segment antibody B12E were combined to construct a PCSK9 non-heterodimer 60 kDa specific sandwich ELISA system (D system). 8H9 was used as the solid phase antibody, and peroxidase-labeled B12E was used as the detection antibody. As the standard, the standard substance of the present invention prepared using the aforementioned “8H9-seph” was used.

  To an immunoplate (Nunc), 5 μg / mL of 8H9 diluted with PBS was added at 100 μL / well, and allowed to stand at 4 ° C. overnight. After washing twice with PBS containing 0.1% Tween 20 (washing solution), PBS containing 1% BSA and 10% Sucrose was added at 200 μL / well, and blocking was performed at room temperature for 2 hours. After removing the liquid in each well, a standard solution (standard substance of the present invention) diluted with PBS containing 0.3% BSA and 0.1% Tween 20 or a measurement sample is added at 100 μL / well, and the mixture is allowed to stand at room temperature for 2 hours. I put it. After the reaction, the plate was washed four times with a washing solution, and 50 ng / mL peroxidase-labeled B12E diluted with PBS containing 10% StabiliZyme (SurModics), 0.3% BSA and 0.1% Tween 20 was added at 100 μL / well. Let stand for 1 hour. After the reaction, the plate was washed 4 times with the washing solution, and the enzyme substrate solution TMB + (Dako) was added at 100 μL / well. After coloring at room temperature for 30 minutes, 0.5 M sulfuric acid was added at 100 μL / well to stop the enzyme reaction. The absorbance of each well was measured at a wavelength of 450 nm (background 560 nm) using Multiskan Ascent (Thermo Labsystems).

  The calibration curve in this ELISA measurement system (D system) is as shown in FIG. This ELISA system was good in the range of 0-4 ng / mL.

  Furthermore, the result of the dilution linearity test of the serum sample in this ELISA measurement system (D system) is as shown in FIG. All samples showed linearity, confirming the accuracy of this ELISA system.

9. Confirmation of reactivity of monoclonal antibody 8H9 to human serum samples (1) Measurement by sandwich ELISA For serum samples S1, S2, and S3 of familial hypercholesterolemia patients, A series, B series, C series and D series sandwich ELISA Each PCSK9 molecular form concentration was measured in the system. Table 3 shows the A system value, the B system value, the C system value, and the D system value of these samples.

(2) Confirmation by immunoprecipitation The above serum samples S1, S2, S3 were immunoprecipitated using 8H9 and B12E as immunoprecipitation antibodies and peroxidase-labeled B12E and G12D as detection antibodies as follows.

For each immunoprecipitated antibody, Dynabeads M-280 Tosyl-activated (Veritas) washed with 0.1 M borate buffer (pH 9.5) was suspended in 0.1 M borate buffer (pH 9.5) containing 3 M ammonium sulfate. 10 μg of antibody per 1.0 × 10 ⁸ beads was added, and the antibody was bound to the beads by inversion mixing at 4 ° C. for 20 hours. 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. The antibody-bound beads were prepared by washing with 1% BSA-containing PBS (pH 7.4). 100 μL of each serum sample was added to 1.0 × 10 ⁷ antibody-bound beads, and reacted at 4 ° C. for 16 hours while mixing by inversion. After washing three times with PBS containing 0.1% Tween 20, the sample 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 reacted overnight at 4 ° C in PBS containing 5% skim milk, washed 5 times with a washing solution, and then exposed to an X-ray film (Kodak) using a chemiluminescence reagent (Perkin Elmer). And a specific band was detected (FIG. 16).

  As shown in FIG. 16, the monoclonal antibody B12E immunoprecipitated a 60 kDa segment, a 53 kDa segment (corresponding to the A system value), and a prosegment (corresponding to the B system value), whereas the monoclonal antibody 8H9 has a 60 kDa segment (D system). Only the corresponding value) was immunoprecipitated. From this, it was confirmed that a 60 kDa segment (non-heterodimer) exists in human serum.

10. Examination of equivalence of purified rh60 kDa PCSK9 (non-heterodimer) with 60 kDa PCSK9 (non-heterodimer) in serum (1) PCSK9 concentration measurement of purified rh60 kDa PCSK9 (non-heterodimer) rh60 kDa PCSK9 (non-heterodimer) For each fraction of the purification process (similar to lanes 1 to 12 in FIG. 3 above), a “total PCSK9 measurement system”, a “heterodimer-specific measurement system”, a “non-heterodimer-specific measurement system”, and a new Each PCSK9 molecular form concentration was measured using the “non-heterodimer 60 kDa segment-specific measurement system” constructed in (1). From the measurement results, it was confirmed that the purified product of rh60 kDa PCSK9 (non-heterodimer) reacts with monoclonal antibodies 1FB, B12E, and B1G that recognize a three-dimensional structure via a disulfide bond (FIG. 17).

(2) Production of rhΔ152PCSK9 using baculovirus-insect cell expression system Complementary strand DNA corresponding to human PCSK9 full-length amino acid sequence (SEQ ID NO: 2) and Δ152PCSK9 (SEQ ID NO: 2 Nos. 153 to 692) was converted into full-length PCSK9 complementary strand Amplification was carried out by PCR using DNA (base sequence of SEQ ID NO: 1) as a template, which was then incorporated into a pVL1392 vector (Life Technologies) to prepare baculovirus transfer vectors pVL / PCSK9 and pVL / Δ152 PCSK9. In the PCR, the following primer sequences were set so that 6 histidine residues were fused to the C-terminus of the protein.

“Gacagatctcagcgacgtcgaggcgctcatggttg” [forward primer (full length) SEQ ID NO: 3]
“Gacagatctatgagcatcccgtggaacctggagcgga” [forward primer (ΔN152) SEQ ID NO: 8]
“Gacgaattctcagtgatggtgatggtgatgctggagctcctgggaggcctgcgcc” [reverse primer (common) SEQ ID NO: 4]

  In this PCR thermal cycle, after denaturation treatment at 94 ° C. for 30 seconds, 23 cycles of “98 ° C., 10 seconds-60 ° C., 5 seconds-72 ° C., 1.5 minutes” were performed, -The reaction was performed for 2 minutes.

  Baculovirus transfer vectors pVL / PCSK9 and pVL / Δ152 PCSK9 were introduced into insect cell line SF21 (Life Technology) together with BaculoGold Linearized Baculovirus DNA (Becton Dickinson) to obtain a recombinant baculovirus.

  The obtained recombinant baculovirus was infected with SF21 cells in a 10 cmφ dish, and cultured at 27 ° C. for 5 days. The cultured cells are suspended in 1 × Complete mini EDTA-free (Roche) and 1% Nonidat P40-containing Tris buffer (TBS: 50 mM Tris-HCl containing 7.5 mM NaCl, pH 7.5), and 30 minutes in ice. Cooled down. Subsequently, the cell suspension was centrifuged (15000 rpm, 4 ° C., 20 minutes), and the supernatant was collected. The supernatant fraction was used as rhPCSK9-containing cell lysate.

  Each lysate was subjected to SDS polyacrylamide electrophoresis and then transferred to a PVDF membrane, and specific bands were detected with peroxidase-labeled anti-histidine tag antibodies, 1FB, and B12E (FIG. 18).

  In the anti-histidine tag antibody that reacts regardless of the presence or absence of a disulfide bond, rhΔ152PCSK9 is detected in 1FB and B12E, whereas rhΔ152PCSK9 produced in this expression system is not detected in 1FB and B12E. It was clarified that a three-dimensional structure different from that was formed.

  This is also true for 60 kDa PCSK9 (non-heterodimer). What is 60 kDa PCSK9 (non-heterodimer) in which the three-dimensional structure of rh60 kDa PCSK9 (non-heterodimer) expressed in an insect cell expression system exists in blood? In contrast, it became clear that it was not suitable as an essential component of the standard substance of the present invention. This is in contrast to the three-dimensional structure of rh60 kDa PCSK9 (non-heterodimer) expressed in animal cell expression systems is substantially the same as 60 kDa PCSK9 (non-heterodimer) present in blood.

11. Examination of LDL receptor-degrading activity of rhPCSK9 non-heterodimer 60 kDa (1) Culture of HepG2 cells HepG2 cells (ATCC) were seeded on a 24-well cell culture plate (BM Instruments) at 1 × 10 ⁵ cells per well. 0.5 mL of DMEM (Life Technology) containing 10% FBS (Nichirei) was added per well and cultured at 37 ° C. and 5% CO ₂ for 24 hours. Next, the medium was changed to 0.3 mL per well of DMEM containing 5% LPDS (prepared according to Lipoprotein Deficient Serum: Havel RJ et al., J Clin Invest, 1955: 34; 1345-1353), 37 ° C., 5% They were cultured for 24 hours in CO ₂ under. Next, the medium was changed to DMEM containing purified rhPCSK9 heterodimer or purified rh60 kDa segment (non-heterodimer) at a concentration of 0 μg / mL, 1.1 μg / mL, 3.3 μg / mL, 10 μg / mL, and 0.25 mL per well. The cells were cultured at 37 ° C. and 5% CO ₂ for 4 hours. The cultured cells were washed once with PBS, 0.2 mL of 1 mM EDTA-containing PBS was added per well, left at 37 ° C. for 10 minutes, and released from the culture plate. The released cells were collected in a microtube (BM device), centrifuged at 400 g at 4 ° C. for 3 minutes to remove the supernatant, and then 30 μL of PBS containing 0.3% BSA and 0.05% sodium azide (buffer) It was suspended in liquid A) and allowed to stand on ice.

(2) Staining of LDL receptor on HepG2 cell surface 20 μL of 5 μg / mL anti-human LDL receptor mouse monoclonal antibody-containing buffer A was added to 30 μL of HepG2 cell suspension and allowed to react on ice for 40 minutes. After the reaction, the cells were washed twice with ice-cooled buffer A, then suspended in buffer A containing 20 μL of 250-fold diluted phycoerythrin-labeled goat anti-mouse IgG antibody (Rockland) for 30 minutes on ice. The LDL receptor on the cell surface was fluorescently labeled by reacting. After the reaction, the cells were washed twice with ice-cold buffer A, suspended in 0.17 mL of ice-cold PBS, the fluorescence intensity was measured with FACSCalibur (Becton Dickinson), and LDL on the cell surface. Receptor expression level was evaluated. The anti-human LDL receptor mouse monoclonal antibody was prepared according to Kosaka S et al., Circulation, 2001: 103; 1142-1147 and Takahashi M et al., Clin Genet, 2001: 59; 290-292. A thing was used.

  Although both rhPCSK9 heterodimer addition and “purified rh60 kDa segment (non-heterodimer)” prepared using “G12D-seph” described above, a decrease in LDL receptor expression on the surface of HepG2 cells was observed. The rh60SK9 segment (non-heterodimer) also had a significantly lower amount of decrease compared to the rhPCSK9 heterodimer (n = 3, significant difference test by Student's t-test) (FIG. 19).

  From this, it was revealed that the LDL receptor degradation promoting activity of the 60 kDa segment (non-heterodimer) was clearly attenuated compared to the PCSK9 heterodimer.

12 Drug Screening System Construction It is known that the PCSK9 heterodimer is dissociated from a 60 kDa segment (non-heterodimer) and a prosegment by treatment with pH 4.0. Using this as a model phenomenon, an assay system for carrying out the measurement method of the present invention was constructed.

  To 30 mL of the above-mentioned rhPCSK9 heterodimer-containing culture supernatant, 4.5 mL of a mixture of 1.8 mL of 1 M citrate buffer (pH 3.0) and 2.7 mL of 2 M Tris solution is added, and left at room temperature for 1 hour. Treatment (no pH 4.0 treatment) or 1.8 mL of 1M citrate buffer (pH 3.0) was added to adjust the pH of the culture supernatant to 4.0, and then allowed to stand at room temperature for 1 hour. Thereafter, 2.7 mL of a 2M Tris solution was added, and the culture supernatant was adjusted to pH 8.0 (with pH 4.0 treatment). Both samples were measured by (a) the above-mentioned “60 kDa segment (non-heterodimer) measurement system (D system)” and (b) “PCSK9 heterodimer-specific measurement system (B system)”. It was possible to clearly detect that the 60 kDa segment (non-heterodimer) increased and the PCSK9 heterodimer decreased in the sample with 0.0 treatment (FIG. 20).

  From this result, the usefulness of the same measurement method using the standard substance of the present invention in screening for a substance that dissociates PCSK9 heterodimer became clear.

Claims (17)

  PCSK9 measurement standard substance consisting of human PCSK9 60 kDa segment (non-heterodimer).   The measurement of human PCSK9 is carried out in a test drug test system that decomposes human PCSK9 heterodimer into a prosegment and a 60 kDa segment (non-heterodimer), and the 60 kDa segment (non-heterodimer) before and after use of the drug. The standard substance for measuring PCSK9 according to claim 1, which is a measurement for determining a ratio to PCSK9 heterodimer or total human PCSK9.   The standard for measuring PCSK9 according to claim 1, wherein the measurement of human PCSK9 is a measurement for analyzing the function of a 60 kDa segment (non-heterodimer).   The standard substance for measuring PCSK9 according to claim 2 or 3, wherein a blood sample is used as a measurement target.   The standard substance for measuring PCSK9 according to claim 2 or 3, wherein a solvent system to which human PCSK9 heterodimer is added is used as a measurement target.   The human PCSK9 60 kDa segment (non-heterodimer) dissociates the prosegment in the human PCSK9 heterodimer produced using the host as an animal cell based on the base sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2. The standard substance for measuring PCSK9 according to any one of claims 1 to 5, which is a human PCSK9 60 kDa segment (non-heterodimer) obtained by removing the prosegment and the mixed heterodimer.   The standard substance for PCSK9 measurement according to claim 6, wherein the dissociation of the prosegment in the human PCSK9 heterodimer is an acidic treatment at pH 5 or lower.   The removal of the prosegment dissociated from the human PCSK9 heterodimer and the mixed heterodimer can be achieved by allowing the antibody specific for the prosegment to capture the prosegment and removing the captured prosegment and the mixed heterodimer. The standard substance for PCSK9 measurement according to claim 6 or 7, which is performed.   Removal of prosegments and intermingled heterodimers dissociated from human PCSK9 heterodimer allows human PCSK9 60 kDa segment (non-heterodimer) to be captured by an antibody specific for human PCSK9 60 kDa segment (non-heterodimer), The standard substance for PCSK9 measurement according to any one of claims 6 to 8, which is carried out by selectively obtaining the captured 60 kDa segment (non-heterodimer).   The standard substance for PCSK9 measurement according to claim 8 or 9, wherein the specific antibody is a monoclonal antibody.   Based on the base sequence encoding the amino acid sequence of all human PCSK9 shown in SEQ ID NO: 2, the prosegment is dissociated in the human PCSK9 heterodimer produced using the host as an animal cell, and the prosegment and the mixed heterodimer are removed. A method for producing a human PCSK9 60 kDa segment (non-heterodimer), whereby a human PCSK9 60 kDa segment (non-heterodimer) is obtained.   Use of human PCSK9 60 kDa segment (non-heterodimer) as a standard for measuring PCSK9.   The measurement of human PCSK9 is carried out in a test drug test system that decomposes human PCSK9 heterodimer into a prosegment and a 60 kDa segment (non-heterodimer), and the 60 kDa segment (non-heterodimer) before and after use of the drug. 13. Use according to claim 12, which is a measurement to determine the ratio to PCSK9 heterodimer or total human PCSK9.   The use according to claim 12, wherein the measurement of human PCSK9 is a measurement for functional analysis of a 60 kDa segment (non-heterodimer).   A hybridoma that produces a monoclonal antibody that reacts with the human PCSK9 60 kDa segment (non-heterodimer) and does not cross-react with the human PCSK9 heterodimer and the human 53 kDa segment.   The hybridoma according to claim 15, wherein the hybridoma is a hybridoma deposited under the accession number NITE P-01582 at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation.   A monoclonal antibody produced by the hybridoma according to claim 15 or 16.