Method for quantifying denatured HDL and analytical reagent used therein

Abstract

The present invention provides a method for quantifying a modified HDL in a sample, the method comprising: combining the modified HDL with a modified-HDL-binding protein; and quantifying a complex between the modified-HDL-binding protein and the modified HDL. The modified-HDL-binding protein includes either a protein having the sequence of a Factor V light chain site, or a variant thereof.

Description

[Technical field]

[0001] The present invention relates to a method for quantifying modified HDL, which can be used to assess the risk of lifestyle-related diseases and the effectiveness of treatments therefor, and an analytical reagent used therefor. [Background technology]

[0002] HDL (High-Density Lipoprotein) is a type of cholesterol with a high density and small particle size. It is known that HDL is responsible for transporting cholesterol from tissues to the liver. HDL is also known as "good cholesterol" because high blood concentrations of HDL reduce the risk of arteriosclerosis. On the other hand, LDL (Low-Density Lipoprotein), which has a low density, is known as "bad cholesterol." The concentrations of HDL and LDL are used as indicators in health checkups.

[0003] The physiological activity of HDL is not only to inhibit the oxidation of LDL, but also to reduce the cytotoxicity caused by oxidized LDL through an increase in NO, exerting an anti-atherosclerotic effect. However, it has been revealed that denatured HDL increases in the blood of patients with coronary artery disease, which acts on blood vessels via LOX-1 to promote atherosclerosis, and that ApoAI, an apoprotein of HDL, accumulates in large amounts in human atherosclerotic lesions. Based on these results, in order to use HDL as an indicator for health checkups, it is necessary not only to simply measure the concentration of HDL, but also to separately quantify HDL with its original physiological activity and HDL whose activity has been changed due to denaturation, etc. In particular, if denatured HDL can be quantified, it is thought that it can be used to verify diseases related to the circulatory system and blood, including the above-mentioned coronary artery disease, and their risks.

[0004] Patent Document 1 discloses a method for measuring dysfunctional HDL contained in a sample, in which the sample is brought into contact with a receptor for dysfunctional HDL, dysfunctional HDL in the sample is bound to the receptor, and dysfunctional HDL bound to the receptor is detected, the receptor being LOX-1 or a modified form thereof, the receptor being immobilized on a carrier, and a method for measuring dysfunctional HDL in which an antibody that recognizes dysfunctional HDL is further bound to the dysfunctional HDL bound to the receptor. This technology provides a measurement technology for dysfunctional HDL that can widely detect dysfunctional HDL of various molecular forms, and also provides a detection technology for lifestyle-related diseases using said technology.

[0005] In addition, the present inventors have disclosed in Patent Document 2 a fusion protein in which the entire protein sequence or a partial fragment protein sequence of ApoA1 is linked directly or via a spacer to a LOX-1 binding protein sequence that binds to LOX-1, and a high density lipoprotein measurement kit using the same. This technology can measure HDL that has undergone various denaturations by measuring HDL that is simultaneously recognized by LOX-1 and an anti-ApoA1 antibody, and uses a fusion protein in which ApoA1 is fused to a protein that specifically binds to LOX-1, such as the LOX-1 binding site of an antibody against LOX-1 (anti-LOX-1 antibody). This fusion protein can simultaneously recognize HDL that has undergone various denaturations, and is used to measure the receptor binding activity and measure the physiological activity itself, and is intended to obtain a fusion protein that does not contain lipids, can be stored for a long time, can be reproducibly adjusted, and can serve as a reference that can reduce variability between measurement tests and increase reliability. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6231307 [Patent Document 2] Patent application 2018-024007 Summary of the Invention [Problem to be solved by the invention]

[0007] Denatured HDL can theoretically be quantified in a laboratory using the technique described in Patent Document 1. The technique described in Patent Document 2 by the present inventors is more sophisticated than conventional techniques. It was expected that these techniques could be applied to establish a method that could be used to quantify denatured HDL, even if it was not very sophisticated.

[0008] However, the technology of Patent Document 1 has not yet been widely adopted. Furthermore, even when the technology of Patent Document 2 of the present inventors is used, the results of quantification of denatured HDL are not always consistent when the technology is used under conditions other than a laboratory or using different samples. These differences are thought to be due to various factors, such as the conditions in the laboratory and the differences in the testing techniques of the people who carry out the technology. Considering the above-mentioned historical background and the fact that measurement should be possible even under somewhat poor conditions for practical clinical application, the inventors have been conducting intensive research with the goal of improving the quantitative technique for denatured HDL into a more versatile quantitative technique.

[0009] The present invention has been made in consideration of the above-mentioned circumstances, and its object is to provide a method for quantifying denatured HDL that can quantify denatured HDL more stably and accurately than conventional methods, and that is useful for determining cardiovascular diseases, diabetes or diabetic diseases, etc., or the degree of progression thereof, and an analytical reagent for use in said method. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention has the following aspects. [1] A method for quantifying denatured HDL contained in a sample, comprising binding the denatured HDL to a denatured HDL-binding protein, and quantifying the bound body of the denatured HDL-binding protein and the denatured HDL, wherein the denatured HDL-binding protein comprises a protein having a sequence of the L chain portion of Factor V or a mutant thereof. [2] The method for quantifying denatured HDL as described above, wherein the denatured HDL-binding protein contains a sequence of a phospholipid recognition site of Factor V. [3] The method for quantifying denatured HDL as described above, wherein the denatured HDL-binding protein comprises a Factor V sequence. [4] The method for quantifying denatured HDL as described above, wherein the denatured HDL-binding protein is a protein having a site that is partially homologous to the C1-C2 domain, which is the phospholipid recognition site of Factor V. [5] The method for quantifying denatured HDL as described above, wherein the protein having a site having partial homology to the C1-C2 domain is MFG-E8, Del-1 or Factor VIII. [6] The method for quantifying denatured HDL, wherein the denatured HDL-binding protein is a recombinant protein. [7] The method for quantifying denatured HDL, comprising forming a complex between the denatured HDL and the denatured HDL-binding protein, and quantifying the complex by immunoassay. [8] The method for quantifying denatured HDL, wherein the quantification by immunoassay is carried out by an ELISA method using an ELISA plate on which the denatured HDL-binding protein or a protein that binds to the denatured HDL-binding protein is immobilized. [9] The method for quantifying denatured HDL, comprising forming a complex of the denatured HDL, the denatured HDL-binding protein and LOX-1 protein, and quantifying the complex by immunoassay.

[10] The method for quantifying denatured HDL, wherein serum or plasma is used as a sample.

[11] The method for quantifying denatured HDL, wherein the quantification comprises a step of using serum as a sample and adding the denatured HDL binding protein to the sample.

[12] An analytical reagent used to quantify denatured HDL to determine cardiovascular disease, diabetes or diabetic disease, the risk thereof, or the degree of progression thereof, the analytical reagent comprising a denatured HDL-binding protein capable of binding to the denatured HDL, the denatured HDL-binding protein comprising a protein having an L-chain sequence of Factor V or a mutant thereof.

[13] The analytical reagent as described above, wherein the denatured HDL-binding protein contains a sequence of a phospholipid recognition site of Factor V.

[14] The analytical reagent as described above, wherein the denatured HDL-binding protein comprises a sequence of Factor V.

[15] The analytical reagent as described above, wherein the denatured HDL-binding protein is a protein having a site partially homologous to the C1-C2 domain, which is a phospholipid recognition site of Factor V.

[16] The analytical reagent as described above, wherein the protein having a site partially homologous to the C1-C2 domain is MFG-E8, Del-1, or Factor VIII.

[17] The analytical reagent as described above, wherein the denatured HDL-binding protein is a recombinant protein.

[18] The analytical reagent as described above, wherein the cardiovascular disease is arteriosclerosis or myocardial infarction or cerebral stroke caused by the progression of arteriosclerosis. Effect of the Invention

[0011] According to the present invention, a method for quantifying denatured HDL can be obtained that can quantify denatured HDL more stably and accurately than conventional methods, and is useful for determining cardiovascular diseases, diabetes or diabetic diseases, etc., or the degree of progression thereof, and an analytical reagent for use in said method is provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing a method for quantifying denatured HDL according to a first embodiment. [Diagram 2] FIG. 1 is a schematic diagram showing a method for quantifying denatured HDL according to a second embodiment. [Diagram 3] FIG. 1 is a graph showing the detection sensitivity of denatured HDL in Test Example 1. [Figure 4] FIG. 1 is a graph showing the detection sensitivity of denatured HDL in plasma in Test Example 2. [Diagram 5] FIG. 1 is a graph showing the detection sensitivity of denatured HDL in serum in Test Example 2. [Figure 6] FIG. 13 is a graph showing the detection sensitivity of denatured HDL in plasma at different Factor V concentrations in Test Example 2. [Figure 7]FIG. 13 is a graph showing the detection sensitivity of denatured HDL in serum at different Factor V concentrations in Test Example 2. [Figure 8] FIG. 13 is a graph showing the amount of immobilized Factor V in Test Example 4. [Figure 9] FIG. 1 is a graph showing a denatured HDL binding test according to Test Example 4. [Figure 10] FIG. 13 is a graph showing the quantification of denatured HDL in plasma and serum in Test Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The quantification method and analytical reagent for denatured HDL according to the present invention will be described below with reference to embodiments, although the present invention is not limited to the following embodiments.

[0014] (First embodiment) (Method for quantifying denatured HDL) In the method for quantifying denatured HDL of the present embodiment, the denatured HDL is bound to a denatured HDL-binding protein when quantifying the denatured HDL contained in a sample, and the complex formed by the binding of the denatured HDL-binding protein to the denatured HDL is quantified.

[0015] In the present embodiment, denatured HDL includes HDL having some structural difference from HDL having inherent physiological activity in vivo, and particularly refers to HDL having reduced physiological activity due to the structural difference. Mechanisms by which HDL becomes denatured HDL include, for example, chemical modification or partial or complete degradation of constituent proteins, genetic defects and mutations, abnormalities or changes in higher-order structure, or binding with molecules that cause loss of activity.

[0016] Examples of such modified HDL include oxidized HDL, in which any part of the protein or lipid constituting HDL is oxidized. Examples of such modified HDL include HClO-HDL, HNE-HDL, carbamylated HDL, and MDA-HDL. These modified HDLs are known to damage vascular endothelial cells in the vascular wall and increase the risk of developing various diseases, such as arteriosclerosis, heart disease, ischemic disease, and other arteriosclerosis-related diseases that cause arteriosclerosis and thrombus formation.

[0017] In the method for quantifying denatured HDL of this embodiment, denatured HDL is bound to a denatured HDL-binding protein, and the bound body of the denatured HDL-binding protein and denatured HDL is quantified. The denatured HDL-binding protein of this embodiment broadly refers to a protein that can directly interact with denatured HDL, for example, a protein that itself can bind to denatured HDL. Denatured HDL may acquire the ability to interact with receptors that cause adverse reactions in the body. In this case, denatured HDL may bind to the receptor via denatured HDL-binding protein, i.e., denatured HDL may bind directly to denatured HDL-binding protein, and this denatured HDL-binding protein may bind to the receptor. Denatured HDL-binding proteins include, for example, Factor V, its mutants, and proteins having partial homology thereto. An example of a receptor that can bind to denatured HDL via another protein is the LOX-1 protein described below.

[0018] In this embodiment, the denatured HDL-binding protein comprises a protein having the sequence of the L chain portion of Factor V or a mutant thereof. Factor V is known as a protein that constitutes the coagulation system, and is a protein of approximately 330 kDa composed of the A1-A2-B-A3-C1-C2 domains. It is roughly divided into an H chain (heavy chain) composed of the A1-A2 domains and an L chain (light chain) composed of the A3-C1-C2 domains. It is known that the H chain mainly binds to cell membranes, and the L chain recognizes and binds to phospholipids via the C1-C2 domains. A mutant refers to one in which a portion of the sequence has been substituted, and in this embodiment, it is preferable that a site other than the L chain site of Factor V has been substituted.

[0019] Since the L-chain site of Factor V is the site that directly binds to denatured HDL, if the denatured HDL-binding protein is a protein containing this site or a mutant thereof, it can bind denatured HDL and the denatured HDL-binding protein and be used for quantification. Furthermore, by using a protein having the sequence of the L-chain portion, which is a part of Factor V, as the denatured HDL-binding protein, a protein having a portion that binds to denatured HDL but has a structure different from that of Factor V can be used. That is, a protein that is more easily available than Factor V or a protein that is easier to test can be used. As a protein that is more easily available than Factor V, for example, a protein that uses only the L-chain portion and a minimum sequence, has a shorter total length than Factor V, and is easier to produce as a recombinant protein can be used. As a protein that is easier to test, a protein that is easier to store or immobilize on a plate by changing the sequence other than the L-chain can be used.

[0020] It is also preferable that the denatured HDL-binding protein contains the sequence of a phospholipid recognition site of Factor V. Among the sequences of Factor V, the phospholipid recognition site in the L chain can recognize and effectively bind to denatured HDL.

[0021] The denatured HDL-binding protein preferably contains the sequence of Factor V. Here, the sequence of Factor V refers to the full-length Factor V protein, and Factor V itself may be used, or a fusion or bond with other proteins, a modified protein, etc. may also be used.

[0022] The denatured HDL-binding protein is also preferably a protein having a site partially homologous to the C1-C2 domain, which is the phospholipid recognition site of Factor V. Examples of such proteins having a site partially homologous to the C1-C2 domain include MFG-E8, Del-1, and Factor VIII. In addition, the protein may be any of these proteins or mutants, or a protein partially comprising the sequence of any of these proteins.

[0023] As the denatured HDL-binding protein, for example, a recombinant protein is preferably used. Here, the recombinant protein is a protein obtained by expression through genetic recombination. The recombinant protein may be fused with other structures or mutated as necessary.

[0024] In this embodiment, the amount of denatured HDL contained in a sample is quantified. Here, the sample broadly refers to a sample containing components extracted from an organism. It is preferable to use a liquid sample of the components, or to add a liquid to the components to make a liquid sample. For example, a biological body fluid can be used directly as a sample. For example, human blood or saliva can be used as a biological body fluid. For example, serum or plasma can be used as a blood sample. In this embodiment, these biological fluids are used as samples for the above-mentioned quantification.

[0025] In this embodiment, it is preferable to use serum as a sample. In this embodiment, a denatured HDL-binding protein having a sequence of the L chain portion of Factor V that directly binds to denatured HDL is bound to denatured HDL in a sample. Therefore, even if serum, which may not contain a factor that directly binds to denatured HDL, is used as a sample, denatured HDL can be stably and accurately quantified. In addition, serum is easier to obtain and store than other body fluid samples such as plasma. In fact, as blood samples, samples frozen and stored in the form of serum are common internationally, so this is a useful technique.

[0026] The present inventors have focused on the fact that, in conventional techniques, when proteins that interact with denatured HDL, such as LOX-1, are used to quantify denatured HDL, denatured HDL can be detected when plasma is used as a sample, but when serum is used as a sample, denatured HDL is detected in small amounts and accurate quantification is not possible. Therefore, they predicted that measurement using serum as a sample would be possible by supplementing the measurement system with substances that are decomposed and lost during the process of forming serum from blood or plasma. The present inventors focused on Factor V as a supplementary substance. In a biological reaction, denatured HDL binds to LOX-1 via Factor V. Specifically, the phosphatidylserine (PS) recognition site of Factor V binds to denatured HDL, and the LOX-1 recognition site binds to LOX-1.

[0027] As described below, when Factor V is added to serum as a sample, the detection sensitivity of denatured HDL in serum increases almost in proportion to the amount of Factor V added. Therefore, denatured HDL can be quantified by adding a denatured HDL-binding protein such as Factor V to a sample such as serum.

[0028] The above-mentioned quantification can be carried out using known means for protein quantification (measuring protein amount), for example, an immunological method can be used for the above-mentioned quantification. Immunological methods for quantification and immunoanalysis can use enzymes (enzyme immunoassay, EIA, ELISA), fluorescent substances (fluorescent immunoassay, FIA), or radioactive substances (radioimmunoassay, RIA), etc. Among these, ELISA is preferable because it is relatively inexpensive, simple, and can analyze a large number of samples.

[0029] For example, in this embodiment, the quantification can be performed using an ELISA method. Specifically, the denatured HDL-binding protein is immobilized on an ELISA plate. A known method can be appropriately used to immobilize (fix) the denatured HDL-binding protein. For example, a receptor can be bound to a known carrier (support). A sample is added to this ELISA plate, the denatured HDL contained in the sample is allowed to bind to the denatured HDL-binding protein, the ELISA plate is washed to remove unbound material, and the denatured HDL or the complex is detected using an antibody that can detect the denatured HDL or the complex, allowing the subject to be quantified and the complex of the denatured HDL and the denatured HDL-binding protein to be quantified. As the antibody that binds to denatured HDL or the complex, an antibody that binds to an HDL sequence can be appropriately used. In this embodiment, since the complex of denatured HDL and denatured HDL-binding protein on the ELISA plate is detected, it is denatured HDL that is detected on the plate. Note that, as the antibody that binds to denatured HDL in this embodiment, either a monoclonal or polyclonal antibody can be used.

[0030] More specifically, in this embodiment, a complex is formed by binding denatured HDL and a denatured HDL-binding protein, and this complex can be quantified by immunoassay. This immunoassay will be described with reference to a schematic diagram in FIG. 1. For example, a denatured HDL-binding protein 20 is immobilized on a plate 40. In this embodiment, the denatured HDL-binding protein 20 is, for example, a protein having an L-chain portion 21 and an H-chain portion 22 and having homology to a Factor V protein. The plate 40 is an ELISA plate. When the protein is immobilized on a known carrier, a structure is generated on the plate 40 in which the H-chain portion 22 is immobilized on the plate 40. The plate 40 is then washed to remove unbound denatured HDL-binding protein 20. A sample derived from a body fluid is injected into this plate 40. Denatured HDL 10 in the sample binds to the L-chain site 21 on the plate 40. The complex on the plate 40 is quantified using denatured HDL or an antibody 30 that binds to the complex. In this embodiment, an anti-apolipoprotein AI antibody (anti-ApoA1) is used as the antibody 30.

[0031] In this embodiment, a denatured HDL-binding protein that directly binds to denatured HDL is immobilized on a plate instead of using a denatured HDL receptor such as LOX-1 as in the conventional technology, so that a denatured HDL receptor is not required. For example, if an easily available protein, such as a protein with an even shorter full length than Factor V as described above, is used as the denatured HDL-binding protein, quantification can be performed more easily than when a denatured HDL receptor such as LOX-1 is used for quantification.

[0032] (Analytical reagent for quantifying denatured HDL) Next, an analytical reagent used in the above-mentioned method for quantifying denatured HDL in body fluids will be described. This analytical reagent can be used to quantify modified HDL in body fluids, and can be used to quantify modified HDL in body fluids to determine cardiovascular diseases, diabetes or diabetic diseases, or the degree of progression thereof. The analytical reagent contains the above-mentioned denatured HDL-binding protein, which makes it possible to quantify denatured HDL having physiological activity.

[0033] (Other configurations) The analytical reagent of this embodiment may contain other components contained in reagents for immune analysis.

[0034] The analytical reagent of this embodiment may be provided as a kit including a plurality of the above-mentioned components. For example, the kit may include an analytical reagent including a denatured HDL-binding protein for quantifying denatured HDL. The kit may also include a carrier, an ELISA plate, a color-developing substrate, an antibody, or the like, for use in the above-mentioned quantification operation.

[0035] The plate may also include an ELISA plate on which the above-mentioned protein is immobilized. For example, the plate may be provided as an analytical plate used for quantifying denatured HDL to determine cardiovascular disease, diabetes or diabetic disease, or the risk or progress thereof, the analytical plate comprising an ELISA plate on which a denatured HDL-binding protein capable of binding to denatured HDL is immobilized, the denatured HDL-binding protein containing a protein having an L-chain sequence of Factor V or a mutant thereof.

[0036] (Effects of this embodiment) The method for quantifying denatured HDL of the present embodiment can stably and accurately quantify denatured HDL compared to the conventional technology. The stably and accurately quantification of denatured HDL of the present embodiment means that, whereas the conventional methods may have differences in detection sensitivity themselves depending on the sample or have low detection sensitivity, the method of the present embodiment can quantify denatured HDL with a constant high sensitivity regardless of the sample conditions, for example, contents other than denatured HDL, etc.

[0037] The method for quantifying denatured HDL according to the present embodiment can stably and accurately quantify denatured HDL, and can therefore be used to assess diseases, their risks, and their progression. Diseases include a wide range of diseases that are thought to be related to HDL, such as cardiovascular diseases, diabetes, and diabetic diseases. Cardiovascular diseases in particular include arteriosclerosis or myocardial infarction or stroke caused by its progression.

[0038] (Application of this embodiment) The quantification method and analytical reagent for denatured HDL of this embodiment can be used, for example, in a method for diagnosing the occurrence of the above-mentioned cardiovascular diseases and / or diabetes or diabetic diseases by measuring denatured HDL in the quantification of HDL in body fluids.

[0039] As a diagnostic method, for example, in measuring denatured HDL in the sample, the risk of contracting the disease and / or the degree of progression thereof can be diagnosed from a change in the ratio of denatured HDL to the total HDL amount. This diagnostic method also includes the steps of using standard protein samples, healthy body fluid samples, etc. as standards, creating calibration curves, comparison tables, etc., and comparing the quantitative results from the specimen sample to be diagnosed with the calibration curves, comparison tables, etc. for use in diagnosis. The present invention can also be used to determine and diagnose the incidence, risk of incidence, and / or degree of progression of obesity-related diseases, such as hypertension and sleep apnea syndrome.

[0040] Second embodiment In the second embodiment, a complex is formed of denatured HDL, denatured HDL binding protein and LOX-1 protein, and this complex is quantified by immunoassay. In addition, the same components as those in the first embodiment are denoted by the same reference numerals in the drawings and the description thereof will be omitted.

[0041] In other words, the present embodiment aims to detect denatured HDL-binding protein more stably and reliably than conventional techniques that attempt quantification by detecting a complex of denatured HDL and LOX-1 protein. In this embodiment, quantification can be performed by improving the techniques and equipment used in conventional techniques that use LOX-1 protein.

[0042] LOX-1 is a molecule discovered by the present inventors (Sawamura T et al. Nature 386, 73-77, 1997), and is known as a type of lectin-like oxidized LDL receptor. The detailed structure of LOX-1 has been elucidated, and it is known that it is a single-pass membrane protein with a lectin-like domain, which is the recognition site for oxidized LDL (JP Patent Publication No. 9-98787, etc.), that it also has soluble components in blood, and that modified high density lipoprotein (HDL) acts as a ligand for LOX-1 (JP Patent Publication No. 2012-100585, Japanese Patent No. 6231307, etc.).

[0043] As the LOX-1 protein, for example, a recombinant LOX-1 protein is preferably used. Here, the recombinant LOX-1 protein is a LOX-1 protein obtained by expression through genetic recombination. The recombinant LOX-1 protein may be fused with other structures or mutated as necessary.

[0044] In the above-mentioned immunoassay, the quantification of the complex of denatured HDL, denatured HDL-binding protein and LOX-1 protein can be carried out using the so-called sandwich ELISA. An outline of the quantification method of this embodiment is shown in Figure 2. In this quantification method, for example, LOX-1 protein 50 is immobilized on plate 40A. Denatured HDL-binding protein 20 is bound to LOX-1 protein 50 on plate 40A. Denatured HDL 10 is bound to the complex of LOX-1 protein 50 and denatured HDL-binding protein 20 by injecting a sample derived from a body fluid into plate 40A. Next, the complex on plate 40A can be quantified using the above-mentioned denatured HDL or antibody 30 that binds to the complex. An anti-Factor-V antibody can also be used as antibody 30.

[0045] In this embodiment, a step of using serum as a sample and adding the denatured HDL-binding protein to the sample may be included. By adding this sample to the ELISA plate on which LOX-1 is immobilized, a complex is formed in which the denatured HDL-binding protein and denatured HDL bind to LOX-1 on the ELISA plate, and the bound body on the ELISA plate can be quantified with the denatured HDL or an antibody that binds to the complex.

[0046] The analytical reagent used in the quantitative determination method of this embodiment may contain recombinant LOX-1 protein for quantifying denatured HDL having pathological activity. As described above, the analytical reagent contains recombinant LOX-1 protein that interacts with denatured HDL, and therefore, the denatured HDL can be quantified by immunoassay of the complex of denatured HDL, denatured HDL-binding protein, and LOX-1.

[0047] The analytical reagent of this embodiment may be provided as a kit comprising a plurality of the above components. For example, the kit may comprise an analytical reagent containing a denatured HDL-binding protein for quantifying denatured HDL and an analytical reagent containing LOX-1 protein. EXAMPLES

[0048] Examples will be given below, but the present invention is not limited to the Examples.

[0049] (Test Example 1) (Verification of the sensitivity of Factor V to detect denatured HDL) First, we investigated the effect of Factor V on the binding of denatured HDL to LOX-1, a receptor for denatured HDL. A plasma or serum sample containing denatured HDL is added to a plate on which LOX-1 is immobilized, and the complex between LOX-1 and denatured HDL is detected with an antibody against denatured HDL (anti-apоA1 antibody). If the addition of Factor V to the sample improves the detection sensitivity depending on the amount of Factor V added and if stable detection sensitivity is obtained, then the binding mechanism between Factor V and denatured HDL can be used to quantify denatured HDL.

[0050] The ELISA was carried out as follows. Recombinant human LOX-1 (61-273aa) was immobilized on a 384-well plate at 0.15μg / well. After washing twice with PBS, blocking was performed with 1% casein-Na. After washing three times with PBS, plasma or serum samples containing Factor V were incubated at room temperature for 2 hours. Sample diluent was 1% casein-Na, 10mM HEPES, 150mM NaCl, pH7.4, 100μM APMSF. After washing three times with PBS, anti-apоA1 antibody (chicken #1) adjusted to 1μg / ml was added and incubated at room temperature for 1 hour. After washing three times with PBS, HRP-labeled anti-chicken IgY antibody diluted 4000-fold was added and incubated at room temperature for 1 hour. After antibody reaction, the plate was washed five times with PBS, and TMB solution (Bio-Rad) was added to the plate and reacted at room temperature. The reaction was stopped with 2 M sulfuric acid, and the absorbance at 450 nm was measured. The concentration of denatured HDL (ng / ml) was calculated using a calibration curve prepared using oxidized HDL oxidized with copper sulfate as a standard.

[0051] The detection results by ELISA are shown in Figure 3. Figure 3 (a), (b), (c), (d), and (e) show the results for plasma and serum collected from different subjects (A, B, C, D, and E), respectively. Complex on the vertical axis indicates the concentration detected by anti-apоA1 antibody for denatured HDL that formed some kind of complex with immobilized LOX-1 (including those in which the binding of LOX-1-denatured HDL is mediated by other proteins), and the horizontal axis indicates the concentration of Factor V added. As shown in Figures 3(a), (b), (c), (d), and (e), in serum, the detection sensitivity of denatured HDL increased depending on the amount of Factor V added in all five samples taken from subjects A, B, C, D, and E. In plasma, depending on the sample taken, there were cases where there was no significant difference depending on the amount of Factor V added, and cases where the increase was almost dependent on the amount of Factor V added. These results suggest that serum, which contains fewer substances than plasma, contains almost no proteins such as Factor V that mediate the binding between LOX-1 and denatured HDL, and that this binding is mediated by the addition of Factor V. On the other hand, plasma may contain proteins such as Factor V that mediate the binding between LOX-1 and denatured HDL, but the amount varies depending on the specimen, its collection method, storage method, etc., and the effect of adding Factor V is thought to be different. These results demonstrated that Factor V binds to denatured HDL, that the binding of denatured HDL to LOX-1 occurs via Factor V, and that the detection sensitivity of this bound complex improves depending on the amount of Factor V added.

[0052] (Test Example 2) By examining the detection sensitivity depending on whether LOX-1 was immobilized on the plate, it was examined whether the increase in detection sensitivity due to Factor V occurred for the complex of LOX-1, Factor V, and denatured HDL. FIG. 4 shows the results of detection by ELISA using plasma collected from each of subjects A, B, C, D, and E as samples with Factor V protein added to 1 μg / mL (plasma+FV) or without (plasma), and using LOX-1 immobilized (LOX-1 immobilized) or non-immobilized (non-coated) plates, with the rest being the same as in Test Example 1. Figure 5 shows the results of detection performed in the same manner as in Figure 4, using serum collected from each of subjects A, B, C, D, and E as samples, with Factor V protein added to 1 μg / mL (serum+FV) and without Factor V protein added (serum). Figure 6 shows the results of detection performed in the same manner as in Figure 4, using plasma collected from each of A, B, C, D, and E as samples, with and without Factor V protein added at 10 μg / mL. Figure 7 shows the results of detection performed in the same manner as in Figure 5, using the serum collected from each of subjects A, B, C, D, and E as samples, with and without Factor V protein added at 10 μg / mL.

[0053] 4 to 7, when a plate on which LOX-1 is not immobilized is used, no difference in the amount of detection is observed regardless of whether Factor V is added to the sample or not. This result shows that Factor V increases the amount of detection of the complex formed by the binding of LOX-1, Factor V, and denatured HDL. Comparing Figures 4 and 5, and Figures 6 and 7, Figures 4 and 6 show that plasma was used, while Figures 5 and 7 show that serum was used, and the amount of detection was higher by adding Factor V. As in Test Example 1, this result is also thought to be due to the addition of Factor V compensating for substances thought to be absent from serum.

[0054] (Test Example 3) Since Factor V binds to denatured HDL, we attempted to determine whether Factor V could be directly immobilized on a plate to quantify denatured HDL.

[0055] First, Factor V dissolved in PBS(-) at a concentration of 0 to 10 μg / mL was added to the plate, and an attempt was made to determine whether it could be immobilized on the plate by ELISA using an anti-Factor V antibody. Solid phase: Human Factor V each concentration Blocking: 1% casein-Na, 2hr at room temperature Detection: Primary antibody anti-FV-LC (AHV-5101) 1μg / ml, 1hr, room temperature Secondary antibody anti-mouse IgG-HRP, 1:4000 in 1% casein-Na, 1 hr at room temperature

[0056] The results are shown in Figure 8. It was shown that Factor V could be immobilized on the plate in an almost concentration-dependent manner.

[0057] Next, the binding of non-denatured HDL (nHDL) and denatured HDL to the Factor V-immobilized ELISA plate was tested. Denatured HDL is oxidized HDL by copper sulfate (Cu 2+ -оxHDL was used. Factor V was immobilized on a 384-well plate (Greiner). After washing twice with PBS, blocking was performed with 1% casein-Na. After washing three times with PBS, nHDL or Cu was prepared at 1 μg / ml in sample diluent (1% casein-Na, 10 mM HEPES, 150 mM NaCl, pH 7.4, 100 μM APMSF). 2+ -оxHDL was incubated at room temperature for 2 hours. After washing three times with PBS, anti-apоA1 antibody (chicken #1) adjusted to 1μg / ml was added and incubated at room temperature for 1 hour. After washing three times with PBS, HRP-labeled anti-chicken IgY antibody diluted 4000-fold was added and incubated at room temperature for 1 hour. After the antibody reaction, the plate was washed five times with PBS, and TMB solution (Bio-Rad) was added to the plate and reacted at room temperature. The reaction was stopped with 2M sulfuric acid, and the absorbance at 450nm was measured.

[0058] The results are shown in Figure 9. The amount of denatured HDL detected increased depending on the amount of Factor V immobilized on the plate. On the other hand, the amount of nHDL detected did not change significantly. This result indicates that this plate can be used to detect denatured HDL.

[0059] (Test Example 4) Denatured HDL in human plasma and serum was detected using a Factor V-immobilized plate prepared in the same manner as in Test Example 3. Denatured HDL was quantified by ELISA using plasma and serum samples collected from subjects X and Y. Factor V was immobilized on a 384-well plate (Greiner). After washing twice with PBS, blocking was performed with 1% casein-Na. After washing three times with PBS, plasma or serum samples were incubated at room temperature for 2 hours. After washing three times with PBS, anti-apоA1 antibody (chicken #1) prepared at 1 μg / ml was added and incubated at room temperature for 1 hour. After washing three times with PBS, HRP-labeled anti-chicken IgY antibody diluted 4000 times was added and incubated at room temperature for 1 hour. After the antibody reaction, the plate was washed five times with PBS, and TMB solution (Bio-Rad) was added to the plate and reacted at room temperature. The reaction was stopped with 2 M sulfuric acid, and the absorbance at 450 nm was measured.

[0060] The results are shown in Figure 10. The sample taken from subject X is shown in Figure 10(a), and the sample taken from subject Y is shown in Figure 10(b). Unlike the conventional technique of immobilizing LOX-1, there was almost no difference in the measured values ​​between plasma and serum, i.e., stable high detection sensitivity was observed for plasma, regardless of the sample. This technique is more sensitive than the technique of immobilizing LOX-1, and not only can quantitative analysis be performed more easily than the sandwich method using LOX-1 and Factor V, but it can also stably and reliably perform quantitative analysis with high sensitivity for both plasma and serum.

[0061] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents described in the claims, as well as in the scope and spirit of the invention. [Industrial Applicability]

[0062] According to the present invention, there is provided a method for quantifying denatured HDL, which can quantify denatured HDL more stably and accurately than conventional methods, and an analytical reagent for use in said method. This method and analytical reagent can be used, in particular, for determining the risk or degree of progression of cardiovascular disease, diabetes, and diabetic diseases. [Explanation of symbols]

[0063] 10 Denatured HDL 20 Denatured HDL-binding protein 21 L chain site 22 H chain site 30 Antibodies 40, 40A Plate 50 LOX-1 protein

Claims

1. A method for quantifying denatured HDL contained in a sample, comprising: adding a denatured HDL-binding protein capable of binding to the denatured HDL to the sample; allowing the denatured HDL to bind to a denatured HDL-binding protein, and quantifying the amount of the denatured HDL-binding protein bound to the denatured HDL; A method for quantifying denatured HDL, wherein the denatured HDL-binding protein comprises a protein having a sequence of the L chain portion of Factor V or a mutant thereof.

2. 2. The method for quantifying denatured HDL according to claim 1, wherein the denatured HDL-binding protein comprises a sequence of a C1-C2 domain, which is a phospholipid recognition site of Factor V.

3. The method for quantifying denatured HDL according to claim 1 or 2, wherein the denatured HDL-binding protein comprises a Factor V sequence.

4. 3. The method for quantifying denatured HDL according to claim 2, wherein the denatured HDL-binding protein is MFG-E8, Del-1 or Factor VIII.

5. The method for quantifying denatured HDL according to claim 1 , wherein the denatured HDL-binding protein is a recombinant protein.

6. 6. The method for quantifying denatured HDL according to claim 1, further comprising forming a complex between the denatured HDL and the denatured HDL-binding protein, and quantifying the complex by immunoassay.

7. 7. The method for quantifying denatured HDL according to claim 6, wherein the quantification by immunoassay is carried out by an ELISA method using an ELISA plate on which the denatured HDL-binding protein or a protein that binds to the denatured HDL-binding protein is immobilized.

8. 8. The method for quantifying denatured HDL according to claim 1, wherein the quantification comprises forming a complex of the denatured HDL, the denatured HDL binding protein and LOX-1 protein, and quantifying the complex by immunoassay.

9. The method for quantifying denatured HDL according to claim 1 , wherein serum or plasma is used as a sample in the quantification.

10. The method for quantifying denatured HDL according to claim 1 , wherein serum is used as a sample in the quantification.

11. An analytical reagent for use in quantifying modified HDL for determining cardiovascular disease, diabetes, or diabetic disease, or the risk thereof, or the degree of progression thereof, comprising: a modified HDL-binding protein capable of binding to the modified HDL; The denatured HDL-binding protein is an analytical reagent comprising a protein having an L-chain sequence of Factor V or a mutant thereof.

12. The analytical reagent according to claim 11, wherein the denatured HDL-binding protein comprises a sequence of a C1-C2 domain, which is a phospholipid recognition site of Factor V.

13. The analytical reagent according to claim 11 or 12, wherein the denatured HDL-binding protein comprises a Factor V sequence.

14. The analytical reagent according to claim 12, wherein the denatured HDL-binding protein is MFG-E8, Del-1 or Factor VIII.

15. The analytical reagent according to claim 11 , wherein the denatured HDL-binding protein is a recombinant protein.

16. 16. The analytical reagent according to claim 11, wherein the cardiovascular disease is myocardial infarction or cerebral stroke caused by arteriosclerosis or its progression.

Images