Quantitative method for modified hdl and analytical reagents for the same

Abstract

Provided is a method for quantifying modified HDL, which is a method for quantifying modified HDL contained in a sample, wherein the modified HDL is allowed to bind to a modified HDL binding protein comprising a protein having the sequence of the L-chain site of factor V or a mutant thereof, and the complex of the modified HDL binding protein and the modified HDL is quantified.

Description

Technical Field

[0001] This invention relates to a quantitative method for modified HDL that can be used to determine the risk of lifestyle-related diseases and their curative effects, as well as analytical reagents for this quantitative method. Background Technology

[0002] HDL (High-Density Lipoprotein) is a type of cholesterol with a high density and small particle size. HDL is known to be responsible for transporting cholesterol from tissues to the liver. Higher concentrations of HDL in the blood reduce the risk of arteriosclerosis and are therefore sometimes referred to as "good cholesterol." On the other hand, LDL (Low-Density Lipoprotein), with its lower density, is known as "bad cholesterol." The concentrations of HDL and LDL are used as indicators for health assessment.

[0003] As a physiological activity, HDL not only inhibits LDL oxidation but also reduces the cytotoxicity caused by oxidized LDL through increased NO levels, thus exerting an anti-atherosclerotic effect. However, in patients with coronary artery disease, serum modified HDL increases, which acts on blood vessels via LOX-1, promoting atherosclerosis. In atherosclerotic lesions, substances modified from ApoAI, the apolipoprotein of HDL, accumulate in large quantities. Due to these results, in order to use HDL as an indicator for health diagnosis, it is necessary not only to measure HDL concentration but also to quantify both HDL with its original physiological activity and HDL whose activity has changed due to modification. In particular, it is believed that if modified HDL can be quantified, it can be used to verify the risks of circulatory system and blood-related diseases, including the aforementioned coronary artery disease.

[0004] Patent Document 1 discloses a method for measuring HDL of dysfunction, which involves contacting the sample with a receptor for HDL of dysfunction, causing the HDL in the sample to bind to the receptor, and detecting the HDL bound to the receptor. The receptor is LOX-1 or a variant thereof, and the receptor is immobilized on a carrier, allowing an antibody that recognizes HDL of dysfunction to further bind to the HDL bound to the receptor. This technology aims to provide a method for measuring HDL of dysfunction capable of broadly detecting various molecular forms of HDL, and to provide a method for detecting lifestyle-related diseases using this technology.

[0005] Furthermore, the inventors of this invention disclosed in Patent Document 2 a fusion protein in which the entire protein sequence or a partial fragment of the ApoA1 protein sequence is directly or via a spacer linked to the LOX-1 binding protein sequence that binds to LOX-1, and a kit for measuring high-density lipoprotein using this fusion protein. This technology can measure HDL that has undergone various modifications by measuring HDL simultaneously recognized by LOX-1 and anti-ApoA1 antibodies, as well as fusion proteins formed by fusing proteins specifically binding to LOX-1 with ApoA1, such as the LOX-1 binding site of an antibody against LOX-1 (anti-LOX-1 antibody). This fusion protein allows for the simultaneous recognition of various modified HDLs, enabling the measurement of receptor binding activity and physiological activity itself. Therefore, it is desirable to obtain a lipid-free, long-term preservative, reproducible, and reliable fusion protein that reduces bias between individual assays.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent No. 6231307

[0009] Patent Document 2: Japanese Patent Application 2018-024007 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] Modified HDL can theoretically be quantified in the laboratory using the technique described in Patent Document 1. The technique in Patent Document 2 of the inventors of this invention is further refined compared to the prior art. It is conceivable that, by applying these methods, it is easy to establish a method for quantifying modified HDL even if it is not fully refined.

[0012] However, the technology in Patent Document 1 has not yet achieved widespread adoption. Furthermore, even when using the technology in Patent Document 2 by the inventors of this invention, the quantitative results of modified HDL are sometimes inconsistent when performed under conditions outside the laboratory and with different samples. These differences are due to various factors, including variations in laboratory conditions and the experimental techniques of the individuals performing the technology.

[0013] The inventors of this invention, taking into account the aforementioned historical context and the fact that measurements should be possible even under slightly less favorable conditions in clinical applications, conducted in-depth research with the goal of improving the quantitative technique for modified HDL into a more universal quantitative technique.

[0014] The present invention was made in view of the above circumstances, and its object is to provide a method for quantifying modified HDL that is more stable and accurate than the conventional method, useful for determining the degree of cardiovascular / vascular diseases, diabetes or diabetic diseases, or their development, as well as analytical reagents for the quantitative method.

[0015] means for solving problems

[0016] To solve the above problems, the present invention has the following provisions.

[0017] [1] A method for quantifying modified HDL, which is a method for quantifying modified HDL contained in a sample, wherein the modified HDL is bound to a modified HDL binding protein, and the binding of the modified HDL binding protein to the modified HDL is quantified, wherein the modified HDL binding protein comprises a protein having a sequence of an L chain region having factor V or a mutant thereof.

[0018] [2] Based on the quantitative method of the modified HDL, the modified HDL binding protein contains the sequence of the phospholipid recognition site of factor V.

[0019] [3] Based on the quantitative method of the modified HDL, the modified HDL binding protein contains the sequence of factor V.

[0020] [4] Based on the quantitative method of the modified HDL, the modified HDL binding protein is a protein having a portion of the C1-C2 domain that is the phospholipid recognition site of factor V.

[0021] [5] Based on the quantitative method of the modified HDL, the protein having a part homology with the C1-C2 domain is MFG-E8, Del-1 or factor VIII.

[0022] [6] Based on the quantitative method of the modified HDL, the modified HDL binding protein is a recombinant protein.

[0023] [7] Based on the quantitative method of the modified HDL, the quantification is to form a complex of the modified HDL and the modified HDL binding protein and to quantify the complex by immunoassay.

[0024] [8] Based on the quantitative method of the modified HDL, the quantification based on the immunoassay is performed by using an ELISA method that uses an ELISA plate that immobilizes the modified HDL-binding protein or the protein bound thereto.

[0025] [9] Based on the quantitative method of the modified HDL, the quantification is to form a complex of the modified HDL, the modified HDL binding protein and the LOX-1 protein and quantify the complex by immunoassay.

[0026]

[10] Based on the quantitative method for modified HDL, serum or plasma is used as the sample in the quantitative method.

[0027]

[11] Based on the quantitative method of the modified HDL, the quantitative method includes a step of using serum as a sample and adding the modified HDL binding protein to the sample.

[0028]

[12] An analytical reagent for quantifying modified HDL for determining cardiovascular disease, diabetes or diabetic disease, their risk or degree of development, wherein the analytical reagent comprises a modified HDL-binding protein capable of binding to the modified HDL, the modified HDL-binding protein comprising a protein having an L-chain site sequence of factor V or a mutant thereof.

[0029]

[13] Based on the analytical reagents, the modified HDL binding protein contains the sequence of the phospholipid recognition site of factor V.

[0030]

[14] Based on the analytical reagents, the modified HDL binding protein contains the sequence of factor V.

[0031]

[15] Based on the analytical reagents, the modified HDL binding protein is a protein having a portion of the C1-C2 domain that is the phospholipid recognition site of factor V.

[0032]

[16] Based on the analytical reagents, the protein having a part of the C1-C2 domain is MFG-E8, Del-1 or factor VIII.

[0033]

[17] Based on the analytical reagents, the modified HDL-binding protein is a recombinant protein.

[0034]

[18] Based on the reagents used in the analysis, the cardiovascular disease is myocardial infarction or stroke caused by arteriosclerosis or its development.

[0035] Invention Effects

[0036] According to the present invention, a quantitative method for modified HDL that can more stably and accurately quantify it compared with the past, and which is useful for determining the degree of cardiovascular / vascular diseases, diabetes or diabetic diseases, or their development, can be obtained, as well as analytical reagents for the quantitative method. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the quantitative method for modified HDL according to the first embodiment.

[0038] Figure 2 This is a schematic diagram illustrating the quantitative method for modified HDL according to the second embodiment.

[0039] Figure 3 This is a graph representing the detection sensitivity of the modified HDL involved in Experiment Example 1.

[0040] Figure 4 This is a graph representing the detection sensitivity of modified HDL in the plasma involved in Experiment Example 2.

[0041] Figure 5 This is a graph representing the detection sensitivity of modified HDL in the serum involved in Experiment Example 2.

[0042] Figure 6 This is a graph representing the detection sensitivity of modified HDL in plasma at the concentration of another factor V involved in Experiment Example 2.

[0043] Figure 7 This is a graph representing the detection sensitivity of modified HDL in serum at the concentration of another factor V involved in Experiment Example 2.

[0044] Figure 8 This is a graph representing the amount of solidification of factor V involved in Experiment Example 4.

[0045] Figure 9 This is a graph representing the modified HDL binding test involved in Experiment Example 4.

[0046] Figure 10 This is a graph representing the quantitative levels of modified HDL in plasma and serum involved in Experiment Example 5. Detailed Implementation

[0047] The following describes the quantitative method for modified HDL according to the present invention, as well as the analytical reagents. However, the present invention is not limited to the following embodiments.

[0048] (First Implementation)

[0049] (Quantitative methods for modified HDL)

[0050] The quantitative method for modified HDL in this embodiment involves binding the modified HDL to a modified HDL-binding protein when quantifying the modified HDL contained in the sample. The complex formed by the binding of the modified HDL-binding protein and the modified HDL is then quantified.

[0051] The modified HDL in this embodiment includes HDLs that have some structural differences from HDLs that are physiologically active in organisms, and in particular, HDLs whose physiological activity is reduced due to these structural differences. Mechanisms by which HDLs become modified HDLs include, for example, chemical modifications of the proteins, partial or complete degradation, genetic defects and mutations, abnormalities or changes in higher-order structures, or binding to molecules that render them inactive.

[0052] Examples of such modified HDLs include oxidized HDLs, which involve the oxidation of any part of the proteins or lipids that constitute HDL. Examples of such modified HDLs include HClO-HDL, HNE-HDL, carbamylated HDL, and MDA-HDL. These modified HDLs are known to inhibit the growth of vascular endothelial cells in the blood vessel wall, increasing the risk of various diseases such as atherosclerotic diseases, thrombotic heart disease, and ischemic diseases.

[0053] In the quantitative method for modified HDL in this embodiment, modified HDL is bound to modified HDL-binding protein, and the binding of modified HDL-binding protein to modified HDL is quantified.

[0054] In this embodiment, the term "modified HDL-binding protein" broadly refers to a protein that can directly interact with modified HDL, such as a protein that can bind to modified HDL itself.

[0055] Modified HDL sometimes acquires the activity of interacting with receptors that cause adverse reactions in organisms. In such cases, modified HDL sometimes binds to the receptor via a modified HDL-binding protein, i.e., the modified HDL binds directly to the modified HDL-binding protein, which in turn binds to the receptor.

[0056] Examples of modified HDL-binding proteins include factor V, its mutants, and proteins with partial homology. LOX-1 protein, described later, is a receptor that sometimes binds to modified HDL via other proteins.

[0057] In this embodiment, the modified HDL binding protein comprises a protein or a mutant thereof having a sequence of the L chain region of factor V.

[0058] Factor V (factor V) is known as a protein constituting the coagulation system. It is a protein of approximately 330 kDa composed of A1-A2-B-A3-C1-C2 domains, and is broadly divided into the H chain (heavy chain) composed of the A1-A2 domain and the L chain (light chain) composed of the A3-C1-C2 domain. It is known that the H chain mainly binds to the cell membrane, while the L chain recognizes and binds to phospholipids via the C1-C2 domain.

[0059] A mutant is a mutant in which a portion of the sequence has been replaced. In this embodiment, it is preferred to be a mutant in which the portion other than the L chain region of factor V has been replaced.

[0060] Since the L-chain region of factor V is the site that directly binds to modified HDL, by making the modified HDL binding protein a protein containing this region or a mutant thereof, it is possible to make modified HDL bind to the modified HDL binding protein and use it for quantification.

[0061] Furthermore, as a modified HDL-binding protein, by using a protein having a sequence containing the L-chain region as part of factor V, it is possible to use a protein that has a site for binding to modified HDL while possessing a structure different from factor V. That is, a protein that is easier to obtain and test than factor V can be used. As a protein easier to obtain than factor V, for example, by using only the L-chain region and a minimal sequence, a protein that is shorter in full length than factor V and easier to manufacture as a recombinant protein can be used. As a protein easier to test, a protein that is easier to preserve and immobilize on a plate can be used by modifying sequences other than the L-chain.

[0062] The modified HDL-binding protein preferably also includes the sequence of the phospholipid recognition site of factor V. The phospholipid recognition site of the L chain in the factor V sequence can recognize modified HDL and bind to it effectively.

[0063] The modified HDL-binding protein preferably contains the sequence of factor V. Here, the sequence of factor V refers to the full length of the factor V protein, which can be factor V itself, a fusion with other proteins, or a modified protein.

[0064] The modified HDL-binding protein is preferably a protein having a portion of the C1-C2 domain that is the phospholipid recognition site of factor V. Examples of such proteins having a portion of the C1-C2 domain include MFG-E8, Del-1, or factor VIII. Alternatively, proteins that contain a portion of these proteins, mutants, or sequences of these proteins may also be used.

[0065] Modified HDL-binding proteins, for example, preferably recombinant proteins. Here, recombinant proteins refer to proteins obtained through gene recombination expression. Recombinant proteins can be fused or mutated with other structures as needed.

[0066] In this embodiment, the modified HDL contained in the sample is quantified. Here, "sample" broadly refers to a sample containing components extracted from a biological organism. The sample is preferably a liquid containing the aforementioned components, or a sample prepared by adding liquid to the aforementioned components. For example, bodily fluids of a biological organism can be used directly as the sample. For example, human blood or saliva can be used as bodily fluids. For blood samples, serum or plasma can be used. In this embodiment, these bodily fluids are used as samples for the aforementioned quantification.

[0067] In this embodiment, serum is preferably used as the sample. In this embodiment, a modified HDL-binding protein having the sequence of the L chain portion of factor V, which directly binds to modified HDL, is used to bind to the modified HDL in the sample. Therefore, even if serum that does not contain a factor that directly binds to modified HDL is used as the sample, the modified HDL can be quantified stably and accurately. Furthermore, compared to other bodily fluid samples such as plasma, serum is easier to obtain and preserve. In fact, freezing and preserving serum samples as blood samples is common internationally, and therefore a beneficial technique.

[0068] The inventors of this invention focused on the following aspect: In the prior art, when proteins that interact with modified HDL, such as LOX-1, are used for the quantification of modified HDL, modified HDL can be detected when plasma is used as a sample, but when serum is used as a sample, the detection rate of modified HDL is low, and accurate quantification is not possible. Therefore, it was predicted that by filling the assay system with substances lost during the process of forming serum from blood and plasma, it would be possible to perform assays using serum as a sample.

[0069] As the filling material mentioned above, the inventors of this invention focus on factor V. As a reaction within the organism, the modified HDL binds to LOX-1 via factor V. Specifically, the phosphatidylserine (PS) recognition site of factor V binds to the modified HDL, and the LOX-1 recognition site binds to LOX-1.

[0070] As described later, when serum is used as a sample, the detection sensitivity of modified HDL in serum increases approximately proportionally with the amount of factor V added. Therefore, by adding a modified HDL-binding protein such as factor V to samples like serum, modified HDL can be quantified.

[0071] In the quantification described above, known methods for protein quantification (protein quality determination) can be used. For example, immunological methods can be used in the quantification described above.

[0072] As a quantitative and immunoassay method utilizing immunological techniques, enzymes (enzyme immunoassay, EIA, ELISA), fluorescent substances (fluorescent immunoassay, FIA), or radioactive substances (radioimmunoassay, RIA) can be appropriately used. Among these, ELISA is relatively inexpensive, simple, and capable of analyzing multiple samples, making it the preferred choice.

[0073] For example, in this embodiment, the ELISA method can be used in the above quantification. Specifically, the modified HDL-binding protein is immobilized on an ELISA plate. The immobilization (stabilization) of the modified HDL-binding protein can be performed using known methods. For example, the receptor can be bound to a known carrier (support).

[0074] The sample is added to the ELISA plate, allowing the modified HDL contained in the sample to bind with the modified HDL-binding protein. The ELISA plate is then washed to remove unbound material. When an antibody capable of detecting modified HDL or the complex is used for detection, the target and the complex of modified HDL and modified HDL-binding protein can be quantified.

[0075] As the antibody binding to modified HDL or the aforementioned complex, an antibody binding to the HDL sequence can be appropriately used. In this embodiment, since the complex of modified HDL and modified HDL-binding protein on the ELISA plate is detected, modified HDL is detected on the plate. It should be noted that in this embodiment, the antibody binding to modified HDL can be either a monoclonal or polyclonal antibody.

[0076] In this embodiment, more specifically, a complex formed by the combination of modified HDL and modified HDL-binding protein can be formed, and the complex can be quantified by immunoassay.

[0077] Regarding this immunoassay, Figure 1 A schematic diagram is shown below for illustration. For example, modified HDL-binding protein 20 is immobilized on plate 40. In this embodiment, as an example, modified HDL-binding protein 20 is a protein homologous to factor V protein, possessing an L-chain region 21 and an H-chain region 22. Plate 40 is an ELISA plate. If immobilization is performed using a known carrier, the aforementioned structure with the H-chain region 22 immobilized on plate 40 is generated. Next, plate 40 is washed to remove unbound modified HDL-binding protein 20.

[0078] A sample from bodily fluids is injected into the plate 40. The modified HDL10 in the sample binds to the L-chain site 21 on the plate 40. The complex on the plate 40 is quantified using an antibody 30 that binds to the modified HDL or the aforementioned complex. In this embodiment, the antibody 30 used is an anti-apolipoprotein AI antibody (anti-ApoA1).

[0079] In this embodiment, instead of using modified HDL receptors such as LOX-1 as in the prior art, a modified HDL-binding protein that directly binds to modified HDL is immobilized on a plate, thus eliminating the need for a modified HDL receptor. For example, if an easily obtainable protein, such as one with a shorter full-length protein than the aforementioned factor V, is used as the modified HDL-binding protein, quantification can be performed more easily compared to using modified HDL receptors such as LOX-1 for quantification.

[0080] (Analytical reagents used for the quantification of modified HDL)

[0081] Next, the analytical reagents used in the above-mentioned method for quantifying modified HDL in body fluids will be described.

[0082] By using this analytical reagent for the quantification of modified HDL in body fluids, it is possible to quantify modified HDL in body fluids for the purpose of determining cardiovascular / vascular disease, diabetes or diabetic disease, or the degree of its development.

[0083] The analytical reagents contain the modified HDL-binding protein described above. By including the modified HDL-binding protein, it is possible to quantify physiologically active modified HDL.

[0084] (Other components)

[0085] The analytical reagents used in this embodiment may also contain components found in other immunoassay reagents.

[0086] Furthermore, the analytical reagents of this embodiment can also be provided as kits having multiple of the above-described configurations. For example, the kit may include analytical reagents containing a quantitative modified HDL-binding protein for modifying HDL. Additionally, the kit may include a carrier, ELISA plate, chromogenic substrate, or antibody for the aforementioned quantitative operations.

[0087] Alternatively, an ELISA plate may be provided with the aforementioned proteins immobilized. For example, it may also be provided as an analytical plate for quantifying modified HDL to determine cardiovascular / vascular disease, diabetes or diabetic disease, their risk, or the extent of their development, comprising an ELISA plate immobilized with a modified HDL-binding protein capable of binding to modified HDL, the modified HDL-binding protein comprising a protein having the L-chain site sequence of factor V or a mutant thereof.

[0088] (Effects of this implementation method)

[0089] Compared with existing technologies, the quantitative method for modified HDL in this embodiment can stably and accurately quantify modified HDL. The ability to stably and accurately quantify modified HDL in this embodiment means that, unlike conventional methods where detection sensitivity varies depending on the sample and is relatively low, the method of this embodiment can quantify modified HDL with a consistently high sensitivity, regardless of sample conditions, such as inclusions other than modified HDL.

[0090] The quantitative method for modified HDL in this embodiment enables stable and accurate quantification of modified HDL, which can then be used to determine diseases, their risks, and the degree of their development. Diseases considered to be related to HDL are broadly included, such as cardiovascular diseases, diabetes, or diabetic disorders. Cardiovascular diseases particularly include myocardial infarction or stroke resulting from arteriosclerosis or its progression.

[0091] (Purpose of this embodiment)

[0092] The quantitative method for modified HDL and the analytical reagents of this embodiment can be used, for example, in the quantitative determination of modified HDL in body fluids to diagnose the aforementioned cardiovascular / vascular diseases and / or diabetes or diabetic diseases.

[0093] As a diagnostic method, for example, the risk of having the above-mentioned diseases and / or the degree of their development can also be diagnosed based on the change in the ratio of modified HDL to total HDL in the above-mentioned sample.

[0094] This diagnostic method also includes the process of using protein standards, healthy bodily fluid samples, etc. as standards to create standard curves, comparison tables, etc., and comparing the quantitative results of the specimens based on the diagnostic subjects with the standard curves, comparison tables, etc. for diagnosis.

[0095] In addition, it can also be used to determine and diagnose obesity-related diseases such as hypertension and sleep apnea syndrome, as well as their prevalence, risk, and / or progression.

[0096] (Second Implementation)

[0097] In the second embodiment, a complex of modified HDL, modified HDL-binding protein, and LOX-1 protein is formed, and the complex is quantified by immunoassay.

[0098] It should be noted that for configurations identical to those in the first embodiment, the same reference numerals are used in the accompanying drawings and descriptions are omitted.

[0099] In other words, compared to existing techniques that attempt to quantify the complex of modified HDL and LOX-1 protein, this embodiment attempts to achieve detection with greater stability and reliability by further utilizing modified HDL-binding proteins. In this embodiment, quantification can be achieved by improving the methods and equipment used in existing techniques that utilize LOX-1 protein.

[0100] LOX-1 is a molecule discovered by the inventors of this invention (Sawamura T et al. Nature 386, 73-77, 1997), and is known to be a lectin-like oxidative LDL receptor. The detailed structure of LOX-1 has also been determined; it is known to be a single-pass membrane protein of the cell membrane, possessing a lectin-like domain, which is the recognition site for oxidative LDL (Japanese Patent Application Publication No. 9-98787, etc.); soluble components are also present in blood; modified high-density lipoprotein (HDL) functions as a ligand for LOX-1, etc. (Japanese Patent Application Publication No. 2012-100585, Japanese Patent No. 6231307, etc.).

[0101] The aforementioned LOX-1 protein is preferably recombinant LOX-1 protein. Here, recombinant LOX-1 protein refers to LOX-1 protein obtained through gene recombination expression. Recombinant LOX-1 protein can also be fused or mutated with other structures as needed.

[0102] In the above immunoassays, the quantification of the complex of modified HDL, modified HDL-binding protein and LOX-1 protein can be performed using a so-called sandwich ELISA.

[0103] A summary of the quantitative method of this embodiment is shown below. Figure 2 As a quantitative method, for example, LOX-1 protein 50 is immobilized on plate 40A. Modified HDL-binding protein 20 is then bound to LOX-1 protein 50 on plate 40A. By injecting a sample from body fluid into plate 40A, modified HDL 10 binds to the complex of LOX-1 protein 50 and modified HDL-binding protein 20. The complex on plate 40A can then be quantified using antibody 30, which binds to the modified HDL or the complex. Antibody 30 can also be an anti-factor-V antibody.

[0104] In this embodiment, a step of using serum as a sample and adding the modified HDL binding protein to the sample can be included. The sample can be added to an ELISA plate immobilized with LOX-1, forming a complex of the modified HDL binding protein and modified HDL bound to the LOX-1 on the ELISA plate. The complex on the ELISA plate can then be quantified using an antibody that binds to the modified HDL or the complex.

[0105] It should be noted that the analytical reagents used in the quantitative method of this embodiment may contain recombinant LOX-1 protein for the quantification of modified HDL with pathological activity. As described above, the recombinant LOX-1 protein in this analytical reagent interacts with modified HDL, thus enabling the quantification of modified HDL, the complex of modified HDL-binding protein and LOX-1, by immunoassay.

[0106] Furthermore, the analytical reagents of this embodiment can also be provided as kits having multiple of the above-described configurations. For example, the kit may include analytical reagents containing quantitative modified HDL-binding proteins for modifying HDL and analytical reagents containing LOX-1 protein.

[0107] Example

[0108] The following are examples. It should be noted that the present invention is not limited to these examples.

[0109] (Experimental Example 1)

[0110] (Validation of the detection sensitivity of modified HDL based on factor V)

[0111] First, we investigated the effect of factor V on the binding of modified HDL to LOX-1, which acts as a receptor for modified HDL.

[0112] A plasma or serum sample containing modified HDL is added to a plate immobilized with LOX-1, and an experiment is performed to detect the complex of LOX-1 and modified HDL using an antibody against modified HDL (anti-apoA1 antibody). If, by adding factor V to the sample, the detection sensitivity increases depending on the amount of factor V added, and a stable detection sensitivity can be obtained, then the binding mechanism of factor V to modified HDL can be used for the quantification of modified HDL.

[0113] The ELISA method is performed according to the following steps.

[0114] Recombinant human LOX-1 (61-273aa) was immobilized at 0.15 μg / well in a 384-well plate. After washing twice with PBS, the plate was blocked with 1% casein-Na. After washing three times with PBS, plasma or serum samples supplemented with factor V were incubated at room temperature for 2 hours. The sample dilution buffer consisted of 1% casein-Na, 10 mM HEPES, 150 mM NaCl, pH 7.4, and 100 μM MAPMSF. After washing three times with PBS, anti-apoA1 antibody (chicken #1) prepared at 1 μg / ml was added, and the plate was 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 the plate was 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. The reaction was allowed to proceed at room temperature. The reaction was stopped with 2M sulfuric acid, and the absorbance at 450 nm was measured. The modified HDL concentration (ng / ml) was calculated using a standard curve prepared by using oxidized HDL oxidized by copper sulfate as a standard.

[0115] The ELISA-based detection results are shown in Figure 3 . Figure 3 In the figure, (a), (b), (c), (d), and (e) represent the results for plasma and serum collected from different subjects (A, B, C, D, and E), respectively. The vertical axis, "complex," represents the concentration of anti-apoA1 antibody relative to the modified HDL (including LOX-1 modified HDL binding with the aid of other protein-modified HDL) that forms a complex against immobilized LOX-1. The horizontal axis represents the concentration of added factor V.

[0116] Regarding serum, such as Figure 3 As shown in (a), (b), (c), (d), and (e), in all five examples of specimens collected from subjects A, B, C, D, and E, the detection sensitivity of modified HDL increased with the amount of factor V added. Regarding plasma, depending on the specimen collected, there were cases where no significant difference was observed due to the amount added, and cases where the sensitivity increased substantially with the amount added.

[0117] Based on the above results, it is believed that for serum, which contains less substance than plasma, since it almost does not contain proteins such as factor V that bind to modified HDL via LOX-1, the addition of factor V mediates this binding. On the other hand, it is believed that plasma sometimes contains proteins such as factor V that mediate the binding of LOX-1 to modified HDL, but their content varies depending on the sample and its collection and preservation methods, and the effect of adding factor V varies.

[0118] The above results indicate that factor V binds to modified HDL, and the binding of modified HDL to LOX-1 occurs via factor V. The detection sensitivity of the complex with this binding increases with the amount of factor V added.

[0119] (Experimental Example 2)

[0120] The detection sensitivity was investigated based on the presence or absence of LOX-1 solidification relative to the plate, thereby investigating whether the increase in detection sensitivity caused by factor V occurred in the complex of LOX-1, factor V, and modified HDL.

[0121] Figure 4 The results indicate the use of plasma samples collected from A, B, C, D, and E above as samples, with factor V protein added at a concentration of 1 μg / mL (plasma+FV) and without factor V protein added (plasma). The plates used were LOX-1 immobilized and non-immobilized. Other results were detected by ELISA in the same manner as in Experimental Example 1.

[0122] Figure 5 This indicates the cases where factor V protein was added (serum+FV) at a concentration of 1 μg / mL using serums collected from A, B, C, D, and E above, and the cases where factor V protein was not added (serum). Other cases are similar to... Figure 4 The same test results were obtained.

[0123] Figure 6 This indicates the cases where factor V protein was added to plasma samples collected from A, B, C, D, and E above, in a manner achieving a concentration of 10 μg / mL, and the cases where factor V protein was not added. Other cases are similar to... Figure 4 The same test results were obtained.

[0124] Figure 7 This indicates the cases where factor V protein was added to serum samples collected from A, B, C, D, and E above, in a manner achieving a concentration of 10 μg / mL, and the cases where factor V protein was not added. Other cases are similar to... Figure 5 The same test results were obtained.

[0125] according to Figures 4-7 If a plate without LOX-1 solidification is used, no difference in detection volume is observed whether factor V is added to the sample or not. Based on this result, it is shown that, with respect to factor V, LOX-1, factor V, and modified HDL combine to form a complex that increases the detection volume.

[0126] right Figure 4 and Figure 5 , Figure 6 and Figure 7 Compared to those using plasma Figure 4 , Figure 6 After using serum Figure 5 , Figure 7 In this case, the detection rate increased after adding factor V. This result is believed to be similar to that in Experiment 1, indicating that substances not present in serum were replenished through the addition of factor V.

[0127] (Experimental Example 3)

[0128] Since factor V is combined with modified HDL, we are exploring whether it is possible to directly solidify factor V on a plate for quantification of modified HDL.

[0129] First, factor V dissolved in PBS(-) at a concentration of 0–10 μg / mL was added to the plate, and an ELISA method based on anti-factor V antibody was used to test whether immobilization on the plate was possible. The following conditions were applied.

[0130] Solidification: Human Factor V at various concentrations

[0131] Blocking: In 1% casein-Na, incubate for 2 hours at room temperature.

[0132] Detection: Primary antibody anti-FV-LC (AHV-5101) 1 μg / ml, 1hr, room temperature

[0133] Secondary antibody anti-mouse IgG-HRP, 1:4000, in 1% casein-Na solution, incubated at room temperature for 1 hour.

[0134] The results are shown in Figure 8 This demonstrates the ability to achieve solidification with respect to the plate by a factor V that is largely dependent on concentration.

[0135] Next, experiments were conducted on the combination of unmodified HDL (nHDL) and modified HDL on the solid-phase ELISA plate with this factor V. The modified HDL used was oxidized HDL, obtained by oxidizing HDL with copper sulfate, i.e., Cu. 2+ -oxHDL.

[0136] Factor V was immobilized on 384-well plates (Greiner). After washing twice with PBS, the plates were blocked with 1% casein-Na. After washing three times with PBS, nHDL or Cu was prepared at a concentration of 1 μg / ml using sample dilution buffer (1% casein-Na, 10 mM HEPES, 150 mM NaCl, pH 7.4, 100 μM APMSF). 2+-oxHDL was incubated at room temperature for 2 hours. The plate was washed three times with PBS, and 1 μg / ml of anti-apoA1 antibody (chicken #1) was added, followed by incubation at room temperature for 1 hour. The plate was then washed three times with PBS, and 4000-fold diluted HRP-labeled anti-chicken IgY antibody was added, followed by incubation 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, allowing it to react at room temperature. The reaction was stopped with 2M sulfuric acid, and the absorbance at 450 nm was measured.

[0137] The results are shown in Figure 9 The detection limit of modified HDL increases depending on the amount of solidification factor V on the plate. On the other hand, the detection limit of nHDL does not change significantly. Based on these results, it is demonstrated that this plate can be used for the detection of modified HDL.

[0138] (Experimental Example 4)

[0139] Modified HDL in human plasma and serum was detected using the same factor V solid-phase plate adjusted as in Experimental Example 3. Quantification of modified HDL was performed using ELISA samples from plasma and serum collected from subjects X and Y.

[0140] Factor V was immobilized on a 384-well plate (Greiner). After washing twice with PBS, the plate was blocked with 1% casein-Na. After washing three times with PBS, the plasma or serum sample was incubated at room temperature for 2 hours. After washing three times with PBS, 1 μg / ml of anti-apoA1 antibody (chicken #1) was added, and the plate was incubated at room temperature for 1 hour. After washing three times with PBS, 4000-fold diluted HRP-labeled anti-chicken IgY antibody was added, and the plate was 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. The reaction was allowed to proceed at room temperature. The reaction was stopped with 2M sulfuric acid, and the absorbance at 450 nm was measured.

[0141] The results are shown in Figure 10 Regarding the specimens collected from subject X, they are shown in... Figure 10 (a) in the figure, concerning the specimen collected from subject Y, is shown in Figure 10 (b) in the middle.

[0142] Unlike existing techniques involving LOX-1 immobilization, the measured values ​​in plasma and serum show almost no difference; that is, for plasma, a stable level of detection sensitivity is observed regardless of the sample. This technique offers higher sensitivity compared to techniques involving LOX-1 immobilization. Furthermore, it demonstrates that compared to sandwich methods using LOX-1 and factor V, it not only facilitates quantification but also enables stable and reliable quantification of both plasma and serum with high sensitivity.

[0143] The foregoing has described several embodiments of the present invention, but these embodiments are merely illustrative and are not intended to limit the scope of the invention. These embodiments can be implemented in various other ways, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included within the scope and spirit of the invention, and similarly within the scope of the invention as described in the claims and its equivalents.

[0144] Industrial applicability

[0145] According to the present invention, a quantitative method for modified HDL that can quantify modified HDL more stably and accurately than before, as well as analytical reagents for using the quantitative method, are available. This quantitative method and analytical reagents are particularly useful for determining the risk or progression of cardiovascular / vascular diseases, diabetes, and diabetic diseases.

[0146] Explanation of reference numerals in the attached figures

[0147] 10: Modified HDL;

[0148] 20: Modified HDL-binding protein;

[0149] 21: L-chain section;

[0150] 22: H-chain region;

[0151] 30: Antibody;

[0152] 40, 40A: board;

[0153] 50: LOX-1 protein.

Claims

1. The application of a modified HDL-binding protein in the manufacture of a reagent for quantifying modified HDL contained in a sample, wherein, The modified HDL-binding protein contains the full-length sequence of factor V. The modified HDL-binding protein was immobilized on a carrier. A sample is added to the carrier to form a complex of the modified HDL contained in the sample and the modified HDL-binding protein. The complex was quantified by immunoassay, thereby quantifying the modified HDL contained in the sample.

2. The application of a modified HDL-binding protein in the manufacture of a reagent for quantifying modified HDL contained in a sample, wherein, The modified HDL-binding protein contains the full-length sequence of factor V. The modified HDL-binding protein was added to the sample. This allows the modified HDL receptor to be immobilized on a carrier. The sample is added to the carrier to form a complex in which the modified HDL contained in the sample binds to the modified HDL receptor via the modified HDL binding protein. The complex was quantified by immunoassay, thereby quantifying the modified HDL contained in the sample.

3. The application according to claim 1 or 2, wherein, The modified HDL-binding protein is a recombinant protein.

4. The application according to claim 1 or 2, wherein, The quantification method is ELISA.

5. The application according to claim 2 or the above, wherein, The modified HDL receptor is the LOX-1 protein.

6. The application according to claim 1 or 2, wherein, In the quantification, serum or plasma is used as the sample.

7. The application according to claim 1 or 2, wherein, Serum was used as the sample in the quantification.

8. The application according to claim 1 or 2, wherein, By quantifying the modified HDL contained in the sample, the disease, the risk of the disease, or the degree of disease progression can be determined. The disease in question is a cardiovascular disease, diabetes, or a diabetic condition.

9. The application according to claim 8, wherein, The cardiovascular disease mentioned refers to myocardial infarction or stroke caused by arteriosclerosis or its development.

10. The application according to claim 2, wherein, The sample was serum. The modified HDL receptor is the LOX-1 protein.

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