JP6231307B2 - Method for measuring dysfunctional HDL, kit for measuring dysfunctional HDL, and method for measuring markers for lifestyle-related diseases - Google Patents

Description

The present invention relates to a method for measuring dysfunctional HDL, a kit for measuring dysfunctional HDL, and a method for measuring a marker for lifestyle-related diseases, and more specifically, a method for measuring dysfunctional HDL using a receptor for dysfunctional HDL, The present invention relates to a dysfunctional HDL measurement kit used in the method and a marker measurement method using the method.

  In recent years, westernization of eating habits has progressed, and lifestyle-related diseases such as metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, hypertension, and arteriosclerosis, which are considered to be caused by this, are increasing. A common problem with these lifestyle-related diseases is the concentration and quality of blood cholesterol.

Cholesterol is a kind of lipid and classified as a steroid, and is a kind of organic compound belonging to a subgroup called sterol. When this cholesterol is transported through the blood, it takes the molecular form of a lipoprotein that combines with the protein to form a complex. Cholesterol transport from the liver to the periphery is handled by lipoproteins with a relatively low specific gravity and large particle size (low-density lipoprotein, hereinafter referred to as “LDL”). Lipoprotein having a relatively high specific gravity and a small particle diameter ( high -density lipoprotein, hereinafter referred to as “HDL”) is in charge. The protein contained in the LDL molecule is apolipoprotein B. The HDL molecule includes many types of proteins, most of which are apolipoprotein A1 (hereinafter sometimes abbreviated as “ApoA1”).

  Advances in lipoprotein research in the 1960s established the theory that LDL increases the risk of arteriosclerosis and HDL reduces the risk. For this reason, LDL is sometimes called “bad cholesterol” and HDL is sometimes called “good cholesterol”. Currently, in general medical examinations, total cholesterol, HDL, and neutral fat are measured, and LDL is calculated from the measured values of the three using a calculation formula. The Atherosclerotic Disease Prevention Guidelines issued by the Japanese Atherosclerosis Society define desirable blood levels of LDL, HDL, triglycerides, and non-HDL (total cholesterol-HDL), and specify the cholesterol management goals by taking drugs doing.

  As described above, LDL is a cause of arteriosclerosis, and it is said that the blood concentration should be controlled. However, due to research progress from the late 1990s, LDL rather than LDL itself is oxidatively modified in the body. Oxidized modified LDL (oxidized LDL) has been shown to be a molecule that increases the risk of arteriosclerosis. Several methods for measuring oxidized LDL have been developed, and some of these methods are used for in-vitro diagnostic drugs used for prognosis management of patients with severe diabetes.

  However, in recent years, it has become clear that HDL, which has been regarded as good, also has “dysfunctional HDL” which increases the risk of arteriosclerosis by impairing the function by modification such as oxidation. Dysfunctional HDL has been shown to promote or cause arteriosclerosis and inflammation, unlike normal HDL.

  Various types of molecular forms are known for dysfunctional HDL. For example, “MDA-HDL” in which the lysine residue of apolipoprotein A1, which is a component of HDL, is oxidatively modified with lipid peroxide, “MetO-HDL” in which methionine residue is oxidatively modified, and hypothesis by the action of myeloperoxidase. Chlorinated “HOCl-HDL”, and the like. In addition to this, there are molecular species in which the lipid portion of the HDL component is oxidized.

  The measurement of the blood concentration of such dysfunctional HDL is expected to be useful as a test method for determining the risk and progression of arteriosclerosis, and research on the measurement technique has been advanced. As a measurement technique, a sandwich ELISA method using a monoclonal antibody having an epitope of apolipoprotein A1 (MetO-apoA1) whose methionine residue is oxidatively modified and an anti-apolipoprotein A1 antibody has been reported (for example, patents). Documents 1 to 4, Non-Patent Documents 1 and 2).

  However, since these methods utilize monoclonal antibodies, the molecular form of dysfunctional HDL that can be detected is limited. Therefore, there is a common problem that dysfunctional HDL of various molecular forms cannot be widely captured and detected as described above.

Japanese Patent No. 4588053 Japanese Patent No. 3998245 Japanese Patent No. 3436444 Japanese Patent No. 4625812

Wang et al., "A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL", Journal of Lipid Research 50, 2009, 586-594 Ueda et al., "Establishment and evaluation of 2 monoclonal antibodies against oxidized apolipoprotein A-I (apoA-I) and its application to determine blood oxidized apoA-I levels", Clinica Chimica Acta 378, 2007,105-111

  As mentioned above, the cause of arteriosclerosis is not attributed to one type of factor, but various biomolecules such as LDL and HDL have been altered and altered by stress in the in vivo environment in the state of pathological preparation. Recent studies have shown that molecules bind to specific receptors to increase the risk of atherosclerosis. Thus, measuring the concentration of dysfunctional HDL in the circulating blood can be a useful test to determine the degree of risk of arteriosclerosis and support appropriate medical intervention.

  However, in a method using a monoclonal antibody having a specific structure of dysfunctional HDL generated by alteration or oxidation as an epitope, dysfunctional HDL having a structure of a different modifying group cannot be detected. As long as monoclonal antibodies are used, this limitation cannot be overcome.

  Therefore, an object of the present invention is to provide a technique for measuring dysfunctional HDL that can widely detect dysfunctional HDLs in various molecular forms, and to provide a technique for detecting lifestyle-related diseases using the technique.

  In order to solve the above problems, the present inventors have advanced research for measuring dysfunctional HDL without using a monoclonal antibody. As a result, it has been found that the above-described problems can be solved by using a receptor for dysfunctional HDL (receptor protein). That is, it was found that various dysfunctional HDLs can be widely captured by using a receptor. The inventors have found that dysfunctional HDL in blood measured by this method is high in patients with diabetes and non-alcoholic fatty liver, and that it is possible to detect lifestyle-related diseases, thereby completing the present invention.

The above-mentioned one aspect that will be provided based on the findings, a method for measuring dysfunctions HDL contained in a sample, the sample is contacted to a receptor dysfunction HDL, dysfunction HDL in a sample It is a method for measuring dysfunctional HDL, characterized by detecting dysfunctional HDL bound to the receptor and bound to the receptor.

The present invention relates to a method for measuring dysfunctional HDL. In the present invention, a sample containing dysfunctional HDL is contacted with a receptor for dysfunctional HDL, and the dysfunctional HDL in the sample is bound to the receptor. The dysfunctional HDL in the sample is then measured by detecting dysfunctional HDL bound to the receptor. Since the present invention uses a receptor for dysfunctional HDL, various molecular forms of dysfunctional HDL can be captured on the receptor. Therefore, it is possible to catch a wide range of dysfunctional HDL. The measurement object is not limited to a specific dysfunctional HDL as in the prior art using monoclonal antibodies.

“Dysfunctional HDL” refers to HDL that has been modified (modified HDL) and has an abnormality in function due to oxidation or loss of constituent proteins or lipids.
The dysfunctional HDL includes a modified HDL (oxidized HDL) in which a protein or lipid part constituting the HDL is oxidized. In addition, dysfunctional HDL includes modified HDL in which a part of the protein constituting HDL is deleted.
For example, as described above, “MDA-HDL” in which the lysine residue of the apolipoprotein A1, which is a component of HDL, is oxidatively modified with lipid peroxide, “MetO-HDL” in which the methionine residue is oxidatively modified, myeloperoxidase, A specific example of dysfunctional HDL is "HOCl-HDL" that has been hypochlorinated by the action of.

The “receptor of dysfunctional HDL” includes a natural receptor (full length of receptor protein) as long as it retains the function as a receptor, that is, binding to dysfunctional HDL which is a ligand, Such variants are also included. Examples of receptor variants include receptor protein variants (amino acid substitutions, etc.), receptor protein partial fragments, receptor proteins and other proteins fusion proteins (chimeric proteins), and the like.
LOX-1 (also called OLR-1), CD36, etc. are known as receptors for dysfunctional HDL.

  Preferably, the receptor is immobilized on a carrier.

  Such a configuration facilitates detection of dysfunctional HDL bound to the receptor. For example, measurement by a solid phase method becomes possible.

  Preferably, the receptor is LOX-1 or a variant thereof.

LOX-1 is a lectin-like oxidized low-density lipoprotein receptor-1 and is known as a kind of receptor for oxidized LDL (Nippon Pharmacology, Vol. 119). , P145-154, 2002; JP 2011-106847 A; JP 2012-100585 A). Recently, it has been reported that modified HDL acts as a ligand for LOX-1 (Besler C, et al. J Clin Invest. 2011; 121: 2693-708). And this invention uses LOX-1 or its modification as a receptor of dysfunctional HDL.

  Examples of LOX-1 variants include LOX-1 mutants (amino acid substitutions, etc.), LOX-1 partial fragments, LOX-1 and other protein fusion proteins (chimeric proteins), etc. The thing holding the function as a receptor is mentioned.

  Preferably, the dysfunctional HDL is oxidized HDL.

  Oxidized HDL is a modified HDL in which HDL constituent proteins and constituent lipids are oxidized, and is a representative example of dysfunctional HDL.

  Preferably, an antibody recognizing dysfunctional HDL is further bound to dysfunctional HDL bound to the receptor.

  With this configuration, dysfunctional HDL bound to a receptor can be detected with an antibody.

  Preferably, the antibody is an anti-apolipoprotein A1 antibody.

Another aspect is a kit for measuring dysfunctional HDL, which is a kit for use in the above-described method for measuring dysfunctional HDL, comprising a receptor for dysfunctional HDL.

The present invention relates to a kit for measuring dysfunctional HDL, and includes a receptor for dysfunctional HDL. According to the kit of the present invention, it is possible to easily measure dysfunctional HDL using the above-described receptor.

  Preferably, it further comprises an antibody that recognizes dysfunctional HDL.

  With this configuration, it is possible to easily and easily detect dysfunctional HDL bound to a receptor.

  Preferably, it further comprises a dysfunctional HDL standard.

  Such a configuration makes it easy to create a calibration curve.

According to another aspect, the method for measuring dysfunctional HDL measures dysfunctional HDL in a body fluid collected from a subject, and detects the presence or absence of lifestyle-related diseases in the subject based on the measurement result. This is a method for detecting lifestyle-related diseases.

The present invention relates to a method for detecting lifestyle-related diseases, and measures dysfunctional HDL in body fluids by the method for measuring dysfunctional HDL described above, and detects the presence or absence of lifestyle-related diseases based on the measurement results. According to this invention, dysfunctional HDL of various molecular forms present in body fluid can be used as an index, so that lifestyle-related diseases involving dysfunctional HDL can be detected widely and with high sensitivity.

  Here, “lifestyle-related diseases” are defined as diseases in which lifestyle habits such as eating habits, exercise habits, rest, smoking, and drinking are related to the onset and progression of the disease. The lifestyle-related diseases include at least metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, arteriosclerosis, and hypertension, and complications thereof.

  “Detection of the presence or absence of lifestyle-related diseases” is a concept that includes not only the presence or absence of lifestyle-related diseases but also the presence or absence of future onset risks. The presence or absence of a future risk of developing a lifestyle-related disease refers to the presence or absence of a risk (risk) of suffering from a lifestyle-related disease in the future when no lifestyle-related disease has developed.

  Preferably, the body fluid is serum or plasma.

  Preferably, the lifestyle-related disease is at least one selected from the group consisting of metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, arteriosclerosis, and hypertension.

The invention according to claim 1 is a method of measuring dysfunctional HDL contained in a sample, wherein the sample is brought into contact with a receptor of dysfunctional HDL, and the dysfunctional HDL in the sample is bound to the receptor. And detecting dysfunctional HDL bound to the receptor, wherein the receptor is LOX-1 or a variant thereof, and the receptor is immobilized on a carrier and binds to the receptor This is a method for measuring dysfunctional HDL, wherein an antibody recognizing dysfunctional HDL is further bound to the dysfunctional HDL.

The invention according to claim 2 is the method for measuring dysfunctional HDL according to claim 1, wherein the dysfunctional HDL is oxidized HDL.

The invention according to claim 3 is the method for measuring dysfunctional HDL according to claim 1 or 2, wherein the antibody is an anti-apolipoprotein A1 antibody.

The invention according to claim 4 is a kit for use in the method for measuring dysfunctional HDL according to any one of claims 1 to 3, wherein the antibody recognizes LOX-1 or a variant thereof and dysfunctional HDL. A kit for measuring dysfunctional HDL, comprising:

The invention according to claim 5 is the kit for measuring dysfunctional HDL according to claim 4, wherein the antibody is an anti-apolipoprotein A1 antibody.

The invention according to claim 6 is the kit for measuring dysfunctional HDL according to claim 4 or 5, further comprising a standard product of dysfunctional HDL.

The invention according to claim 7 is a method for measuring a lifestyle-related disease marker comprising dysfunctional HDL, which is in a body fluid collected from a subject by the method for measuring dysfunctional HDL according to any one of claims 1 to 3. This is a method for measuring a lifestyle-related disease marker characterized by measuring dysfunctional HDL.

The invention according to claim 8 is the method for measuring a lifestyle-related disease marker according to claim 7, wherein the body fluid is serum or plasma.

The invention according to claim 9 is characterized in that the lifestyle-related disease is at least one selected from the group consisting of metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, arteriosclerosis, and hypertension. It is the measuring method of the lifestyle-related disease marker of Claim 7 or 8.

The invention according to claim 10 includes a step of comparing the obtained measurement value of dysfunctional HDL with a reference value, and measuring the lifestyle-related disease marker according to any one of claims 7 to 9 Is the method.

  According to the method for measuring dysfunctional HDL of the present invention, various types of dysfunctional HDL can be measured.

  According to the kit for measuring dysfunctional HDL of the present invention, the method for measuring dysfunctional HDL using a receptor can be easily carried out.

According to the method for measuring a lifestyle-related disease marker of the present invention, the presence or absence of a lifestyle-related disease involving dysfunctional HDL can be detected widely and with high sensitivity.

It is a graph showing the calibration curve created by the method of the present invention. It is a graph showing the result of having measured the malfunctioning HDL in serum by the method of this invention.

Hereinafter, embodiments of the present invention will be described.
The method for measuring dysfunctional HDL of the present invention comprises contacting a sample with a receptor of dysfunctional HDL, binding dysfunctional HDL in the sample to the receptor, and detecting dysfunctional HDL bound to the receptor. It is a feature.

As described above, “dysfunctional HDL” refers to HDL that has been modified (modified HDL) and has an abnormality in function due to oxidation or loss of constituent proteins and lipids. For example, HDL in which a protein constituting HDL, HDL in which a lipid portion is oxidized (oxidized HDL), or HDL in which a part of the protein constituting HDL is deleted is included in dysfunctional HDL. Specific examples of dysfunctional HDL include MDA-HDL, MetO-HDL, HOCl-HDL, and the like.
In particular, oxidized HDL is a representative example of dysfunctional HDL that is a measurement target of the present invention.

  Of course, the receptor for dysfunctional HDL does not include antibodies (immunoglobulin) against dysfunctional HDL.

  In the present invention, dysfunctional HDL in a sample is bound to a “receptor for dysfunctional HDL”. The receptor for dysfunctional HDL is not particularly limited as long as it is a receptor for which dysfunctional HDL is a ligand. Specific examples of the receptor for dysfunctional HDL include the above-mentioned LOX-1 and CD36.

  CD36 is a family of class B scavenger receptors and is a membrane protein expressed on the surface of cells. It has a molecular weight of 88 kD, and is known to bind to various proteins such as collagen and thrombospondin, play an important role in various signal transduction, and also binds to oxidized HDL.

  In addition, as long as the receptor for dysfunctional HDL retains the binding ability to dysfunctional HDL, in addition to the natural type receptor (the full length of the receptor protein), a modified form thereof (a mutant of the receptor protein, A partial fragment of a receptor protein, a fusion protein of a receptor protein and another protein, etc.).

  In a preferred embodiment, LOX-1 is used as a receptor for dysfunctional HDL. The gene for LOX-1 has already been cloned, and recombinant LOX-1 is available (Japanese Patent Laid-Open No. 9-98787).

  Although there is no limitation in particular as a sample in this invention, For example, the bodily fluid (for example, serum and plasma) extract | collected from the human can be mentioned.

  In the present invention, dysfunctional HDL bound to the receptor is detected. At this time, it is preferable to use a receptor immobilized on a carrier (support). Thereby, measurement by a so-called solid phase method becomes possible. The carrier is not particularly limited as long as the receptor can be immobilized, and a known carrier such as a microtiter plate, beads, latex particles, or the like can be used.

  An antibody that recognizes dysfunctional HDL can be further used. Examples of the antibody include anti-apolipoprotein A1 antibody (anti-ApoA1 antibody). According to this embodiment, for example, dysfunctional HDL can be captured by a receptor, and dysfunctional HDL can be detected by an antibody.

  As an example, LOX-1 (receptor for dysfunctional HDL) immobilized on a carrier captures dysfunctional HDL in a sample and detects dysfunctional HDL with an anti-ApoA1 antibody (an antibody that recognizes dysfunctional HDL). The measurement system will be described. This example belongs to the so-called sandwich method.

First, LOX-1 is immobilized on a carrier such as a microtiter plate. Then, a sample such as serum is added to and brought into contact with the immobilized LOX-1, and the dysfunctional HDL in the sample is bound to LOX-1. After washing, anti-ApoA1 antibody is added to allow anti-ApoA1 antibody to bind to dysfunctional HDL bound to LOX-1. Then, the bound anti-ApoA1 antibody is detected, and dysfunctional HDL in the sample is measured using this as an index.
The anti-ApoA1 antibody may be either labeled or unlabeled. When an unlabeled anti-ApoA1 antibody is used, a labeled antibody (labeled secondary antibody) that binds to the anti-ApoA1 antibody may be used. As a kind of label | marker, well-known things, such as an enzyme, a fluorescent substance, and a radioactive substance, are employable. The anti-ApoA1 antibody may be either polyclonal or monoclonal.

  Instead of the sandwich method, a competitive method can also be used. It is also possible to immobilize anti-ApoA1 antibody and detect dysfunctional HDL with LOX-1.

  In the method for measuring dysfunctional HDL of the present invention, as a standard product (standard substance) used for preparing a calibration curve, for example, an HDL fraction prepared from human blood is oxidized with copper sulfate or the like ( Hereinafter, it may be referred to as “artificial oxidation HDL”). Then, by using the standard product, a dilution series of standard dysfunctional HDL whose concentration is defined by protein concentration measurement or cholesterol measurement can be prepared, and a calibration curve can be created from these measurement results.

  The kit for measuring dysfunctional HDL of the present invention comprises a receptor for dysfunctional HDL. In a preferred embodiment, the antibody further comprises an antibody that recognizes dysfunctional HDL (eg, an anti-ApoA1 antibody). In addition, a standard product of dysfunctional HDL (for example, the above artificial oxidized HDL) is further included.

  Examples of the configuration of the kit of the present invention are given below. The kit having this configuration may further contain a carrier such as a plate or a bead, a chromogenic substrate, and the like.

[Example of kit configuration]
(A) LOX-1 (solution or lyophilized product) Appropriate amount (b) HRP-labeled anti-ApoA1 antibody (solution or lyophilized product) Appropriate amount (c) Artificial oxidized HDL (standard product. Solution or lyophilized product) Appropriate amount

  According to the method for detecting lifestyle-related diseases of the present invention, dysfunctional HDL in body fluid collected from a subject is measured by the method for measuring dysfunctional HDL, and the presence or absence of lifestyle-related diseases in the subject is determined based on the measurement result. It is to detect. Examples of body fluids include serum or plasma.

  For example, dysfunctional HDL can be measured by the above-described method using body fluid of a healthy person, and a reference value (cut-off value) can be set. Then, dysfunctional HDL is measured in the same manner using the body fluid of the subject, and the measured value is compared with a reference value, so that the presence or absence of lifestyle-related diseases in the subject can be detected.

  Examples of lifestyle-related diseases to be detected include metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, arteriosclerosis, hypertension, and complications thereof.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Preparation of dysfunctional HDL standard (artificial oxidized HDL) A HDL fraction was prepared from human blood by a conventional method. The HDL concentration of the obtained fraction was adjusted to 0.5 mg / mL, copper sulfate was added so that the final concentration was 50 μmol, and the mixture was incubated at 37 ° C. for 48 hours to oxidatively modify HDL. Thereafter, EDTA was added to a final concentration of 2 mmol to stop the oxidation, and an artificial oxidized HDL serving as a standard product was obtained.

(2) Construction of measurement system by sandwich method and preparation of calibration curve Human LOX-1 in PBS (0.5 μg / mL) was prepared. 100 μL of this was injected into each well of a microtiter plate and incubated overnight at 4 ° C. (immobilization of LOX-1). After discharging the LOX-1 solution and washing each well with PBST (PBS + 0.05% Tween 20) three times, 200 μL of blocking solution (5% BSA) was injected into each well. Incubated for 2 hours at room temperature (blocking).

  The blocking solution was discharged, and each hole was washed three times with PBST, and then 100 μL of a standard product (artificial oxidation HDL. 0 to 1000 ng / mL) for preparing a calibration curve prepared in (1) above was injected as a sample. Incubated for 2 hours at room temperature. The sample was discharged and each well was washed 3 times with PBST, and then 100 μL of a 3000-fold diluted solution of HRP-labeled goat anti-human ApoA1 antibody was injected into each well. Incubated for 2 hours at room temperature.

The anti-human ApoA1 antibody solution was discharged, each hole was washed 5 times with PBST, and then 100 μL of TMB substrate (Bio-Rad) was injected into each hole. After incubation for 30 minutes in the dark, the enzyme reaction was stopped by injecting 50 μL of 1M sulfuric acid. Absorbance (A450) at 450 nm was measured with a microplate reader. A calibration curve was prepared from the obtained absorbance.
As a control, unoxidized HDL (0 to 1000 ng / mL) was also measured.

  The results are shown in Table 1 and FIG. In Table 1 and FIG. 1, “oxHDL” indicates artificially oxidized HDL, and “HDL” indicates HDL that has not been oxidized. That is, for artificially oxidized HDL, an A450 value correlated with the concentration was obtained. And linearity was obtained at a concentration range of at least 0 to 1000 ng / mL, and dysfunctional HDL (oxidized HDL) could be measured.

(3) Examination using serum samples Using the serum of healthy individuals (25 cases), nonalcoholic fatty liver patients (115 cases), and diabetic patients (415 cases), oxidized HDL was obtained by the method of (2) above. It was measured. Serum was diluted 50-fold with a diluent (5% BSA, 2 mM EDTA in HEPES with 0.05% Tween 20). The artificial oxidation HDL produced in (1) was used as a standard product. The dilution series of the standard product was set to 7 points that were serially diluted by 1/2 times starting from 4000 ng / mL. The measurement results are shown in FIG.

About the average value ± standard deviation (μg / mL) of the dysfunctional HDL concentration (artificial oxidized HDL conversion), 11.9 ± 11.2 μg / mL in healthy subjects and 29.6 ± 21.9 μg in nonalcoholic fatty liver patients It was 85.9 ± 48.0 μg / mL for diabetic patients. And a significant difference was recognized between a healthy person and a non-alcoholic fatty liver patient, and between a healthy person and a diabetic patient. Non-alcoholic fatty liver can be regarded as an aspect of metabolic syndrome.
From the above, it was shown that lifestyle-related diseases such as diabetes can be detected by measuring dysfunctional HDL in serum by the method of the present invention.

Claims (10)

A method for measuring dysfunctional HDL contained in a sample, wherein the sample is brought into contact with a receptor for dysfunctional HDL, the dysfunctional HDL in the sample is bound to the receptor, and the dysfunction is bound to the receptor. HDL is detected ,
The receptor is LOX-1 or a variant thereof;
The receptor is immobilized on a carrier;
A method for measuring dysfunctional HDL, further comprising binding an antibody that recognizes dysfunctional HDL to dysfunctional HDL bound to the receptor . The method for measuring dysfunctional HDL according to claim 1, wherein the dysfunctional HDL is oxidized HDL. The method for measuring dysfunctional HDL according to claim 1 or 2 , wherein the antibody is an anti-apolipoprotein A1 antibody. A kit for use in the method for measuring dysfunctional HDL according to any one of claims 1 to 3 , comprising LOX-1 or a variant thereof and an antibody that recognizes dysfunctional HDL. Incomplete HDL measurement kit. The dysfunctional HDL measurement kit according to claim 4, wherein the antibody is an anti-apolipoprotein A1 antibody. The kit for measuring dysfunctional HDL according to claim 4 or 5 , further comprising a standard product of dysfunctional HDL. A method for measuring a lifestyle-related disease marker comprising dysfunctional HDL,
A method for measuring a lifestyle-related disease marker , comprising measuring dysfunctional HDL in a body fluid collected from a subject by the method for measuring dysfunctional HDL according to any one of claims 1 to 3 . The method for measuring a lifestyle-related disease marker according to claim 7 , wherein the body fluid is serum or plasma. The life according to claim 7 or 8 , wherein the lifestyle-related disease is at least one selected from the group consisting of metabolic syndrome, impaired glucose tolerance, diabetes, dyslipidemia, arteriosclerosis, and hypertension. A method for measuring markers of habitual diseases. The method for measuring a lifestyle-related disease marker according to any one of claims 7 to 9, comprising a step of comparing the obtained measured value of dysfunctional HDL with a reference value.

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