JP7084590B2 - Selection method and selection reagent for subjects in need of treatment for dyslipidemia - Google Patents

Description

The present invention relates to a method for selecting a subject who needs treatment for dyslipidemia and a reagent for selection.
This application claims priority based on Japanese Patent Application No. 2016-213872 filed in Japan on October 31, 2016, the contents of which are incorporated herein by reference.

Dyslipidemia is a disease in which serum lipid levels show abnormal values, and depending on the type of lipid showing abnormal values, "high-low-density lipoprotein cholesterolemia (high-LDL cholesterolemia)" and "low-density lipoprotein cholesterol" It is classified into "blood (low HDL cholesterolemia)" and "hypertriglyceridemia".
In recent years, changes in high-density lipoprotein (hereinafter referred to as HDL) function have been reported as a pathological condition of dyslipidemia, and HDL in familial CETP (cholesterol ester transfer protein) deficient patients present with high HDL cholesterolemia. Dyslipidemia has been reported (see Non-Patent Document 1).

HDL is a lipoprotein with a specific density of 1.063 to 1.21. It is a lipoprotein that has the function of removing excess cholesterol accumulated in peripheral tissues by the cholesterol reverse transfer system and transporting it to the liver for processing. Many epidemiological studies have revealed that cholesterol in HDL (hereinafter referred to as HDL-C) is a negative risk factor for arteriosclerosis, and HDL-C has come to be called good cholesterol, and HDL-C concentration. It was simply thought that high cholesterol was desirable for the body.
However, since it was found that drugs that increase HDL-C concentration, such as CETP inhibitors, are not effective for arteriosclerosis, HDL-C concentration, which is a quantitative factor of HDL, alone causes arteriosclerosis, etc. It has become clear that cardiovascular diseases cannot be explained well, and it has been reported that the qualitative factor of HDL, which is the ability of HDL to extract cholesterol, is involved in cardiovascular diseases such as arteriosclerosis (Non-Patent Document 2). reference).

As a therapeutic agent for dyslipidemia, statins, probucol and the like are used in clinical practice. Statins are known to have a mild effect on HDL-C levels and have been reported to have a positive effect on HDL function. However, it is also known that dyslipidemia is not improved despite administration of statins. Probucol, a cholesterol catabolism promoter, has a strong antioxidant effect and an LDL cholesterol lowering effect. It has been reported that in patients with familial hypercholesterolemia heterozygotes, the incidence of cardiovascular events is significantly lower in the probucol-administered group than in the non-administered group (see Non-Patent Document 3).

In recent years, it has been pointed out that oxidized lipoproteins such as oxidized high-density lipoprotein (hereinafter referred to as oxidized HDL) and oxidized low-density lipoprotein are related to the development of porphyritic sclerosis lesions (see Non-Patent Document 4). An immunological measurement method for lipoprotein has been reported (see Patent Document 1).

International Publication No. 2003/006989

The Lipid, Vol.25 (3), p.36-50 (2014). New England J Medicine, Vol.367, p.2089-2099 (2012). J Atheroscler Thromb, Vol.15 (3), p.292-303 (2008). New England J Medicine, Vol.320, p.915-924 (1989).

An object of the present invention is to provide a method and a reagent for efficiently selecting a subject in need of treatment for dyslipidemia.

The present inventors have found that by measuring oxidized HDL in a sample collected from a subject, a subject who needs treatment for dyslipidemia can be efficiently selected, and the present invention has been completed.

That is, the present invention relates to the following [1] to [17].
[1] A method for selecting a subject who requires treatment for dyslipidemia, which comprises measuring the concentration of oxidized HDL in a sample collected from the subject.
[2] The method according to [1], which comprises the following steps (1) to (3).
(1) Step of collecting a sample from a subject;
(2) A step of measuring the concentration of oxidized HDL in the sample collected in step (1);
(3) If the concentration of oxidized HDL measured in step (2) is higher than the concentration of oxidized HDL in a healthy person, the subject determines that treatment for dyslipidemia is necessary, and the oxidized HDL in a healthy person. If the concentration is lower than the concentration, the subject determines that treatment for dyslipidemia is not necessary.

[3] The method according to [1] or [2], wherein the measurement of the concentration of oxidized HDL is performed by an immunological measurement method.
[4] The method according to [3], wherein the immunological measurement method is performed using an antibody that binds to oxidized phosphatidylcholine or an antibody that binds to phosphocholine and an antibody that binds to high-density lipoprotein. ..
[5] The method according to [4], wherein the antibody that binds to the high-density lipoprotein is an antibody that binds to the apoA protein.
[6] The method according to any one of [1] to [5], wherein the treatment for dyslipidemia is treatment with a therapeutic agent for dyslipidemia.

[7] The method according to [6], wherein the therapeutic agent for dyslipidemia is a cholesterol catabolism promoter.
[8] The method according to [7], wherein the cholesterol catabolism promoter is probucol.
[9] The method according to any one of [1] to [8], wherein the sample is blood.

[10] A reagent for selecting a subject in need of treatment for dyslipidemia, which comprises a reagent for measuring oxidized HDL.
[11] The reagent according to [10], wherein the oxidized HDL measuring reagent is an immunological measuring reagent.
[12] The reagent according to [11], wherein the immunological measurement reagent is a reagent containing an antibody that binds to phosphatidyl oxide or an antibody that binds to phosphocholine and an antibody that binds to a high-density lipoprotein.
[13] The reagent according to [12], wherein the antibody that binds to the high-density lipoprotein is an antibody that binds to the apoA protein.

[14] The reagent according to any one of [10] to [13], wherein the treatment for dyslipidemia is treatment with a therapeutic agent for dyslipidemia.
[15] The reagent according to [14], wherein the therapeutic agent for dyslipidemia is a cholesterol catabolism promoter.
[16] The reagent according to [15], wherein the cholesterol catabolism promoter is probucol.
[17] The reagent according to any one of [10] to [16], wherein the oxidized HDL measuring reagent is a reagent for measuring oxidized HDL in blood.

The present invention provides methods and reagents for efficiently selecting subjects in need of treatment for dyslipidemia.

It is a dot plot showing the oxidized HDL concentration of each subject in the group which took probucol and the group which did not take probucol. “Probucol (-)” on the horizontal axis represents a non-probucol group, and “probucol (+)” represents a probucol group. The vertical axis represents the oxidized HDL concentration (unit: U / L). It is a box plot showing the oxidized HDL concentration of each subject in the group which took probucol and the group which did not take probucol. “Probucol (-)” on the horizontal axis represents a non-probucol group, and “probucol (+)” represents a probucol group. The vertical axis represents the oxidized HDL concentration (unit: U / L). The box part represents the range from the first quartile to the third quartile, the thick line of the box part represents the median, the upper part of the beard part is the upper 10th percentile, and the lower part of the beard part is. Represents the lower 10th percentile. It is a dot plot showing the ratio of the oxidized HDL concentration to the phospholipid (HDL-PL) concentration in HDL in the group which took probucol and the group which did not take probucol. “Probucol (-)” on the horizontal axis represents a non-probucol group, and “probucol (+)” represents a probucol group. The vertical axis represents the ratio of the oxidized HDL concentration to the phospholipid (HDL-PL) concentration in HDL (unit: U · dL / mg · L). It is a box whiskers showing the ratio of the oxidized HDL concentration to the phospholipid (HDL-PL) concentration in HDL in the group which took probucol and the group which did not take probucol. “Probucol (-)” on the horizontal axis represents a non-probucol group, and “probucol (+)” represents a probucol group. The vertical axis represents the ratio of the oxidized HDL concentration to the phospholipid (HDL-PL) concentration in HDL (unit: U · dL / mg · L). The box part represents the range from the first quartile to the third quartile, the thick line of the box part represents the median, the upper part of the beard part is the upper 10th percentile, and the lower part of the beard part is. Represents the lower 10th percentile.

1. 1. Method for selecting subjects requiring treatment for dyslipidemia The method for selecting subjects requiring treatment for dyslipidemia of the present invention is to measure the concentration of oxidized HDL in a sample collected from a subject. This is a characteristic method.
In the present invention, the sample collected from the subject is not particularly limited as long as it is a sample in which oxidized HDL can be measured, and examples thereof include blood. Examples of blood include whole blood, serum, plasma and the like.

In the present invention, oxidized HDL refers to HDL having apoA protein, which is an apoprotein constituting HDL, and exhibiting dysfunction due to oxidation of a lipid portion constituting HDL.
In the present invention, the measurement of oxidized HDL in a sample collected from a subject includes not only the measurement of the oxidized HDL concentration in the sample but also the measurement of the ratio of the oxidized HDL concentration in the sample to the total HDL concentration. do. Total HDL concentration is determined by measuring HDL-specific substances such as apoA protein concentration in HDL, phospholipid (HDL-PL) concentration in HDL, and phosphatidylcholine (HDL-PC) concentration in HDL. be able to.

In the present invention, the method for selecting a subject who needs treatment for dyslipidemia can be performed by a method including the following steps.
(1) Step of collecting a sample from a subject;
(2) A step of measuring oxidized HDL in the sample collected in step (1) and obtaining a measured value;
(3) Using a known concentration of oxidized HDL as a sample, a step of obtaining a measured value by the same method as in step (2) and creating a calibration curve showing the relationship between the oxidized HDL concentration and the measured value;
(4) The oxidized HDL concentration in the sample is determined from the measured value obtained in the step (2) and the calibration curve showing the relationship between the oxidized HDL concentration and the measured value prepared in the step (3). Process;
(5) If the concentration of oxidized HDL determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the subject needs to be treated for lipid dysfunction. Yes, if the concentration is less than the oxidized HDL concentration in a healthy person, the step to compare with the criterion that the subject does not need treatment for dyslipidemia;
(6) As a result of comparison in step (5), if the concentration of oxidized HDL in the sample determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the subject has dyslipidemia. A step in which it is determined that treatment is necessary, and if the oxidized HDL concentration in a healthy person is lower than that, the subject is determined not to require treatment for dyslipidemia.

<Process (1)>
Examples of the sample collected from the subject in the step (1) include the above-mentioned sample and the like.

<Process (2)>
In the step (2), the measurement of the oxidized HDL in the sample collected in the step (1) is performed by any method as long as the method can measure the oxidized HDL in the sample collected in the step (1). It can be done, for example using a method such as an immunological measurement method. Examples of the immunoassay method include any known immunoassay method, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), indirect fluorescent antibody method. (Indirect Fluorescence assay), Luminescent immunoassay, Physicochemical assay [turbidity immunoassay (TIA), Latex agglutination (LAPIA), Fine particle count immunoassay (PCIA)], Western blotting The method and the like are mentioned, and the ELISA method is preferable. In the immunological measurement method, a sandwich method, a competitive method, or the like can be used, and a homodianis method, a heterogeneous method, or the like can also be used.

A specific embodiment of the method for measuring oxidized HDL in the sample collected in step (1) using the immunological measurement method is shown below.
・ Measurement method 1
A method for measuring oxidized HDL in a sample collected in step (1), which comprises the following steps.
(I) A step of reacting the sample collected in step (1) with the first antibody that binds to the oxidized lipoprotein to generate an immune complex of the first antibody and the oxidized lipoprotein;
(II) The immune complex generated in step (I) is reacted with a labeled second antibody in which a label is bound to a second antibody that binds to HDL, and the first antibody, oxidized lipoprotein and labeled second antibody are reacted. Steps to generate immune complexes of antibodies;
(III) Step (III) A step of measuring the labeled amount in the immune complex produced in (II).

As the antibody (first antibody, second antibody), either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable. As the antibody (first antibody, second antibody), Fc portion such as Fab obtained by papain treatment, F (ab') ₂ obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment. Antibodies fragments from which have been removed can also be used.

Examples of the first antibody that binds to the oxidized lipoprotein include an antibody that binds to oxidized phosphatidylcholine, an antibody that binds to phosphocholine, and the like. Examples of the antibody that binds to oxidized phosphatidylcholine include a monoclonal antibody (hereinafter referred to as DLH3 antibody) produced by mouse-mouse hybridoma cell line FOH1a / DLH3 (FERM BP-7171). As an antibody that binds to phosphocholine, for example, it is produced by T-15 antibody [J. Exp. Med., Vol. 132, p.737-751 (1970).], Hybridoma KTM-285 (FERM BP-7589). Examples thereof include the monoclonal antibody KTM-285 and the recombinant antibody KTM-2001 produced by the transformed cell KTM-2001 (FERM BP-7549).

Examples of the second antibody that binds to HDL include an antibody that binds to the apoA protein. Examples of the antibody that binds to the apo A protein include an antibody that binds to the apo AI protein, an antibody that binds to the apo AI II protein, and the like.
The reaction temperature in the step (I) and the step (II) is not particularly limited as long as it is a reaction temperature capable of measuring the oxidized HDL, and examples thereof include 0 to 50 ° C., preferably 4 ° C. to 40 ° C. The reaction time in the step (I) and the step (II) is not particularly limited as long as it is a reaction time that enables the measurement of oxidized HDL, and is, for example, 1 minute to 72 hours, preferably 5 minutes to 20 hours.

Steps (I)-(III) can also be performed in an aqueous medium. The aqueous medium is not particularly limited as long as it is an aqueous medium capable of measuring oxidized HDL, and examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable. The pH of the aqueous medium is, for example, 4 to 10, and when a buffer solution is used as the aqueous medium, it is desirable to use a buffering agent suitable for the pH to be set. The buffer used for preparing the buffer is not particularly limited as long as it has a buffering ability, and for example, a lactic acid buffer, a citrate buffer, an acetate buffer, a succinic acid buffer, a phthalate buffer, and phosphorus. Acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, apple acid buffer, oxalic acid buffer, glycine buffer, borate buffer, carbon dioxide buffer, Examples include glycine buffer, Tris buffer, Good buffer and the like.

Good buffers include, for example, 2-morpholinoetan sulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, Tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamide) iminodiacetic acid (ADA) buffer, piperazin-N, N'-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamide) Amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) buffer Agent, 3-morpholinopropane sulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N'- (2-Sulfoethyl) piperazine (HEPES) buffer, 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N -Tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPSO) buffer, piperazine-N, N'-bis (2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N- (2-hydroxy) Ethyl) -N'-(2-hydroxy-3-sulfopropyl) piperazin (HEPPSO) buffer, N- (2-hydroxyethyl) -N'-(3-sulfopropyl) piperazin (EPPS) buffer, tricin [ N-Tris (Hydroxymethyl) Methyl Glycin] Buffer, Bicin [N, N-Bis (2-Hydroxyethyl) Glycin] Buffer, 3- [N-Tris (Hydroxymethyl) Methyl] Amino Propane Sulfonic Acid (TAPS) Buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propane Examples include sulfonic acid (CAPS) buffers.

The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and particularly preferably 0.01 to 0.1 mol / L.
Step (I) and step (II) can also be performed at the same time. A cleaning step may or may not be provided between the step (I) and the step (II), but it is preferable to provide a cleaning step. Further, a cleaning step may or may not be provided between the step (II) and the step (III), but it is preferable to provide a cleaning step. The first antibody may or may not be immobilized on the insoluble carrier, but is preferably immobilized. When the first antibody is immobilized on the insoluble carrier, the immune complex of the first antibody produced in step (I) and the component to be measured is unreacted by washing the insoluble carrier after step (I). It can be separated from the components (components derived from the sample, excess first antibody, etc.). Similarly, when the first antibody is immobilized on an insoluble carrier, the first antibody, oxidized HDL and labeled second antibody produced in step (II) are washed by washing the insoluble carrier after step (II). Immune complex can be separated from unreacted components (excessive labeled second antibody, etc.). The cleaning solution includes phosphate buffered saline [10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2 (hereinafter referred to as PBS)], PBS containing a surfactant, and the above. Aqueous medium and the like can be mentioned. Examples of the surfactant include nonionic surfactants such as Tween 20 and the like.

The insoluble carrier is not particularly limited as long as it is an insoluble carrier capable of measuring oxidized HDL. Preferred materials for the insoluble carrier include polymer materials such as polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, nitrocellulose, cellulose acetate, cellulose acetate and polyethylene terephthalate. Examples thereof include glass, ceramics, magnetic particles and metals. Preferred shapes of the insoluble carrier include tubes, beads, plates, fine particles such as latex, sticks and the like, and 96-well / sheet polystyrene microtiter plates and the like are preferable.

As a method for immobilizing the first antibody on an insoluble carrier, a known method such as a method using a physical bond, a method using a chemical bond, or a combination thereof is used. Examples of the physical bond include an electrostatic bond, a hydrogen bond, a hydrophobic bond and the like. Examples of the chemical bond include a covalent bond and a coordinate bond.
The first antibody may be immobilized on the insoluble carrier directly or indirectly on the insoluble carrier. As an indirect immobilization method, for example, a solution of a biotinylated first antibody is added to an insoluble carrier on which avidin is immobilized, and the first antibody becomes an insoluble carrier through a specific binding between biotin and avidin. Examples thereof include a method of immobilization, a method of adding a solution of the first antibody to an insoluble carrier on which Fc is immobilized, and a method of immobilizing the first antibody on the insoluble carrier by the interaction between Fc and the first antibody. Furthermore, the first antibody may be immobilized on an insoluble carrier by covalent bonding via a linker. Examples of the linker include a molecule capable of covalently binding to both the functional group of the first antibody and the functional group held on the surface of the insoluble carrier, and can react with the functional group of the first antibody. A molecule having the reaction active group 1 and the second reaction active group capable of reacting with the functional group held on the surface of the insoluble carrier in the same molecule is preferable, and among them, the first reaction activity Molecules in which the group and the second reactive group are different are particularly preferable. The functional group of the first antibody and the functional group retained on the surface of the insoluble carrier include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group and an N-hydroxysuccinimide (NHS) group. , Maleimide group and the like. Reaction active groups in the linker include aryl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, solarene, etc. Examples thereof include groups such as pyridyl disulfide and vinyl sulfone.

・ Measurement method 2
(I) A step of reacting the sample collected in step (1) with a first antibody that binds to HDL to generate an immune complex of the first antibody and HDL;
(II) The immune complex produced in step (I) is reacted with a labeled second antibody to which a label is bound to a second antibody that binds to the oxidized lipoprotein to cause the first antibody, the oxidized lipoprotein and labeling. Steps to generate immune complexes of the second antibody;
(III) Step (III) A step of measuring the labeled amount in the immune complex produced in (II).

As the antibody (first antibody, second antibody), either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable. As the antibody (first antibody, second antibody), Fc portion such as Fab obtained by papain treatment, F (ab') ₂ obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment. Antibodies fragments from which have been removed can also be used.

Examples of the first antibody that binds to HDL include an antibody that binds to the apoA protein. Examples of the antibody that binds to the apo A protein include an antibody that binds to the apo AI protein, an antibody that binds to the apo AI II protein, and the like. Examples of the second antibody that binds to the oxidized lipoprotein include an antibody that binds to oxidized phosphatidylcholine, an antibody that binds to phosphocholine, and the like.
Examples of the antibody that binds to oxidized phosphatidylcholine include a monoclonal antibody produced by the mouse-mouse hybridoma cell line FOH1a / DLH3 (FERM BP-7171). As an antibody that binds to phosphocholine, for example, it is produced by T-15 antibody [J. Exp. Med., Vol. 132, p.737-751 (1970).], Hybridoma KTM-285 (FERM BP-7589). Examples thereof include the monoclonal antibody KTM-285 and the recombinant antibody KTM-2001 produced by the transformed cell KTM-2001 (FERM BP-7549).

The reaction temperature in the step (I) and the step (II) is not particularly limited as long as it is a reaction temperature capable of measuring the oxidized HDL, and examples thereof include 0 to 50 ° C., preferably 4 ° C. to 40 ° C. The reaction time in the step (I) and the step (II) is not particularly limited as long as it is a reaction time that enables the measurement of oxidized HDL, and is, for example, 1 minute to 72 hours, preferably 5 minutes to 20 hours.
Steps (I)-(III) can also be performed in an aqueous medium. The aqueous medium is not particularly limited as long as it is an aqueous medium capable of measuring oxidized HDL, and examples thereof include the above-mentioned aqueous medium.
Step (I) and step (II) can also be performed at the same time. A cleaning step may or may not be provided between the step (I) and the step (II), but it is preferable to provide a cleaning step. Further, a cleaning step may or may not be provided between the step (II) and the step (III), but it is preferable to provide a cleaning step. The first antibody may or may not be immobilized on the insoluble carrier, but is preferably immobilized. When the first antibody is immobilized on the insoluble carrier, the immune complex of the first antibody produced in step (I) and the component to be measured is unreacted by washing the insoluble carrier after step (I). It can be separated from the components (components derived from the sample, excess first antibody, etc.). Similarly, when the first antibody is immobilized on an insoluble carrier, the first antibody, oxidized HDL and labeled second antibody produced in step (II) are washed by washing the insoluble carrier after step (II). Immune complex can be separated from unreacted components (excessive labeled second antibody, etc.). The cleaning solution includes phosphate buffered saline [10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2 (hereinafter referred to as PBS)], PBS containing a surfactant, and the above. Aqueous medium and the like can be mentioned. Examples of the surfactant include nonionic surfactants such as Tween 20 and the like.

The insoluble carrier is not particularly limited as long as it is an insoluble carrier capable of measuring oxidized HDL. Preferred materials for the insoluble carrier include, for example, the above-mentioned materials and the like.
Examples of the method for immobilizing the first antibody on the insoluble carrier include the above-mentioned immobilization method.

Examples of the labeling substance for labeling the second antibody in the measuring method 1 and the measuring method 2 include enzymes, fluorescent substances, luminescent substances, radioisotopes, biotin, digoxigenin, polypeptides containing tag sequences, metal colloidal particles, and colored latex particles. And so on. Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate). Other fluorescent substances include, for example, quantumdot (Science, 281, 2016-2018, 1998), phycobilly protein such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein), BFP. Examples thereof include proteins that emit fluorescence such as (Blue fluorescent Protein). Examples of the luminescent substance include acridinium and its derivatives, ruthenium complex compounds, loffin and the like. Further, as the ruthenium complex compound, those shown in Clin. Chem. 37, 9, 1534-1539, 1991, which emit light electrochemically together with an electron donor, are preferable. Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, ¹³¹ I and the like. Examples of the polypeptide containing the tag sequence include FLAG peptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Lys), polyhistidine (His tag, His His His His His), and myc epitope peptide (myc tag, Glu Gln Lys Leu). Ile Ser Glu Glu Glu Asp Leu), hemaglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) and the like.

Labeling of the second antibody can be carried out by a reaction that causes a covalent bond between the functional group of the second antibody and the functional group of the labeling substance with or without a linker. Examples of the functional group include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinimide (NHS) ester group, a maleimide group, an isocyanate group and an isothiocyanate group. It is possible to carry out a condensation reaction between these functional groups.

Examples of the binding method not mediated by a linker include a method using a carbodiimide compound such as EDC. In this case, it is also possible to use an active ester such as NHS or a derivative thereof. Further, the condensation reaction between the isothiocyanate group and the amino group does not require other reagents and proceeds only by mixing under neutral to weakly alkaline conditions, which is preferable.
As the linker, for example, a linker having both a reactive active group capable of reacting with the functional group of the second antibody and a reactive active group capable of reacting with the functional group of the labeling substance in the molecule. A molecule having a first reactive group capable of reacting with an amino acid residue of a second antibody and a second reactive group capable of reacting with a functional group of a labeling substance in the same molecule can be mentioned. Among them, a molecule in which the first reactive group and the second reactive group are different is particularly preferable. Examples of the reaction active group in the linker include the above-mentioned reaction active groups.

Examples of the method for chemically binding the radioisotope to the second antibody include the methods described in the literature (Antibody Immunoconj. Radiopharm., 3, 60, 1990).
When the labeling substance is a polypeptide such as an enzyme, avidin, a protein that emits fluorescence, a phycobili protein, or a polypeptide containing a tag sequence, a known gene recombination technique (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring) According to Harbor Laboratory Press, 2001), an expression vector containing a DNA encoding a fusion protein of a labeling substance and an antibody can be prepared, the expression vector can be introduced into an appropriate host, and the host can be cultured. The DNA encoding the fusion protein can be obtained by cloning the DNA encoding the antibody and the labeling substance by PCR or the like and ligating the respective DNAs by a ligase reaction.

For the measurement of the labeled amount, an appropriate method can be selected depending on the labeling substance. When the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength, a spectrophotometer, a multi-well plate reader, or the like can be used. When the labeling substance is a fluorescent substance, a fluorometer, a fluorescent multi-well plate reader, or the like can be used. When the labeling substance is a luminescent substance, a luminescent photometer, a luminescent multi-well plate reader, or the like can be used. When the labeling substance is a radioisotope, the amount of the radioisotope can be measured by a scintillation counter, a γ-well counter, or the like for the radioactivity.

When the label is an enzyme, measuring the labeled amount means measuring the enzyme activity. The labeled amount can be measured by reacting the substrate of the enzyme with the enzyme and measuring the produced substance. When the enzyme is peroxidase, the peroxidase activity can be measured by, for example, an absorptiometry, a fluorescence method, a luminescence method or the like. As a method for measuring peroxidase activity by the absorbance method, for example, a combination of peroxidase and its substrate hydrogen peroxide and an oxidative color-developing chromogen are reacted, and the absorbance of the reaction solution is measured by a spectrophotometer, a multi-well plate reader, or the like. The method of measuring with is mentioned. Examples of the oxidative color-developing chromogen include a leuco-type chromogen, an oxidative-coupling chromogen, and the like.

The leuco-type chromogen is a substance that is independently converted into a dye in the presence of an active peroxide substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4'-bis (dimethylamino) ) Diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA), 10-N- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) -10H-phenothiazine Examples thereof include sodium salt (DA-67).

The oxidative coupling color-developing chromogen is a substance in which two compounds oxidatively couple to form a dye in the presence of an active peroxide substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and anilines (Trinder's reagent), a combination of a coupler and phenols, and the like. Examples of the coupler include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazine and the like. Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), and N-ethyl-N- (2-hydroxy). -3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3 , 5-Dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl- N- (3-Methylphenyl) -N'-Succinylethylenediamine (EMSE), N-Ethyl-N- (3-Methylphenyl) -N'-Acetylethylenediline, N-Ethyl-N- (2-Hydroxy-3-) Sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS) and the like can be mentioned. Examples of the phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

As a method for measuring peroxidase activity by the fluorescence method, for example, a combination of peroxidase and its substrate hydrogen peroxide and a fluorescent substance is reacted, and the intensity of fluorescence generated by a fluorometer, a fluorescent multi-well plate reader, or the like is measured. The method of doing this can be mentioned. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, coumarin and the like.
As a method for measuring peroxidase activity by the luminescence method, for example, peroxidase is reacted with a combination of hydrogen peroxide as a substrate and a luminescent substance, and the intensity of luminescence generated by a luminescence intensity meter, a luminescence multi-well plate reader, or the like is measured. How to do it, etc. Examples of the luminescent substance include luminol compounds and lucigenin compounds.

When the enzyme is alkaline phosphatase, the alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring the alkaline phosphatase activity by the luminescence method include a method of reacting alkaline phosphatase with a substrate thereof and measuring the luminescence intensity of the generated luminescence with a luminescence intensity meter, a luminescence multi-well plate reader, or the like. Examples of the substrate for alkaline phosphatase include 3- (2'-spirodamantan) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane / disodium salt (AMPPD), 2-chloro-. 5- {4-Methylspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo [3.3.1.1 ^3.7 ] decane] -4-yl} phenylphosphate disodium salt ( CDP-Star ^TM ), 3- {4-Methylspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo [ ^3.3.1.13.7 ] decane] -4'-yl} Phenylphosphate disodium salt (CSPD ^TM ), 9-[(phenyloxy) (phosphoryloxy) methylidene] -10-methylacridan disodium, 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene]- Examples thereof include 10-methylacridan disodium (Lumigen ^TM APS-5).

When the enzyme is β-D-galactosidase, the β-D-galactosidase activity can be measured by, for example, an absorptiometry (colorimetric method), a luminescence method, a fluorescence method, or the like. As a method for measuring β-D-galactosidase activity by the absorbance method (colorimetric method), β-D-galactosidase is reacted with its substrate, and the absorbance of the reaction solution is measured with a spectrophotometer, a multi-well plate reader, or the like. The method of doing this can be mentioned. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by an absorptiometry (colorimetric method) include o-nitrophenyl-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by the luminescence method, for example, β-D-galactosidase is reacted with its substrate and the luminescence of the reaction solution is measured with a luminescence intensity meter, a luminescence multi-well plate reader, or the like. And so on. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by the luminescence method include Galacton-Plus [Galacton-Plus, manufactured by Applied Biosystems] or a similar compound thereof. Can be mentioned. As a method for measuring β-D-galactosidase activity by the fluorescence method, for example, a method of reacting β-D-galactosidase with its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescence multi-well plate reader, or the like. And so on. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by the fluorescence method include 4-methylumbelliferyl-β-D-galactopyranoside.

When the enzyme is luciferase, the luciferase activity can be measured by, for example, a luminescence method. Examples of the method for measuring the luciferase activity by the luminescence method include a method in which luciferase is reacted with its substrate and the luminescence degree of the reaction solution is measured with a luminescence intensity meter, a luminescence multi-well plate reader, or the like. Examples of the substrate for luciferase include luciferin and coelenterazine.
In the method for measuring oxidized HDL, the antigen-antibody reaction, that is, the steps (I) and (II) in the measuring method 1, the steps (I) and the step (II) in the measuring method 2, are subjected to metal ions, salts, saccharides, and antiseptic. It can be carried out in the coexistence of an agent, a protein, a protein stabilizer, a surfactant and the like. Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like. Examples of salts include sodium chloride, potassium chloride and the like. Examples of the saccharide include mannitol, sorbitol and the like. Examples of the preservative include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), bioace, Proclin 300, Proxel GXL, and the like. Examples of the protein include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, block ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) and the like. Examples of the protein stabilizer include peroxidase stabilizing buffer [Peroxidase Stabilizing Buffer, manufactured by DakoCytomation], polyethylene glycol (PEG) and the like. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, amphoteric surfactants and the like.

<Process (3)>
In the step (3), the calibration curve showing the relationship between the oxidized HDL concentration and the measured value is measured by the above-mentioned oxidized HDL measuring method using a standard product having a known oxidized HDL concentration as a sample. , Can be made from the obtained measurements and the oxidized HDL concentration.

<Process (4)>
In the step (4), the oxidized HDL concentration in the sample collected in the step (1) was measured by the above-mentioned method for measuring oxidized HDL using the sample, and the obtained measured value and the above-mentioned calibration curve were measured. Can be determined from.

<Process (5)>
In step (5), when the concentration of oxidized HDL in the sample determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the patient needs treatment for dyslipidemia, and the patient needs treatment for dyslipidemia. If it is below the oxidized HDL concentration, it is compared with the criterion that the patient does not need treatment for dyslipidemia.

<Step (6)>
In step (6), as a result of comparison in step (5), if the concentration of oxidized HDL in the sample determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the subject is It is determined that treatment for dyslipidemia is necessary, and if the concentration is lower than the oxidized HDL concentration in a healthy person, the subject is determined to not require treatment for dyslipidemia.

The oxidized HDL concentration in a healthy person in the present invention is, for example, 30 U / L, and the ratio of the oxidized HDL concentration to the total HDL-PL concentration is, for example, 0.22 U · dL / mg · L. The unit "U / L" here is defined as 1 U / L oxidized HDL obtained by copper oxidation of 1 mg / L HDL-PL.
The treatment for dyslipidemia in the present invention is not particularly limited as long as it is a treatment that lowers the oxidized HDL of the subject, and examples thereof include treatment with a therapeutic agent for dyslipidemia. The therapeutic agent for dyslipidemia is not particularly limited as long as it is a therapeutic agent that lowers the oxidized HDL of the subject, and examples thereof include cholesterol catabolism promoters and the like. Examples of the cholesterol catabolism promoter include probucol and the like.

2. 2. Reagent for selecting subjects requiring treatment for dyslipidemia The reagent for selecting subjects requiring treatment for dyslipidemia of the present invention is a reagent characterized by containing an oxidized HDL measuring reagent. The reagent for selecting a subject who needs treatment for dyslipidemia of the present invention can be used in the method for selecting a subject who needs treatment for dyslipidemia of the present invention. Further, in the reagent for selecting a subject who needs treatment for dyslipidemia of the present invention, when the oxidized HDL concentration in the sample collected from the subject is equal to or higher than the oxidized HDL concentration in a healthy person. There is a standard table that describes the criteria that the subject needs treatment for dyslipidemia and that the subject does not need treatment for dyslipidemia if the oxidized HDL concentration is lower than that in healthy subjects. It may be included.

The reagent for measuring oxidized HDL in the reagent for selecting a subject who needs treatment for dyslipidemia of the present invention is a reagent used in the above-mentioned method for measuring oxidized HDL. The reagent for measuring oxidized HDL is not particularly limited as long as it is a reagent capable of measuring oxidized HDL in a sample, and examples thereof include an immunological measuring reagent. Examples of the immunological measurement reagent include reagents based on the above-mentioned immunological measurement method.
Specific embodiments of the oxidized HDL measuring reagent based on the immunological measuring method are shown below.

・ Measuring reagent 1
An oxidized HDL measurement reagent containing a first antibody that binds to an oxidized lipoprotein and a labeled second antibody that has a label bound to a second antibody that binds to HDL.
・ Measuring reagent 2
An oxidized HDL measurement reagent containing a first antibody that binds to HDL and a labeled second antibody that has a label bound to a second antibody that binds to an oxidized lipoprotein.

Examples of the antibody that binds to HDL, the antibody that binds to the oxidized lipoprotein, and the label in the labeled antibody include the above-mentioned antibody that binds to HDL, the antibody that binds to the oxidized lipoprotein, and the label in the labeled antibody. Be done.
In the measurement reagent 1, the first antibody that binds to the oxidized lipoprotein may or may not be immobilized on an insoluble carrier, but it is preferably immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like. In the measurement reagent 2, the first antibody that binds to HDL may or may not be immobilized on an insoluble carrier, but it is preferable that it is immobilized. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.
The oxidized HDL measuring reagent may contain the above-mentioned aqueous medium, metal ions, salts, sugars, preservatives, proteins, protein stabilizers, surfactants and the like.

In the present invention, as a method for selecting a subject who needs treatment for dyslipidemia by using an oxidized HDL measuring reagent, for example, a method including the following steps and the like can be mentioned.
(1) Step of collecting a sample from a subject;
(2) A step of measuring the oxidized HDL in the sample collected in the step (1) using an oxidized HDL measuring reagent and obtaining a measured value;
(3) Using a known concentration of oxidized HDL as a sample, a step of obtaining a measured value by the same method as in step (2) and creating a calibration curve showing the relationship between the oxidized HDL concentration and the measured value;
(4) The oxidized HDL concentration in the sample is determined from the measured value obtained in the step (2) and the calibration curve showing the relationship between the oxidized HDL concentration and the measured value prepared in the step (3). Process;
(5) When the concentration of oxidized HDL determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the patient needs treatment for lipid dysfunction. A step to compare with the criterion that if the oxidized HDL concentration in a healthy person is less than that, the patient does not need treatment for dyslipidemia;
(6) As a result of comparison in step (5), if the concentration of oxidized HDL in the sample determined in step (4) is equal to or higher than the oxidized HDL concentration in a healthy person, the subject has dyslipidemia. A step in which it is determined that treatment is necessary, and if the oxidized HDL concentration in a healthy person is lower than that, the subject is determined not to require treatment for dyslipidemia.

Examples of the sample include the above-mentioned sample and the like. The measurement of oxidized HDL in the sample collected in the step (1) is performed using an oxidized HDL measuring reagent. Examples of the oxidized HDL measuring reagent include the above-mentioned oxidized HDL measuring reagent. A calibration curve showing the relationship between the oxidized HDL concentration and the measured value can be created by the above-mentioned method or the like. The oxidized HDL concentration in the sample collected in the step (1) can be determined by the above-mentioned method or the like. Examples of the oxidized HDL concentration in a healthy person to be compared with the determined oxidized HDL concentration include the above-mentioned oxidized HDL concentration in a healthy person. By comparing the determined oxidized HDL concentration with the oxidized HDL concentration in a healthy person, it is possible to select a subject who needs treatment for dyslipidemia.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to this Example.
[Example 1]
Determination of Oxidized HDL Concentration in Serum Collected from Subjects (1) Oxidized HDL Measuring Reagents Oxidized HDL measuring reagents containing the following DLH3 antibody-immobilized plate and peroxidase-labeled anti-apoAI antibody solution were prepared.

<DLH3 antibody-immobilized plate>
In each well of the 96-well microplate (manufactured by Nunk), a 10 μg / mL Tris-HCl buffer (pH 8.0) solution of DLH3 antibody prepared by the method described in JP-A-7-238098 is added to 100 μL / well. Incubate at 4 ° C for 16 hours, then remove the Tris-HCl buffer solution and add 350 μL of Tris-HCl buffer (pH 8.0) containing 1% (w / v) BSA to each well. Was blocked by incubating at 25 ° C. for 2 hours, and then washed 4 times with PBS (pH 7.4) containing 0.05% (w / v) Tween20 to prepare a plate on which DLH3 antibody was immobilized. ..

<Peroxidase-labeled anti-apo AI antibody solution>
Using the Peroxidase Labeling Kit-NH ₂ (manufactured by Dojin Kagaku Kenkyusho), the anti-apo AI mouse monoclonal antibody (manufactured by Abnova) was labeled with peroxidase according to the protocol attached to the kit, and the peroxidase-labeled anti-apo AI was labeled. Antibodies were prepared.

(2) Preparation of calibration curve 1-calibration curve showing the relationship between the oxidized HDL concentration and the measured value (absorbance)
Oxidized HDL reaction buffer with concentrations of 2 mU / L, 4 mU / L, and 8 mU / L obtained by oxidizing HDL-PL with copper sulfate [0.1 g / L Tween 20, 48 g. / L PEG6000 and PBS (pH 7.4) containing 10 g / L BSA] solution and reaction buffer (oxidized HDL concentration: 0 U / L) were used as standard solutions. Here, 1 mU / L oxidized HDL means oxidized HDL obtained by copper-oxidizing 1 μg / L of HDL-PL.

100 μL of each standard solution was added to each well of the DLH3 antibody-immobilized plate prepared in (1) above, and the reaction was carried out at 37 ° C. for 2 hours (primary reaction). After each well of the plate after the primary reaction was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, each well was filled with the peroxidase-labeled anti-apo AI antibody solution prepared in (1) above. 100 μL was added and the reaction was carried out at 37 ° C. for 1 hour (secondary reaction). After washing each well of the plate after the secondary reaction with PBST, 100 μL of TMB (3,3', 5,5'-tetramethylbenzidine) aqueous solution was added to each well, and the reaction was carried out at 37 ° C. for 20 minutes. , 0.5 mol / L Sulfuric acid 50 μL was added and the reaction was stopped (color development reaction). The absorbance of the reaction solution obtained by the color development reaction at 450 nm was measured, and a calibration curve showing the relationship between the oxidized HDL concentration and the absorbance was prepared.

(3) Preparation of calibration curve 2-A calibration curve showing the relationship between the phospholipid concentration and the measured value (absorptiometry) Using the phospholipid measurement kit "Detaminer L PL" (manufactured by Kyowa Medex Co., Ltd.), the kit According to the method described in the attached document, measurement is performed using the Detaminer standard serum lipid measurement (manufactured by Kyowa Medex Co., Ltd.) as a standard product, and a calibration curve showing the relationship between the phospholipid concentration and the measured value (absorptiometry) is created. did.

(4) Measurement of oxidized HDL in serum collected from subjects-Comparison between probucol oral group and probucol non-administrative group A patient who was visiting the Osaka University Hospital and had a dyslipidemia took probucol. The group was divided into a group (39 patients) and a group that did not take probucol (138 patients).
Measurement was performed by the same method as in (2) above, except that serum obtained from each subject in the group (39 subjects) who took probucol was used instead of the standard solution in (2) above, and each subject was measured. The absorbance of the serum obtained from the examiner was measured. From the measured absorbance and the calibration curve prepared in (2) above, the oxidized HDL concentration in the serum collected from each subject was determined. Similarly, the oxidized HDL concentration in the serum collected from each subject in the group (138 subjects) who did not take probucol was determined. The results are shown in FIG.

As is clear from FIG. 1, the average oxidized HDL concentration in serum obtained from each subject in the group not taking probucol was 30.0 ± 8.1 U / L, whereas the average concentration of oxidized HDL was 30.0 ± 8.1 U / L, whereas probucol was taken. The average serum oxidized HDL concentration obtained from each subject in the group was 18.7 ± 6.6 U / L, which was more significant in the group who took probucol than in the group who did not take probucol. In addition, the serum of oxidized HDL was low (P <0.05). As described above, the oxidized HDL concentration was significantly different between the probucol oral group and the non-administrative group, and it was clarified that probucol, which is a cholesterol catabolism promoter, has an effect of reducing the oxidized HDL concentration. rice field. Probucol is one of the therapeutic agents for dyslipidemia. Therefore, if a subject having a high concentration of oxidized HDL is selected and a therapeutic agent for dyslipidemia is administered to the subject, the oxidized HDL can be reduced and the therapeutic effect of dyslipidemia can be expected.

(5) Measurement of oxidized HDL and HDL-PL in serum collected from subjects-Comparison between probucol oral group and probucol non-oral group The group (39 subjects) who took the same probucol as in (4) above was DCM. According to (Designated Comparison Method), the HDL fraction was separated from the serum collected from each subject, and the phospholipids in the separated HDL fraction were measured using "Detaminer L PL" (manufactured by Kyowa Medex Co., Ltd.). .. The HDL-PL concentration was determined from the measured value (absorbance) obtained by the measurement and the calibration curve prepared in (3) above.

Serum derived from the same patient from the oxidized HDL concentration in the serum obtained from each patient in the patient group (39 patients) who took probucol determined in (4) above and the HDL-PL concentration determined above. The ratio of the oxidized HDL concentration to the HDL-PL concentration (HDL / HDL-PL) was determined.
Similarly, instead of the group that took the same probucol as in (4) above, the group that did not take the same probucol as in (4) above was used, and the same subjects in the group that did not take probucol were collected. The ratio of serum oxidized HDL concentration to HDL-PL concentration (oxidized HDL / HDL-PL) was determined. The results are shown in FIG.

As is clear from FIG. 2, the average value of oxidized HDL / HDL-PL in the serum obtained from each subject in the group not taking probucol was 0.224 ± 0.049 U · dL / mg · L. On the other hand, the average value of oxidized HDL / HDL-PL in the serum obtained from each subject in the group who took probucol was 0.206 ± 0.045 U · dL / mg · L, and did not take probucol. The group receiving probucol had significantly lower oxidized HDL / HDL-PL in serum (P <0.05) than the group. Thus, since the oxidized HDL / HDL-PL was significantly different between the probucol oral group and the non-oral group, probucol, which is a cholesterol catabolic agent, may have an effect of reducing the oxidized HDL concentration. It became clear. Probucol is one of the therapeutic agents for dyslipidemia. Therefore, if a subject having a high concentration of oxidized HDL is selected and a therapeutic agent for dyslipidemia is administered to the subject, the oxidized HDL can be reduced and the therapeutic effect of dyslipidemia can be expected.

The method for selecting a subject who needs treatment for dyslipidemia and the reagent for selecting a subject who needs treatment for dyslipidemia according to the present invention are useful in clinical diagnosis.

Claims (13)

The concentration of oxidized high-density lipoprotein (hereinafter referred to as oxidized HDL) in the sample collected from the subject is determined by an antibody that binds to phosphatidyloxide oxide or an antibody that binds to phosphocholine, and an antibody that binds to high-density lipoprotein. If the concentration of oxidized HDL is higher than or higher than the oxidized HDL concentration in a healthy person, the subject needs treatment for lipid dysfunction and is less than the oxidized HDL concentration in a healthy person. In the case of, a test method for selecting a subject who needs treatment for dyslipidemia, which is characterized by comparing with a criterion that the subject does not need treatment for dyslipidemia. The method according to claim 1, which comprises the following steps (1) to (3).
(1) Step of collecting a sample from a subject;
(2) A step of measuring the concentration of oxidized HDL in the sample collected in step (1);
(3) When the concentration of oxidized HDL measured in step (2) is equal to or higher than the oxidized HDL concentration in a healthy person, the subject needs treatment for dyslipidemia and is less than the oxidized HDL concentration in a healthy person. In the case of, the step of comparing with the criterion that the subject does not need treatment for dyslipidemia. The method according to claim 1 or 2 , wherein the antibody that binds to the high-density lipoprotein is an antibody that binds to the apoA protein. The method according to any one of claims 1 to 3 , wherein the treatment for dyslipidemia is a treatment with a therapeutic agent for dyslipidemia. The method according to claim 4 , wherein the therapeutic agent for dyslipidemia is a cholesterol catabolism promoter. The method according to claim 5 , wherein the cholesterol catabolism promoter is probucol. The method according to any one of claims 1 to 6 , wherein the sample is blood. Oxidized high-density lipoprotein measuring reagent (hereinafter referred to as oxidized HDL measuring reagent), which is an immunological measurement reagent containing an antibody that binds to phosphatidylcholine oxide or an antibody that binds to phosphocholine and an antibody that binds to high-density lipoprotein. A reagent for selecting a subject in need of treatment for dyslipidemia, which is characterized by containing. The reagent according to claim 8 , wherein the antibody that binds to the high-density lipoprotein is an antibody that binds to the apoA protein. The reagent according to claim 8 or 9 , wherein the treatment for dyslipidemia is a treatment with a therapeutic agent for dyslipidemia. The reagent according to claim 10 , wherein the therapeutic agent for dyslipidemia is a cholesterol catabolism promoter. The reagent according to claim 11 , wherein the cholesterol catabolism promoter is probucol. The reagent according to any one of claims 8 to 12 , wherein the oxidized HDL measuring reagent is a reagent for measuring oxidized HDL in blood.

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