JP7084589B2 - Oxidized high-density lipoprotein measurement method - Google Patents

Description

The present invention relates to a method for measuring oxidized high-density lipoprotein, a measuring reagent, and a measuring kit.
The present application claims priority based on Japanese Patent Application No. 2016-213873 filed in Japan on October 31, 2016, the contents of which are incorporated herein by reference.

Arteriosclerosis occurs frequently in muscular arteries such as aorta, coronary arteries, cerebral arteries, and carotid arteries, and is a major cause of angina, myocardial infarction, and cerebral infarction. Elevated serum cholesterol, platelet aggregation, and endothelial damage have been proposed as the causes, but the causes have not been analyzed.

Serum lipids include coronary diseases such as myocardial infarction and angina, cerebral arterial diseases such as cerebral infarction and cerebrovascular dementia, renal arterial diseases such as nephropathy and diabetic nephropathy, and peripheral arterial occlusion. It is strongly suggested that it is related to various cardiovascular diseases such as peripheral arterial diseases, and its measurement is considered to be extremely important for diagnosis of these diseases, elucidation of pathological conditions, detection of therapeutic effects, etc. ..

However, in a recent study, a comparison of the absolute amounts of serum lipids between the diseased patient group and the healthy subject group showed that there was not much difference between the two groups, but rather in the serum of denatured lipoproteins. It was reported that the abundance was clearly different between the two groups (see Non-Patent Document 1).

In recent years, as a method for measuring oxidized high-density lipoprotein for diagnosing cardiovascular diseases such as arteriosclerosis, 9F5-3a, which is an antibody that recognizes oxidized phospholipids (particularly lysophosphatidylcholine) of high-density lipoprotein, has been used. A method for measuring oxidized high-density lipoprotein using an anti-apo AI antibody (see Patent Document 1), a measurement method using an antibody that binds to phosphocholine and an antibody that binds to apoprotein (see Patent Document 2), etc. Has been reported.

Japanese Unexamined Patent Publication No. 9-33525 International Publication No. 2003/006989

Circulation, 94, Suppl. I, 1288 (1996).

An object of the present invention is to provide a simple and highly sensitive method for measuring oxidized high-density lipoprotein in a sample, a measuring reagent, and a measuring kit.

As a result of diligent studies to solve the above problems, the present inventors have made a polyoxyethylene-based surfactant with a sample and an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine. It was found that the oxidized high-density lipoprotein in a sample can be measured easily and with high sensitivity by reacting in an aqueous medium containing at least one selected from the group consisting of an activator and a cationic surfactant. , The present invention has been completed.

That is, the present invention relates to the following [1] to [28].
[1] A method for measuring oxidized high-density lipoprotein in a sample, which comprises the following steps.
(1) A sample and an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine are selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants. An immune complex consisting of an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof, which is reacted in an aqueous medium containing at least one of these. Step to generate 1;
(2) An antibody or an antibody fragment thereof that binds to a high-density lipoprotein by reacting it with the immune complex 1 produced in the step (1) in an aqueous medium and binds to phosphatidylcholine oxide, or the antibody fragment thereof. , A step of producing an immune complex 2 comprising an antibody or antibody fragment that binds to phosphocholine, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to high-density lipoprotein;
(3) A step of measuring the immune complex 2 produced in the step (2).

[2] A method for measuring oxidized high-density lipoprotein in a sample, which comprises the following steps.
(1') An aqueous medium containing a sample and an antibody that binds to a high-density lipoprotein or an antibody fragment thereof, at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant. A step of reacting in the process to generate an immune complex 3 consisting of an oxidized high-density lipoprotein and an antibody or antibody fragment thereof that binds to the high-density lipoprotein;
(2') An antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine is reacted with the immune complex 3 produced in the step (1') in an aqueous medium. An immune complex consisting of an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, an antibody or an antibody fragment thereof that binds to an oxidized high-density lipoprotein and phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine. Step to generate 4;
(3') Step of measuring the immune complex 4 produced in the step (2').

[3] In [1] or [2], the aqueous medium of the step (1) or the step (1') further contains at least one substance selected from the group consisting of protein and polyethylene glycol. The method described.
[4] The method according to [3], wherein the protein is albumin.
[5] The method according to [4], wherein the albumin is bovine serum albumin.
[6] The polyoxyethylene-based surfactant is a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, a polyoxyethylene alkyl ether, or a polyoxyethylene alkyl ether. To any one of [1] to [5], which is phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or a salt thereof, or polyoxyethylene fatty acid glyceryl. The method described.

[7] The method according to any one of [1] to [6], wherein the cationic surfactant is a quaternary ammonium salt.
[8] The method according to any one of [1] to [7], wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein.
[9] The method according to [8], wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein.

[10] A reagent used for the measurement method according to [1] or [2], which comprises an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine, and a high-density lipoprotein. A reagent for measuring high-density oxidized lipoprotein, which comprises an antibody or an antibody fragment thereof that binds to, and at least one selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants.
[11] The reagent according to [10], further containing at least one substance selected from the group consisting of proteins and polyethylene glycols.
[12] The reagent according to [11], wherein the protein is albumin.
[13] The reagent according to [12], wherein the albumin is bovine serum albumin.
[14] The polyoxyethylene-based surfactant is a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, a polyoxyethylene alkyl ether, or a polyoxyethylene alkyl ether. To any one of [10] to [13], which is phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or a salt thereof, or polyoxyethylene fatty acid glyceryl. The described reagents.

[15] The reagent according to any one of [10] to [14], wherein the cationic surfactant is a quaternary ammonium salt.
[16] The reagent according to any one of [10] to [15], wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein.
[17] The reagent according to [16], wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein.

[18] At least one selected from the group consisting of an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine, and a polyoxyethylene-based surfactant and a cationic surfactant. A kit for measuring an oxidized high-density lipoprotein, which comprises a first reagent containing and, and a second reagent containing an antibody or an antibody fragment thereof that binds to the high-density lipoprotein.
[19] A first reagent containing an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, and at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and a first reagent. A kit for measuring high-density oxidized lipoprotein, which comprises an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or a second reagent containing an antibody that binds to phosphocholine or the antibody fragment thereof.
[20] The above-mentioned [18] or [19], wherein at least one substance selected from the group consisting of protein and polyethylene glycol is contained in at least one reagent of the first reagent and the second reagent. kit.

[21] An antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or a first reagent containing the antibody fragment, an antibody that binds to a high-density lipoprotein or a second reagent containing the antibody fragment. A kit for measuring high-density oxidized lipoprotein, which comprises a third reagent containing at least one selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants.
[22] The kit according to [21], wherein at least one substance selected from the group consisting of protein and polyethylene glycol is contained in at least one reagent of the first to third reagents.
[23] The kit according to [20] or [22], wherein the protein is albumin.
[24] The kit according to [23], wherein the albumin is bovine serum albumin.

[25] The polyoxyethylene-based surfactant is a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, a polyoxyethylene alkyl ether, or a polyoxyethylene alkyl ether. To any one of [18] to [24], which is phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or a salt thereof, or polyoxyethylene fatty acid glyceryl. The kit described.
[26] The kit according to any one of [18] to [25], wherein the cationic surfactant is a quaternary ammonium salt.
[27] The kit according to any one of [18] to [26], wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein.
[28] The kit according to [27], wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein.

INDUSTRIAL APPLICABILITY The present invention provides a simple and highly sensitive method for measuring oxidized high-density lipoprotein in a sample, a measuring reagent, and a measuring kit.

1. 1. Measurement method <Measurement method 1>
The method for measuring oxidized high-density lipoprotein in a sample of the present invention is characterized by including the following steps.
(1) A sample and an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine are selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants. An immune complex consisting of an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof, which is reacted in an aqueous medium containing at least one of these. Step to generate 1;
(2) An antibody or an antibody fragment thereof that binds to a high-density lipoprotein by reacting it with the immune complex 1 produced in the step (1) in an aqueous medium and binds to phosphatidylcholine oxide, or the antibody fragment thereof. , A step of producing an immune complex 2 comprising an antibody or antibody fragment that binds to phosphocholine, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to high-density lipoprotein;
(3) A step of measuring the immune complex 2 produced in the step (2).

≪Process (1)≫
In the step (1), the sample and the antibody or the antibody fragment thereof that binds to phosphatidylcholine oxide, or the antibody or the antibody fragment thereof that binds to phosphocholine is composed of a polyoxyethylene-based surfactant and a cationic surfactant. The method of reacting in an aqueous medium containing at least one selected from the group includes an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof. There is no particular limitation as long as it is a method that enables the production of the immune complex 1 comprising the same.

The antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine may or may not be immobilized on an insoluble carrier, but it must be immobilized. preferable.
If the antibody or antibody fragment that binds to phosphatidylcholine oxide or the antibody or antibody fragment that binds to phosphocholine is immobilized on an insoluble carrier, the antibody or antibody fragment that binds to phosphatidylcholine oxide or the antibody fragment thereof binds to phosphocholine. The antibody or the insoluble carrier on which the antibody fragment is immobilized may be produced in the reaction solution of the antigen-antibody reaction, and in this case, it binds to oxidized phosphatidylcholine to which one (A) of a combination of a pair of affinity substances is bound. The antibody or the antibody fragment thereof, or the antibody that binds to phosphocholine or the antibody fragment, and the insoluble carrier to which the other (a) of the combination of the set of affinity substances is bound are reacted in the reaction solution of the antigen-antibody reaction. By doing so, an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody that binds to phosphocholine or an insoluble carrier on which the antibody fragment is immobilized can be generated in the reaction solution of the antigen-antibody reaction.

Examples of the combination of Aa include the following combinations and the like.
・ Combination of biotin and avidins (avidin, neutral avidin, streptavidin, etc.);
・ Combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
-An antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or a combination of an Fc region of the antibody fragment and an antibody that binds to the Fc region.

The insoluble carrier may be an antibody that binds to phosphatidylcholine oxide or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof and an insoluble carrier that enables the method for measuring an oxidized high-density lipoprotein of the present invention. However, there are no particular restrictions, for example, polystyrene plates such as microtiter plates, granules (beads) made of glass or synthetic resin, spherical objects (balls) made of glass or synthetic resin, latex, magnetic particles, nitrocellulose film and the like. Examples include various membranes and test tubes made of synthetic resin.

The binding between the antibody that binds to phosphatidylcholine oxide or the antibody fragment thereof, or the antibody that binds to phosphocholine or the antibody fragment thereof and the insoluble carrier is a bond that enables the method for measuring the high-density oxidized lipoprotein of the present invention. If there is, there is no particular limitation, and examples thereof include physical adsorption and chemical bonding. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordinate bond.

The antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the antibody or antibody fragment thereof that binds to phosphocholine may be directly immobilized on an insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. It may be indirectly immobilized on an insoluble carrier. As an indirect immobilization method, for example, an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or phosphocholine is bound via a specific binding between biotin and avidins (avidin, streptavidin, neutral avidin, etc.). Examples thereof include a method of immobilizing an antibody or the antibody fragment on an insoluble carrier. Further, the antibody that binds to oxidized phosphatidylcholine or the antibody fragment thereof, or the antibody that binds to phosphocholine or the antibody fragment thereof may be immobilized on an insoluble carrier by covalent bonding via a linker.

The linker may be, for example, a molecule capable of covalently binding both a functional group on the surface of an insoluble carrier and an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody that binds to phosphocholine or a functional group of the antibody fragment. There are no particular restrictions, for example, a first reactive group capable of reacting with an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody that binds to phosphocholine or a functional group of the antibody fragment, and an insoluble carrier surface. It is a molecule having a second reactive active group capable of reacting with the functional group of the above, and it is preferable that the first reactive active group and the second reactive active group are different groups. Examples of the functional group of the antibody or the antibody fragment thereof that binds to phosphatidylcholine oxide, the functional group of the antibody or the antibody fragment thereof that binds to phosphocholine, and the functional group held on the surface of the insoluble carrier include a carboxyl group, an amino group and a glycidyl. Examples thereof include a group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinimide group (NHS group), a maleimide group and the like. Examples of the reaction active group in the linker include aryl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, and solarene. , Pyridyl disulfide, vinyl sulfone and the like.

Further, the sample may be mixed in advance with at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and then the obtained mixture may be subjected to an antigen-antibody reaction.

The reaction temperature of the reaction between the sample and the antibody or the antibody fragment thereof that binds to phosphatidylcholine oxide or the antibody or the antibody fragment thereof that binds to phosphocholine in the step (1) is the method for measuring the oxidized high-density lipoprotein of the present invention. The temperature is not particularly limited as long as it enables the above, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the antibody that binds to phosphatidylcholine oxide or the antibody fragment thereof, or the antibody that binds to phosphocholine or the antibody fragment thereof in the reaction solution is particularly high as long as it is a concentration that enables the method for measuring the high-density oxidized lipoprotein of the present invention. There is no limitation, usually 0.01 to 100 μg / mL, preferably 0.1 to 20 μg / mL.

≪Process (2)≫
In the step (2), an antibody that binds to a high-density lipoprotein or an antibody fragment thereof may be added and reacted with the immune complex 1 produced in the step (1), or an antibody that binds to a high-density lipoprotein may be added and reacted. Alternatively, the immune complex 1 produced in step (1) may be added to the antibody fragment and reacted.

In step (2), an antibody or an antibody fragment thereof that binds to phosphatidyloxide oxide, an antibody or an antibody fragment thereof that binds to phosphocholine, an oxidized high-density lipoprotein, and an antibody or an antibody fragment thereof that binds to a high-density lipoprotein. The reaction temperature of the reaction for producing the immune complex 2 consisting of It is preferably to 45 ° C, particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the antibody that binds to the high-density lipoprotein or the antibody fragment thereof in the reaction solution is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL. It is preferably 0.1 to 20 μg / mL.

Step (1) and step (2) may be performed sequentially or at the same time.
Further, a step of washing the insoluble carrier after the reaction of the step (1) may be added between the steps (1) and the step (2), if necessary. The cleaning solution used for cleaning the insoluble carrier after the reaction in step (1) is not particularly limited as long as it is a cleaning solution that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is, for example, phosphate buffering. Examples thereof include physiological saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS) and PBS containing a surfactant. Examples of the surfactant include nonionic surfactants such as Tween 20 and the like.

The above-mentioned cleaning step may be added after the step (2). The cleaning solution used for cleaning the insoluble carrier after the step (2) is not particularly limited as long as it is a cleaning solution that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include the above-mentioned cleaning solution.

≪Process (3) ≫
In step (3), the concentration of oxidized high-density lipoprotein in the sample is determined by measuring the amount of immune complex 2 produced in step (2) using the following method.

(I) When an antibody that binds to a high-density lipoprotein or the antibody fragment thereof is not labeled An antibody that binds to a high-density lipoprotein or an antibody that binds to the antibody fragment (hereinafter referred to as a third antibody) or the first antibody. The labeled third antibody or the antibody fragment in which the label is bound to the antibody fragment of the 3 antibody is combined with the immune complex 2 (an antibody that binds to phosphatidylcholine oxide or the antibody fragment thereof, or an antibody that binds to phosphocholine or the antibody fragment thereof. It binds to oxidized phosphatidylcholine by reacting with an antibody or an antibody fragment that binds to a high-density lipoprotein in an immune complex consisting of an oxidized high-density lipoprotein and an antibody or an antibody fragment thereof that binds to the high-density lipoprotein. An antibody or the antibody fragment thereof, or an antibody or the antibody fragment that binds to phosphocholine, an oxidized high-density lipoprotein, an antibody or the antibody fragment that binds to the high-density lipoprotein, and a labeled third antibody or the antibody fragment. The amount of the immune complex 2 produced in the step (2) can be measured by forming the immune complex 5 composed of the above and measuring the label in the immune complex by the method described below. Examples of the third antibody include an antibody that binds to high-density lipoprotein, an antibody that binds to the Fc region of the antibody fragment, an antibody fragment thereof, and the like. Examples of the sign include a sign described later.
The reaction temperature of the reaction between the labeled third antibody or the antibody fragment and the antibody or the antibody fragment that binds to the high-density lipoprotein in the immune complex 2 enables the method for measuring the oxidized high-density lipoprotein of the present invention. The temperature is not particularly limited, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the labeled third antibody or the antibody fragment in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL. It is preferably 0.1 to 20 μg / mL.

(Ii) When an antibody or an antibody fragment thereof that binds to a high-density lipoprotein is labeled, the amount of the immune complex 2 produced in the step (2) is measured by measuring the label in the immune complex 2. can do.
Examples of the label include enzymes, fluorescent substances, luminescent substances, radioisotopes, biotin, digoxygenin, polypeptides containing tag sequences, metal colloidal particles, colored latex particles and the like.

Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine B-isothiocyanate (RITC). Other fluorescent substances include, for example, quantum dot (Science, 281, 2016-2018, 1998), phycobilly protein such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein). , BFP (Blue fluorescent Protein) and other proteins that emit fluorescence.
Examples of the luminescent substance include acridinium and its derivatives, ruthenium complex compounds, loffin and the like. Examples of the ruthenium complex compound include the ruthenium complex compounds shown in Clin. Chem. 37, 9, 1534-1539, 1991.
Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, ¹³¹ I and the like.

An antibody that binds to phosphatidylcholine oxide or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof, an oxidized high-density lipoprotein, and a labeled antibody or antibody fragment that binds to high-density lipoprotein. Measurement of the label in the immune complex consisting of, and binding to an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine, an oxidized high-density lipoprotein, and a high-density lipoprotein. The measurement of the label in the immune complex comprising the antibody or the antibody fragment thereof and the labeled third antibody or the antibody fragment thereof can be appropriately selected depending on the label to be used.
When the label is a coloring substance, that is, a substance that absorbs light of a certain wavelength, the absorbance can be measured using a spectrophotometer, a multi-well plate reader, or the like.
When the label is a fluorescent substance, the fluorescence intensity can be measured using a fluorometer, a fluorescent multi-well plate reader, or the like.
When the label is a luminescent substance, the luminescence intensity can be measured using a luminescence photometer, a luminescence multi-well plate reader, or the like.
When the label is a radioisotope, the radioactivity can be measured by a scintillation counter, a γ-well counter, or the like.
If the label is an enzyme, the labeled amount can be quantified by measuring the enzyme activity. For example, the labeled amount can be measured by reacting the substrate of an 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, or the like. As a method for measuring peroxidase activity by the absorbance method, for example, peroxidase is reacted with a combination of hydrogen peroxide as a substrate and an oxidative color-developing chromogen, and the absorbance of the reaction solution is measured by a spectrophotometer or a multi-well plate reader. A method of measuring with or the like can be 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), 10-N-carboxymethylcarbamoyl-3 , 7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] Amine (BCMA) and the like can be mentioned.

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-bis (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'-Succinylethylene Diamine (EMSE), N-Ethyl-N- (3-Methylphenyl) -N'-Acetylethylene Diamine, 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, peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as a substrate thereof, and the intensity of fluorescence generated by a fluorometer, a fluorescent multi-well plate reader, or the like is measured. Examples include a measuring method. 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. Examples include a measuring method. 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 [3.3.1.1 ^3.7 ] decane] -4'-yl} phenylphosphate disodium salt (CSPD) ^TM ), 9-[(phenyloxy) (phosphoryloxy) methylidene] -10-methylacridan disodium, 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium (Lumigen ^TM APS-5) and the like.

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-nitrofell-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by the luminescence method, for example, a method of reacting β-D-galactosidase with its substrate and measuring the luminescence of the reaction solution 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] and similar compounds thereof. Can be mentioned. As a method of 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.

When the label is other than a fluorescent substance, a luminescent substance, a radioactive isotope and an enzyme, a labeled substance that specifically binds to the label is labeled with a fluorescent substance, a luminescent substance, a radioactive isotope, an enzyme or the like, and an immune complex. A fluorescent substance, a luminescent substance, a radioactive isotope or an enzyme that binds to the label in 2 or the label in the immune complex 5 and labels the substance that specifically binds to the label is produced by the above method. By measuring, the label can be measured. Examples of the substance that specifically binds to the label include an antibody that specifically binds to the label, and avidins and the like when the label is biotin.

By performing the following steps (4) and (5) after the step (3), the concentration of the oxidized high-density lipoprotein in the sample can be determined.
(4) As a sample, the steps (1) to (3) are carried out using the oxidized high-density lipoprotein having a known concentration, and a calibration curve showing the relationship between the oxidized high-density lipoprotein concentration and the measured value of the label is prepared. Process;
(5) A step of determining the concentration of oxidized high-density lipoprotein in a sample from the calibration curve prepared in step (4) and the measured value of the label measured in step (2).

<Measurement method 2>
The method for measuring oxidized high-density lipoprotein in a sample of the present invention is characterized by including the following steps.
(1') A sample and an antibody or an antibody fragment thereof or an antibody fragment thereof or an antibody fragment thereof that binds to a high-density lipoprotein are selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant. A step of reacting in an aqueous medium containing at least one to generate an immune complex 3 consisting of an oxidized high-density lipoprotein and an antibody or antibody fragment thereof that binds to the high-density lipoprotein;
(2') An antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine is reacted with the immune complex 3 produced in the step (1') in an aqueous medium. An immune complex consisting of an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, an antibody or an antibody fragment thereof that binds to an oxidized high-density lipoprotein and phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine. Step to generate 4;
(3') Step of measuring the immune complex 4 produced in the step (2').

≪Process (1') ≫
In step (1'), a group consisting of an antibody or an antibody fragment thereof or an antibody fragment thereof or an antibody fragment thereof that binds to a high-density lipoprotein is composed of a polyoxyethylene-based surfactant and a cationic surfactant. The method of reacting in an aqueous medium containing at least one selected from the above can generate an immune complex 3 consisting of an oxidized high-density lipoprotein and an antibody or antibody fragment thereof that binds to the high-density lipoprotein. There is no particular limitation as long as it is the method.

The antibody or antibody fragment thereof that binds to the high-density lipoprotein may or may not be immobilized on an insoluble carrier, but is preferably immobilized.
When the antibody that binds to the high-density lipoprotein or the antibody fragment thereof is immobilized on an insoluble carrier, the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized is contained in the reaction solution of the antigen-antibody reaction. In this case, an antibody or an antibody fragment thereof that binds to a high-density lipoprotein to which one of a set of affinity substances (B) is bound, and the other of a combination of a set of affinity substances. By reacting the insoluble carrier to which (b) is bound in the reaction solution of the antigen-antibody reaction, the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized is placed in the reaction solution of the antigen-antibody reaction. Can be generated with.

Examples of the combination of B and b include the following combinations and the like.
・ Combination of biotin and avidins (avidin, neutral avidin, streptavidin, etc.);
・ Combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
-A combination of an antibody that binds to high-density lipoprotein or an Fc region of the antibody fragment and an antibody that binds to the Fc region.

The insoluble carrier is not particularly limited as long as it is an insoluble carrier that immobilizes an antibody or an antibody fragment thereof that binds to high-density lipoprotein and enables the method for measuring oxidized high-density lipoprotein of the present invention. For example, the above-mentioned insoluble carrier is used. Examples include carriers.

The binding between the antibody or the antibody fragment thereof that binds to the high-density lipoprotein and the insoluble carrier is not particularly limited as long as it is a binding that enables the method for measuring the oxidized high-density lipoprotein of the present invention. The method can be mentioned.
The antibody or the antibody fragment thereof that binds to the high-density lipoprotein may be directly immobilized on the insoluble carrier or indirectly immobilized on the insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. good. Examples of the indirect immobilization method include the above-mentioned methods.
Further, the antibody or the antibody fragment thereof that binds to the high-density lipoprotein may be immobilized on the insoluble carrier by covalent bonding via a linker. The linker is not particularly limited as long as it is a molecule capable of covalently binding both a functional group on the surface of an insoluble carrier and an antibody that binds to a high-density lipoprotein or a functional group of the antibody fragment, and for example, the above-mentioned linker and the like can be used. Can be mentioned.
Further, the sample may be mixed in advance with at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and then the obtained mixture may be subjected to an antigen-antibody reaction.

The reaction temperature between the sample and the antibody or the antibody fragment thereof that binds to the high-density lipoprotein in the step (1') is not particularly limited as long as it is a temperature that enables the method for measuring the oxidized high-density lipoprotein of the present invention. Usually, it is 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the antibody that binds to the high-density lipoprotein or the antibody fragment thereof in the reaction solution is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL. It is preferably 0.1 to 20 μg / mL.

≪Process (2') ≫
In step (2'), an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof is added to the immune complex 3 produced in step (1') and reacted. Alternatively, the immune complex 3 produced in step (1') may be added and reacted with the antibody or antibody fragment thereof that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment thereof that binds to phosphocholine.
In step (2'), an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, an antibody or an antibody fragment thereof that binds to an oxidized high-density lipoprotein and phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine. The reaction temperature of the reaction for producing the immune complex 4 comprising the above is not particularly limited as long as it is a temperature that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 0 to 50 ° C. 4 to 45 ° C is preferable, and 20 to 40 ° C is particularly preferable. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the antibody that binds to phosphatidylcholine oxide or the antibody fragment thereof, or the antibody that binds to phosphocholine or the antibody fragment thereof in the reaction solution is particularly high as long as it is a concentration that enables the method for measuring the high-density oxidized lipoprotein of the present invention. There is no limitation, usually 0.01 to 100 μg / mL, preferably 0.1 to 20 μg / mL.

The step (1') and the step (2') may be performed sequentially, but may be performed at the same time.
When step (1') and step (2') are carried out at the same time, an antibody or an antibody fragment thereof that binds the oxidized high-density lipoprotein in the sample to the high-density lipoprotein, and an antibody that binds to phosphatidylcholine oxide or React with the antibody fragment or an antibody that binds to phosphocholine or the antibody fragment. Even when the step (1') and the step (2') are performed at the same time, as described above, the antibody or the antibody fragment thereof that binds to the high-density lipoprotein is immobilized on the insoluble carrier, but is not immobilized. Also, immobilization of an antibody or an antibody fragment thereof that binds to high-density lipoprotein on an insoluble carrier can be performed by the above-mentioned method.

Further, a step of washing the insoluble carrier after the reaction of the step (1') may be added between the steps (1') and the step (2'), if necessary. The cleaning solution used for cleaning the insoluble carrier after the reaction in step (1') is not particularly limited as long as it is a cleaning solution that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is, for example, the above-mentioned cleaning solution. And so on.
The above-mentioned cleaning step may be added after the step (2'). The cleaning solution used for cleaning the insoluble carrier after the step (2') is not particularly limited as long as it is a cleaning solution that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include the above-mentioned cleaning solution. ..

≪Process (3') ≫
In step (3'), the concentration of oxidized high-density lipoprotein in the sample is determined by measuring the amount of immune complex 4 produced in step (2') using the following method.

(I') When the antibody or antibody fragment that binds to phosphatidylcholine oxide, or the antibody or antibody fragment that binds to phosphocholine is not labeled, the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide or phosphocholine is bound. An antibody that binds to an antibody or the antibody fragment (hereinafter referred to as a fourth antibody) or a labeled fourth antibody or the antibody fragment to which a label is bound to the antibody fragment is bound to the immune complex 4 (high density lipoprotein). Binds to oxidized phosphatidylcholine in an immune complex consisting of an antibody or the antibody fragment, an oxidized high density lipoprotein, an antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or an antibody that binds to phosphocholine or the antibody fragment. An antibody or antibody fragment that binds to high-density lipoprotein by reacting with an antibody or antibody fragment that binds to phosphocholine, an oxidized high-density lipoprotein, and an antibody that binds to oxidized phosphatidylcholine. Alternatively, an immune complex 6 composed of the antibody fragment, an antibody that binds to phosphocholine or the antibody fragment, and a labeled fourth antibody or the antibody fragment is formed, and the labeling in the immune complex is designated by the method described below. By measuring with the above, the amount of the immune complex 4 produced in the step (2') can be measured. Examples of the fourth antibody include an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, an antibody that binds to phosphocholine, an antibody that binds to the Fc region of the antibody fragment, or a fragment thereof.
The reaction temperature of the reaction between the labeled fourth antibody or the antibody fragment and the antibody or the antibody fragment that binds to oxidized phosphatidylcholine in the immune complex 4 or the antibody or the antibody fragment that binds to phosphocholine is the present invention. The temperature is not particularly limited as long as it enables a method for measuring an oxidized high-density lipoprotein, and is usually 0 to 50 ° C, preferably 4 to 45 ° C, and particularly preferably 20 to 40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, preferably 30 minutes. Minutes to 2 hours are particularly preferred. The concentration of the labeled fourth antibody or the antibody fragment in the reaction solution is not particularly limited as long as it is a concentration that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL. It is preferably 0.1 to 20 μg / mL.

(Ii') When an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine is labeled, by measuring the label in the immune complex 4, step (2'). ), The amount of the immune complex 4 can be measured.
Examples of the label include the above-mentioned labeling substance and the like, and examples of the measurement of the label include the above-mentioned measurement method and the like.
When the label is other than a fluorescent substance, a luminescent substance, a radioactive isotope, or an enzyme, a labeled substance that specifically binds to the label is labeled with a fluorescent substance, a luminescent substance, a radioactive isotope, an enzyme, or the like, and an immune complex. A fluorescent substance, a luminescent substance, a radioactive isotope or an enzyme that binds to the label in 4 or the label in the immune complex 6 and labels the substance that specifically binds to the label is produced by the above method. By measuring, the label can be measured. Examples of the substance that specifically binds to the label include an antibody that specifically binds to the label, and avidins and the like when the label is biotin.

By performing the following steps (4') and (5') after the step (3'), the concentration of the oxidized high-density lipoprotein in the sample can be determined.
(4') Perform steps (1') to (3') using a known concentration of oxidized high-density lipoprotein as a sample, and a calibration curve showing the relationship between the oxidized high-density lipoprotein concentration and the measured value of the label. Process to create;
(5') A step of determining the concentration of oxidized high-density lipoprotein in a sample from the calibration curve prepared in step (4') and the measured value of the label measured in step (2').

The oxidized high-density lipoprotein in the present invention is a high-density lipoprotein having apoA protein, which is an apoprotein constituting the high-density lipoprotein, and exhibiting dysfunction due to oxidation of the lipid portion constituting the high-density lipoprotein. Say.

The sample in the present invention is not particularly limited as long as it is a sample that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include biological samples. Examples of the biological sample include whole blood, plasma, serum, urine, spinal fluid, saliva, sheep's water, urine, sweat, pancreatic fluid and the like, and whole blood, plasma, serum, urine and the like are preferable.

The antibody that binds to oxidized phosphatidylcholine in the present invention is not particularly limited as long as it binds to the oxidized high-density lipoprotein and enables the method for measuring the oxidized high-density lipoprotein of the present invention. Both can be used. Examples of the antibody that binds to oxidized phosphatidylcholine in the present invention include a monoclonal antibody (hereinafter referred to as DLH3 antibody) produced by mouse-mouse hybridoma cell line FOH1a / DLH3 (FERM BP-7171).

The antibody that binds to phosphocholine in the present invention is not particularly limited as long as it binds to the oxidized high-density lipoprotein and enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is either a polyclonal antibody or a monoclonal antibody. Can also be used. Examples of the antibody that binds to phosphocholine in the present invention include the monoclonal antibody KTM-285 produced by the T-15 antibody [J.Exp.Med., 132,737 (1970)] and the hybridoma KTM-285 (FERM BP-7589). , KTM-2001, a recombinant antibody produced by the transformed cell KTM-2001 (FERM BP-7549), and the like.

The antibody fragment of the antibody that binds to oxidized phosphatidylcholine in the present invention or the antibody fragment of the antibody that binds to phosphocholine binds to the oxidized high-density lipoprotein and enables the method for measuring the oxidized high-density lipoprotein of the present invention. The fragment is not particularly limited, and for example, Fc moieties such as Fab obtained by treating the antibody with papain, F (ab') ₂ obtained by pepsin treatment, and Fab' obtained by pepsin treatment-reduction treatment were removed. Examples thereof include antibody fragments, antibody fragments from which the Fc portion has been removed by a genetic engineering method, and the like.

The antibody that binds to the high-density lipoprotein in the present invention is not particularly limited as long as it binds to the high-density lipoprotein and enables the method for measuring the oxidized high-density lipoprotein of the present invention. Any of these can be used.

As the antibody that binds to the high-density lipoprotein in the present invention, an antibody that binds to the apoA protein is preferable, and an antibody against the apoAI protein is more preferable. Examples of such an antibody include an anti-Apo AI protein monoclonal antibody [manufactured by Abnova] and the like.
The antibody fragment of the antibody that binds to the high-density lipoprotein in the present invention is not particularly limited as long as it is an antibody fragment that binds to the high-density lipoprotein and enables the method for measuring the oxidized high-density lipoprotein of the present invention. , The above-mentioned antibody fragment and the like.

The polyoxyethylene-based surfactant in the present invention is not particularly limited as long as it is a polyoxyethylene-based surfactant that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is, for example, a polyoxyethylene sorbitan fatty acid ester. , Polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycycle Examples thereof include phenyl ether sulfate ester or a salt thereof, and polyoxyethylene fatty acid glyceryl.

Examples of the fatty acid in the polyoxyethylene sorbitan fatty acid ester include saturated or unsaturated fatty acids having 8 to 24 carbon atoms, and saturated or unsaturated fatty acids having 12 to 18 carbon atoms are preferable. Saturated or unsaturated fatty acids having 8 to 24 carbon atoms include, for example, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, and the like. Examples thereof include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanoic acid, docosahexaenoic acid, tetradocosanic acid, tetracosapentaenoic acid and the like. Examples of saturated or unsaturated fatty acids having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, and polyoxyethylene sorbitan monopalmitate. Commercially available products of polyoxyethylene sorbitan fatty acid ester include, for example, Tween 20 (polyoxyethylene sorbitan monolaurate; manufactured by Sigma-Aldrich), Tween 40 (polyoxyethylene sorbitan monopalmitate; manufactured by Wako Pure Chemical Industries, Ltd.), and the like. Tween 60 (polyoxyethylene sorbitan monostearate; manufactured by Sigma-Aldrich), Tween 80 (polyoxyethylene sorbitan monooleate; manufactured by Wako Pure Chemical Industries, Ltd.), Leodor TW-L106 (polyoxyethylene sorbitan monolaurate; Kao), Leodor TW-L120 (Polyoxyethylene sorbitan monolaurate; Kao), Leodor TW-O106V (Polyoxyethylene sorbitan monooleate; Kao), Leodor TW-O120V (Polyoxyethylene sorbitan) Monooleate; manufactured by Kao, Leodor TW-P120 (polyoxyethylene sorbitan monopalmitate; manufactured by Kao), Leodor TW-S106V (polyoxyethylene sorbitan monostearate; manufactured by Kao), Leodor TW-S120V (polyoxyethylene sorbitan monostearate; manufactured by Kao) Polyoxyethylene sorbitan monostearate; manufactured by Kao Co., Ltd.), BLAUNON OT-106 (polyoxyethylene sorbitan monooleate; manufactured by Aoki Oil & Fat Co., Ltd.), BLAUNON OT-21 (polyoxyethylene sorbitan monooleate; manufactured by Aoki Oil & Fat Co., Ltd.) , Newcol 25 (Polyoxyethylene sorbitan laurate; manufactured by Nippon Emulsifier), Newcol 65 (Polyoxyethylene sorbitan stearate; manufactured by Japan Embroidery), Newcol 85 (Polyoxyethylene sorbitan oleate; manufactured by Japan Embroidery) ) Etc. can be mentioned.

Examples of the fatty acid in the polyoxyethylene fatty acid ester include saturated or unsaturated fatty acids having 8 to 24 carbon atoms, and saturated or unsaturated fatty acids having 12 to 18 carbon atoms are preferable. Saturated or unsaturated fatty acids having 8 to 24 carbon atoms include, for example, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, and the like. Examples thereof include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanoic acid, docosahexaenoic acid, tetradocosanic acid, tetracosapentaenoic acid and the like. Examples of saturated or unsaturated fatty acids having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Examples of the polyoxyethylene fatty acid ester include polyoxyethylene monolaurate, polyoxyethylene monostearate, polyoxyethylene monooleate, and polyoxyethylene distearate. Commercially available products of polyoxyethylene fatty acid ester include, for example, Emanone 1112 (polyoxyethylene monolaurate; manufactured by Kao Corporation), Emanone 4110 (polyoxyethylene monooleate; manufactured by Kao Corporation), and Emanone 3199V (polyoxyethylene monostete). Aleate; manufactured by Kao Corporation, Emanon 3299V (polyoxyethylene distea Aleate; manufactured by Kao Corporation), BLUINON L-400 (polyoxyethylene monolaurate; manufactured by Aoki Oil & Fat Co., Ltd.), BLAUNON S-300A (polyoxyethylene mono) Stearate; manufactured by Aoki Oil & Fat Co., Ltd.), BLAUNON O-600SA (polyoxyethylene monooleate; manufactured by Aoki Oil & Fat Co., Ltd.) and the like.

Examples of the alkyl in the polyoxyethylene alkyl phenyl ether include an alkyl having 8 to 9 carbon atoms. Examples of the alkyl having 8 to 9 carbon atoms include octyl and nonyl. Examples of the polyoxyethylene alkyl phenyl ether include polyoxyethylene octylphenyl ether and polyoxyethylene nonylphenyl ether. Commercially available products of polyoxyethylene alkyl phenyl ether include, for example, Triton X-100 (polyoxyethylene octylphenyl ether; manufactured by Sigma-Aldrich), BLAUNON NK-810 (polyoxyethylene octylphenyl ether; manufactured by Aoki Oil & Fat Co., Ltd.) and the like. Can be mentioned.

Examples of the alkyl in the polyoxyethylene alkylamine include an alkyl having 8 to 24 carbon atoms, and an alkyl having 10 to 20 carbon atoms is preferable. Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl. , Nonadecylic, Icosyl, Heneikosyl, Docosyl (Behenil), Tridecylic, Tetracosyl and the like. Examples of alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned. Commercially available products of polyoxyethylene alkylamine include, for example, Nymine L-201 (oxyethylene dodecylamine; manufactured by Nichiyu Co., Ltd.), Nymeen L-202, Nymeen L-207, and Nymeen L-215 (above, polyoxyethylene dodecylamine). (Nichiyu Co., Ltd.), Nymeen S-202, Nymeen S-204, Nymeen S-210, Nymeen S-215, Nymeen S-220 (above, polyoxyethylene octadecylamine; Nymeen T2-202), Nymeen T2-202 , Nymeen T2-210, Nymeen T2-230 [above, polyoxyethylene alkyl (beef fat) amine; manufactured by Nichiyu Co., Ltd.], Nymeen F-202, Nymeen F-203, Nymeen F-205, Nymeen F-210, Nymeen F -215 [above, polyoxyethylene alkyl (palm oil) amine; manufactured by Nichiyu Co., Ltd.], Brownon L-202, Brownon L-205, Brownon L-207, Brownon L-210, Brownon L-230 (above, polyoxy) Ethylenedodecylamine; manufactured by Aoki Oil & Fat Co., Ltd.), Brownon S-207, Brownon S-210, Brownon S-215, Brownon S-220, Brownon S-230 (above, polyoxyethylene octadecylamine; manufactured by Aoki Oil & Fat Co., Ltd.), Brownon S-205T, Brownon S-208T, Brownon S-210T, Brownon S-215T, Brownon S-230T [above, polyoxyethylene alkyl (beef) amine; manufactured by Aoki Oil & Fat Co., Ltd.], Neucor OD420 (polyoxyethylene octadecylamine) Pionin D3104 (polyoxyethylene dodecylamine; manufactured by Takemoto Yushi), Pionin D3110 (polyoxyethylene dodecylamine; manufactured by Takemoto Yushi), Pionin D3605 [polyoxyethylene alkyl (soy) amine; Takemoto Oil and fat company], Pionin D3615T [polyoxyethylene alkyl (beef fat) amine; Takemoto oil and fat company], BLAUNON O209 (polyoxyethylene oleylamine; Aoki oil and fat company) and the like.

Examples of the alkyl in the polyoxyethylene alkyl ether include alkyl having 8 to 24 carbon atoms, and alkyl having 10 to 20 carbon atoms is preferable. Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl. , Nonadecylic, Icosyl, Heneikosyl, Docosyl (Behenyl), Tridecylic, Tetracosyl and the like. Examples of alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned. Commercially available products of polyoxyethylene alkyl ether include, for example, Nonion K-204, Nonion K-220 (above, polyoxyethylene lauryl ether; manufactured by Nichiyu Co., Ltd.), Nonion E-205, Nonion E-215 (above, polyoxy). Ethylene oleyl ether; manufactured by Nichiyu Co., Ltd.), Nonion S-215, Nonion S-225 (above, polyoxyethylene stearyl ether; manufactured by Nichiyu Co., Ltd.), Emargen 108, Emargen 120 (above, polyoxyethylene lauryl ether; Kao) Emargen 220 (polyoxyethylene cetyl ether; manufactured by Kao), Emargen 320P (polyoxyethylene stearyl ether; manufactured by Kao), Emargen 420 (polyoxyethylene oleyl ether; manufactured by Kao), Adecator LA-875, Adecator LA-975 (polyoxyethylene lauryl ether; manufactured by ADEKA), Adecator OA-7 (polyoxyethylene oleyl ether; manufactured by ADEKA), Emalmin NL-70, Emalmin NL-90, Emalmin NL-100, Emalmin NL-110, Emulmin LS-80, Emulmin LS-90, Emulmin L-380 (above, polyoxyethylene lauryl ether; manufactured by Sanyo Kasei Kogyo Co., Ltd.), BLAUNON EL-1507, BLAUNON EL-1509, BLAUNON EL-1512P, BLAUNON EL-1515, BLAUNON EL-1521 (above, polyoxyethylene lauryl ether; manufactured by Aoki Yushi), BLAUNON CH-310, BLAUNON CH-310L, BLAUNON CH-313 (above, polyoxyethylene cetyl ether; manufactured by Aoki Yushi) ), BLAUNON SR-711 (polyoxyethylene stearyl ether; manufactured by Aoki Oil & Fat Co., Ltd.), BLAUNON EN-1511 (polyoxyethylene oleyl ether; manufactured by Aoki Oil & Fat Co., Ltd.), Neugen SD-60, Neugen SD-70, Neugen SD-80. , Neugen SD-110, Neugen SD-150 (above, polyoxyethylene isodecyl ether; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Neugen TDS-80, Neugen TDS-100, Neugen TDS-200D (above, polyoxyethylene tridecyl). Ether; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and the like.

Examples of the alkyl in the polyoxyethylene alkyl ether phosphoric acid or a salt thereof include alkyl having 8 to 24 carbon atoms, and alkyl having 10 to 20 carbon atoms is preferable. Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl. , Nonadecylic, Icosyl, Heneikosyl, Docosyl (Behenil), Tridecylic, Tetracosyl and the like. Examples of alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned. Examples of the salt include lithium salt, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, monoethanolamine salt and the like. Examples of the polyoxyethylene alkyl ether phosphoric acid or a salt thereof include polyoxyethylene lauryl ether phosphoric acid sodium salts and the like. Commercially available products of polyoxyethylene alkyl ether phosphoric acid or salts thereof include, for example, Plysurf A212C, Plysurf A215C (hereinafter, polyoxyethylene tridecyl ether phosphoric acid; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Plysurf A208F (Polyoxy). Ethylene Octyl Ether Phosphoric Acid; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Plysurf A219B (Polyoxyethylene Lauryl Ether Phosphoric Acid; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Plysurf DB-01 (Polyoxyethylene Lauryl Ether Phosphoric Acid / Monoethanol) Amin salt; manufactured by Daiichi Kogyo Seiyaku Co., Ltd., NIKKOL DLP-10, NIKKOL TLP-4 (above, sodium polyoxyethylene lauryl ether phosphate; manufactured by Nikko Chemicals), NIKKOL TCP-5 (polyoxyethylene cetyl ether phosphoric acid). Sodium; manufactured by Nikko Chemicals Co., Ltd.), NIKKOL DOP-6NV (polyoxyethylene oleyl ether phosphate sodium; manufactured by Nikko Chemicals Co., Ltd.) and the like.

The polycyclic phenyl in the polyoxyethylene polycyclic phenyl ether is a phenyl group in which two or more groups having one aromatic ring (substituent) are substituted in the group, and a group having two or more aromatic rings in the group (substituent). Examples thereof include a phenyl group in which one or more substituents) are substituted. Examples of the group having one aromatic ring in the group include benzyl, 1- (phenyl) ethyl and the like. Examples of the group having two or more aromatic rings in the group include naphthyl and the like. Examples of commercially available products of polyoxyethylene polycyclic phenyl ether include Newcol 704, Newcol 706, Newcol 707, Newcol 708, Newcol 709, Newcol 710, Newcol 711, Newcol 712, and Newcol 714. Examples thereof include Newcol 610, Newcol 2607, Newcol 2609, and Newcol 2614 (all manufactured by Nippon Emulsifier Co., Ltd.).

Polyoxyethylene polycyclic phenyl ether Sulfate ester or a polycyclic phenyl in a salt thereof is a phenyl group in which two or more groups having one aromatic ring (substituent) are substituted in the group, and two or more aromatics in the group. Examples thereof include a phenyl group in which one or more groups having a ring (substituent) are substituted. Examples of the group having one aromatic ring in the group include benzyl, 1- (phenyl) ethyl and the like. Examples of the group having two or more aromatic rings in the group include naphthyl and the like. Examples of the salt include lithium salt, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, monoethanolamine salt and the like. Commercially available products of polyoxyethylene polycyclic phenyl ether sulfate ester or salts thereof include, for example, Newcol 707-SF, Newcol 707-SFC, Newcol 707-SN, Newcol 714-SF, and Newcol 714-SN (above). , Made by Nippon Ester Co., Ltd.) and the like.

Examples of the fatty acid in the polyoxyethylene fatty acid glyceryl include saturated or unsaturated fatty acids having 8 to 24 carbon atoms, and saturated or unsaturated fatty acids having 12 to 18 carbon atoms are preferable. Saturated or unsaturated fatty acids having 8 to 24 carbon atoms include, for example, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, and the like. Examples thereof include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanoic acid, docosahexaenoic acid, tetradocosanic acid, tetracosapentaenoic acid, coconut oil fatty acid and the like. Examples of saturated or unsaturated fatty acids having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and coconut oil fatty acids. Examples of the polyoxyethylene fatty acid glyceryl include polyoxyethylene palm oil fatty acid glyceryl, polyoxyethylene laurate glyceryl, and polyoxyethylene oleate glyceryl. Commercially available products of polyoxyethylene fatty acid glyceryl include, for example, Unigri MK-207, Unigri MK-230, Unigri MK-278 (above, polyoxyethylene palm oil fatty acid glyceryl; manufactured by Nichiyu Co., Ltd.), Unigri ML-212, Unigli ML. -220 (above, glyceryl polyoxyethylene laurate; manufactured by Nichiyu Co., Ltd.), Unigli MO-220, Unigli MO-230 (above, glyceryl polyoxyethylene oleate; manufactured by Nichiyu Co., Ltd.) and the like can be mentioned.

The cationic surfactant in the present invention is not particularly limited as long as it is a cationic surfactant that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include quaternary ammonium salts. Be done. Examples of the quaternary ammonium salt include a quaternary ammonium salt represented by the following general formula (I).

(In the formula, R ¹ represents an alkyl having 8 to 24 carbon atoms, R ² represents an alkyl having 1 to 24 carbon atoms, and R ³ and R ⁴ represent the same or different alkyl having 1 to 6 carbon atoms, respectively. Represents the alkyl of, and X represents the anion)

Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl. , Nonadecylic, Icosyl, Heneikosyl, Docosyl (Behenil), Tridecylic, Tetracosyl and the like.

Examples of alkyl having 1 to 24 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, isooctyl, nonadecylic, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), and pentadecyl. , Hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, icosyl, heneicosyl, docosyl (behenyl), tridecylic, tetracosyl and the like.
Examples of the alkyl having 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl and the like.

Examples of anions include hydroxide ions, halogen ions, anions derived from inorganic acids, anions derived from organic acids, and the like.
Examples of the halogen ion include fluorine ion, chlorine ion, bromine ion, iodine ion and the like.
Examples of the anion ion derived from an inorganic acid include nitrate ion, sulfate ion, phosphate ion, carbonate ion, bicarbonate ion and the like.
Examples of anions derived from organic acids include carboxylic acid ions such as formic acid ion, acetate ion, lactic acid ion, citrate ion and glutamate ion.

Commercially available products of the quaternary ammonium salt include, for example, cation AB (octadecyltrimethylammonium chloride; manufactured by Nichiyu Co., Ltd.), cation BB (dodecyltrimethylammonium chloride; manufactured by Nichiyu Co., Ltd.), and cation 2ABT (distealyldimethylammonium chloride; Cation 2DB-500E (didecyldimethylammonium chloride; manufactured by Nichiyu Co., Ltd.), cation 2-OLR (diorail dimethylammonium chloride; manufactured by Nichiyu Co., Ltd.) and the like.

In the method for measuring an oxidized high-density lipoprotein of the present invention, a surfactant selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant may be used in combination of two or more. good.

The concentration of the polyoxyethylene-based surfactant or the cationic surfactant in the reaction solution in the present invention is not particularly limited as long as it is a concentration that enables the method for measuring the oxidized high-density lipoprotein of the present invention. For example, it is 0.0001 to 2.0% (w / v), preferably 0.001 to 0.1% (w / v), and particularly preferably 0.005 to 0.05% (w / v).
In the present invention, oxidized HDL can be measured with higher sensitivity by further using protein and / or polyethylene glycol.
The protein in the present invention is not particularly limited as long as it is a protein that enables the method for measuring the oxidized high-density lipoprotein of the present invention, for example, albumin, fetal bovine serum (FBS), casein, block ace (DS Pharma Biomedical). (Manufactured by the company) and the like, and albumin is preferable. Examples of albumin include bovine serum albumin (BSA) and the like.
The concentration of the protein in the reaction solution in the present invention is not particularly limited as long as it is a concentration that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is, for example, 0.01 to 10.0% (w / v), 0.1. ~ 2.0% (w / v) is preferable.
The polyethylene glycol in the present invention is not particularly limited as long as it is a polyethylene glycol capable of measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include polyethylene glycol having an average molecular weight of 100 to 25,000 and an average molecular weight of 200. ~ 20,000 polyethylene glycol is preferred.
The concentration of polyethylene glycol in the reaction solution in the present invention is not particularly limited as long as it is a concentration that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is, for example, 0.5 to 15.0% (w / v). It is preferably 1.0 to 10.0% (w / v).

The aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium capable of measuring the oxidized high-density lipoprotein of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution. Is preferable. The pH of the aqueous medium is, for example, 4 to 10. When a buffer solution is used as the aqueous medium, it is preferable to use a buffer solution 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, oxalate buffer, glycine buffer, borate buffer, carbon dioxide buffer, Good buffers and the like can be mentioned.

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, [N -Tris (hydroxymethyl) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-Tris (hydroxymethyl) methyl] aminopropanesulfon Acid (TAPS) buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-) Cyclohexylamino) propanesulfonic acid (CAPS) buffers and the like can be mentioned.

The aqueous medium may contain metal ions, sugars, preservatives, protein stabilizers and the like. Examples of the metal ion include magnesium ion, manganese ion, zinc ion 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 stabilizer include a peroxidase stabilizing buffer [Peroxidase Stabilizing Buffer, manufactured by DakoCytomation] and the like.

2. 2. Measuring Reagent The measuring reagent for the oxidized high-density lipoprotein of the present invention is a reagent used in the method for measuring the oxidized high-density lipoprotein of the present invention, and binds to an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or phosphocholine. A reagent containing an antibody or the antibody fragment, an antibody that binds to a high-density lipoprotein or the antibody fragment, and at least one selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants. be. The reagent of the present invention may further contain at least one substance selected from the group consisting of protein and polyethylene glycol.

The antibody or antibody fragment thereof that binds to phosphatidylcholine oxide in the measuring reagent of the present invention, the antibody or antibody fragment thereof that binds to phosphocholine, the antibody that binds to high-density lipoprotein or the antibody fragment thereof, for example, binds to the above-mentioned phosphatidylcholine oxide. Examples thereof include an antibody or an antibody fragment thereof, an antibody or an antibody fragment thereof that binds to phosphocholine, an antibody that binds to a high-density lipoprotein, or an antibody fragment thereof. The content of the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, the antibody or antibody fragment thereof that binds to phosphocholine, the antibody that binds to high-density lipoprotein, or the antibody fragment thereof in the reagent for measurement of the present invention is high in oxidation of the present invention. The content is not particularly limited as long as it enables a method for measuring the density lipoprotein, and is usually 0.01 to 100 μg / mL in the above-mentioned aqueous medium or in the state of being dissolved in the above-mentioned aqueous medium, and 0.1 to 20 μg. / mL is preferred.

When the measurement reagent of the present invention is used in the above-mentioned measurement method 1, the antibody or antibody fragment thereof that binds to phosphatidyloxide oxide, or the antibody or antibody fragment thereof that binds to phosphocholine is immobilized on an insoluble carrier. It is preferable to have it.
Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.
When the measuring reagent of the present invention is used in the above-mentioned measuring method 1, an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody that binds to phosphocholine or an insoluble carrier on which the antibody fragment is immobilized is immobilized. May be produced in the reaction solution of the reaction between the sample and the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the antibody or antibody fragment thereof that binds to phosphocholine.

In this case, the measuring reagent of the present invention is an oxidized phosphatidylcholine to which one (A) of a set of affinity substances is bound instead of an antibody that binds to phosphocholine or an insoluble carrier on which the antibody fragment is immobilized. Included is an antibody or fragment thereof that binds, or an antibody that binds to phosphocholine or the antibody fragment thereof, and an insoluble carrier to which the other (a) of a combination of a set of affinity substances is bound. Examples of the combination of a set of affinity substances, that is, the combination of A and a include the above-mentioned combinations and the like.

When the measuring reagent of the present invention is used in the above-mentioned measuring method 2, it is preferable that the antibody or the antibody fragment thereof that binds to the high-density lipoprotein is immobilized on an insoluble carrier.
Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.
When the measuring reagent of the present invention is used in the above-mentioned measuring method 2, the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized is used as a sample and the high-density lipoprotein. It may be produced in a reaction solution for reaction with an antibody to be bound or an antibody fragment thereof.

In this case, one (B) of a set of affinity substances was bound to the measurement reagent of the present invention instead of the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment was immobilized. An antibody or fragment thereof that binds to a high-density lipoprotein and an insoluble carrier to which the other (b) of a combination of a set of affinity substances is bound are included. Examples of the combination of a set of affinity substances, that is, the combination of B and b include the above-mentioned combinations and the like.

The measuring reagent of the present invention may be freeze-dried or liquid. When a freeze-dried measurement reagent is used, it is dissolved in an aqueous medium to make it liquid before measurement, and the measurement is performed.
In the liquid measurement reagent, an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, an antibody or an antibody fragment thereof that binds to phosphocholine, an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, and a polyoxyethylene-based surfactant. At least one selected from the group consisting of an activator and a cationic surfactant is in a state of being dissolved in an aqueous medium.
Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, protein stabilizers and the like.

Examples of the polyoxyethylene-based surfactant and the cationic surfactant in the measuring reagent of the present invention include the above-mentioned polyoxyethylene-based surfactant and the cationic surfactant, respectively.
The content of each of the polyoxyethylene-based surfactant and the cationic surfactant in the measuring reagent of the present invention may be any content that enables the method for measuring the oxidized high-density lipoprotein of the present invention. However, there is no particular limitation, and the content is usually 0.0001 to 2.0% (w / v) in the above-mentioned aqueous medium or in the state of being dissolved in the above-mentioned aqueous medium, and 0.001 to 0.1% (w / v). The content is preferably 0.005 to 0.05% (w / v), and particularly preferably 0.005 to 0.05% (w / v).

In the measuring reagent of the present invention, two or more kinds of surfactants selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants may be used in combination.
Examples of the protein in the measuring reagent of the present invention include the above-mentioned proteins. The content of the protein in the measuring reagent of the present invention is not particularly limited as long as it is a content that enables the method for measuring the high-density oxidized lipoprotein of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned aqueous medium. The content is 0.01 to 10.0% (w / v) in the state of being dissolved in, and preferably 0.1 to 2.0% (w / v).
Examples of the polyethylene glycol in the measuring reagent of the present invention include the above-mentioned polyethylene glycol. The content of polyethylene glycol in the measuring reagent of the present invention is not particularly limited as long as it is a content that enables the method for measuring the high-density oxidized lipoprotein of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned aqueous solution. The content is 0.5 to 15.0% (w / v) when dissolved in a medium, and the content is preferably 1.0 to 10.0% (w / v).

3. 3. Measurement kit The measurement reagent for the oxidized high-density lipoprotein of the present invention can be taken in the form of a kit from the viewpoints of storage, transportation, distribution and the like. The measurement kit of the present invention is used in the method for measuring the oxidized high-density lipoprotein of the present invention.

<Measurement kit 1>
The kit for measuring the high-density oxidized lipoprotein of the present invention comprises an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine, and a polyoxyethylene surfactant and cationic surfactant. A kit comprising a first reagent comprising at least one selected from the group consisting of agents, and a second reagent comprising an antibody or antibody fragment thereof that binds to a high density lipoprotein.
In the kit, at least one substance selected from the group consisting of protein and polyethylene glycol may be contained in at least one reagent of the first reagent and the second reagent.

The measurement kit 1 of the present invention is a kit used for the measurement method 1 of the present invention. That is, the method for measuring oxidized high-density lipoprotein using the measurement kit 1 of the present invention is a method including the following steps.
(1) The sample and the first reagent of the measurement kit 1 are reacted in an aqueous medium to react the oxidized high-density lipoprotein with an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody thereof that binds to phosphocholine. A step of producing an immune complex 1 consisting of a fragment;
(2) The second reagent of the measurement kit 1 is reacted with the immune complex 1 produced in the above step (1) in an aqueous medium to bind to an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or phosphocholine. A step of producing an immune complex 2 comprising an antibody or the antibody fragment, an oxidized high-density lipoprotein, and an antibody or the antibody fragment that binds to the high-density lipoprotein;
(3) Step of measuring the immune complex 2 produced in the step (2) In the measurement kit 1 of the present invention, the antibody or the antibody fragment thereof that binds to phosphatidyloxide oxide, or the antibody or the antibody fragment thereof that binds to phosphocholine is used. , It is preferable that it is immobilized on an insoluble carrier. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

The antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the antibody that binds to phosphocholine or the insoluble carrier on which the antibody fragment thereof is immobilized, is a sample and an antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or phosphocholine. It may be produced in the reaction solution of the reaction with the antibody or the antibody fragment thereof that binds to. In this case, instead of the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the insoluble carrier in which the antibody or antibody fragment that binds to phosphocholine is immobilized, one of a combination of affinity substances (A) is used. A bound antibody or antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody that binds to phosphocholine or the antibody fragment thereof and an insoluble carrier to which the other (a) of a combination of a pair of affinity substances is bound are used. Examples of the combination of a set of affinity substances, that is, the combination of A and a include the above-mentioned combinations and the like.

In the measurement kit of the present invention, a set of an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine, to which one (A) of a combination of a pair of affinity substances is bound. The insoluble carrier to which the other (a) of the combination of the affinity substances of the above is bound may be contained in the same reagent or may be contained in different reagents, but is preferably contained in different reagents. An antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine to which one (A) of a combination of a set of affinity substances is bound, or an antibody or an antibody fragment thereof that binds to phosphocholine and a combination of a set of affinity substances. When the insoluble carrier to which the other (a) is bound is contained in a separate reagent, the following kit is also included in the measurement kit of the present invention.

An antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine, to which one (A) of a combination of a pair of affinity substances is bound, and a polyoxyethylene-based surfactant and a positive. A first reagent containing at least one selected from the group consisting of ionic surfactants, a second reagent containing an antibody or antibody fragment thereof that binds to a high-density lipoprotein, and a set of affinity substances. An oxidized high-density lipoprotein measuring kit containing a third reagent containing an insoluble carrier to which the other (a) of the combination of the above is bound.

An antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide to which one (A) of a combination of a pair of affinity substances is bound, or an antibody or an antibody fragment thereof that binds to phosphocholine, and a polyoxyethylene-based surfactant and a positive reagent. A first reagent containing at least one selected from the group consisting of ionic surfactants, a second reagent containing an antibody or antibody fragment thereof that binds to a high-density lipoprotein, and a set of affinity substances. A third reagent containing an insoluble carrier to which the other (a) of the combination is bound is further contained, and at least one substance selected from the group consisting of a protein and polyethylene glycol is added to at least one of the first to third reagents. Included, oxidized high density lipoprotein measurement kit.

<Measurement kit 2>
Further, the measurement kit for the oxidized high-density lipoprotein of the present invention is at least selected from the group consisting of an antibody or an antibody fragment thereof that binds to the high-density lipoprotein, a polyoxyethylene-based surfactant, and a cationic surfactant. It is a kit containing a first reagent containing one kind and a second reagent containing an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or an antibody or an antibody fragment thereof that binds to phosphocholine.
In the kit, at least one substance selected from the group consisting of protein and polyethylene glycol may be contained in at least one reagent of the first reagent and the second reagent.

The measurement kit 2 of the present invention is a kit used for the measurement method 2 of the present invention. That is, the method for measuring oxidized high-density lipoprotein using the measurement kit 2 of the present invention is a method including the following steps.
(1') An immune complex 3 composed of an oxidized high-density lipoprotein and an antibody or an antibody fragment thereof that binds to the high-density lipoprotein by reacting the sample with the first reagent of the measurement kit 2 in an aqueous medium. Process to generate;
(2') The second reagent of the measurement kit 2 is reacted with the immune complex 3 produced in the step (1') in an aqueous medium to oxidize with an antibody or an antibody fragment thereof that binds to a high-density lipoprotein. A step of producing an immune complex 4 consisting of a high-density lipoprotein and an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine;
(3') Step of measuring the immune complex 4 produced in the step (2') In the measurement kit 2 of the present invention, the antibody or the antibody fragment thereof that binds to the high-density lipoprotein is immobilized on an insoluble carrier. It is preferable to have. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.

Even if the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized is produced in the reaction solution of the reaction between the sample and the antibody that binds to the high-density lipoprotein or the antibody fragment. good. In this case, instead of the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized, one (B) of the combination of the affinity substances binds to the high-density lipoprotein. An insoluble carrier to which the antibody or the antibody fragment thereof is bound to the other (b) of a combination of a set of affinity substances is used. Examples of the combination of a set of affinity substances, that is, the combination of B and b include the above-mentioned combinations and the like.

In the measurement kit of the present invention, an antibody or an antibody fragment thereof that binds to a high-density lipoprotein to which one (B) of a combination of a set of affinity substances is bound, and the other (b) of a combination of a set of affinity substances. ) May be contained in the same reagent or in different reagents, but it is preferably contained in different reagents. An antibody or antibody fragment thereof bound to a high-density lipoprotein to which one (B) of a set of affinity substances is bound, and an insoluble carrier to which the other (b) of a combination of a pair of affinity substances is bound. The following kits are also included in the measurement kits of the invention when they are included in separate reagents.

Selected from the group consisting of an antibody or an antibody fragment thereof bound to a high-density lipoprotein to which one (B) of a combination of a pair of affinity substances is bound, and a polyoxyethylene-based surfactant and a cationic surfactant. A first reagent containing at least one of these, an antibody that binds to phosphatidylcholine oxide or a fragment thereof, or an antibody that binds to phosphocholine or a second reagent containing the antibody fragment, and a set of affinity substances. An oxidized high density lipoprotein measuring kit comprising a third reagent containing an insoluble carrier to which the other (b) of the combination is bound.

Selected from the group consisting of an antibody or an antibody fragment thereof bound to a high-density lipoprotein to which one (B) of a combination of a pair of affinity substances is bound, and a polyoxyethylene-based surfactant and a cationic surfactant. A first reagent containing at least one of these, an antibody that binds to phosphatidylcholine oxide or a fragment thereof, or an antibody that binds to phosphocholine or a second reagent containing the antibody fragment, and a set of affinity substances. A third reagent containing an insoluble carrier to which the other (b) of the combination is bound is contained, and at least one substance selected from the group consisting of a protein and polyethylene glycol is contained in at least one of the first to third reagents. Oxidized high density lipoprotein measurement kit.

<Measurement kit 3>
Furthermore, the kit for measuring the high-density oxidized lipoprotein of the present invention binds to an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or a first reagent containing an antibody that binds to phosphocholine or an antibody fragment thereof, or a high-density lipoprotein. It is a kit containing a second reagent containing the antibody or the antibody fragment thereof, and a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant.
In the kit, at least one substance selected from the group consisting of protein and polyethylene glycol may be contained in at least one reagent of the first to third reagents.

The measurement kit 3 of the present invention is a kit used for the measurement method 1 or the measurement method 2 of the present invention.
The method for measuring oxidized high-density lipoprotein when measuring by the measuring method 1 of the present invention using the measuring kit 3 of the present invention is a method including the following steps.
(1) A step of mixing the sample and the third reagent of the measurement kit 3 to prepare a sample diluent;
(2) An antibody or an antibody fragment thereof that binds to an oxidized high-density lipoprotein and oxidized phosphatidylcholine by reacting the sample diluted solution prepared in the above step (1) with the first reagent of the measurement kit 3 in an aqueous medium. Alternatively, a step of producing an immune complex 1 composed of an antibody that binds to phosphocholine or an antibody fragment thereof;
(3) The second reagent of the measurement kit 3 is reacted with the immune complex 1 produced in the step (2) in an aqueous medium to bind to an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, or phosphocholine. A step of producing an immune complex 2 comprising an antibody or the antibody fragment, an oxidized high-density lipoprotein, and an antibody or the antibody fragment that binds to the high-density lipoprotein;
(4) Step of measuring the immune complex 2 produced in the step (3) In the above measuring method, the steps (1) and (2) may be performed sequentially or simultaneously.

In the measurement kit 3 of the present invention, it is preferable that the antibody or the antibody fragment thereof that binds to phosphatidyloxide oxide, or the antibody or the antibody fragment thereof that binds to phosphocholine is immobilized on an insoluble carrier. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.
The antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the antibody that binds to phosphocholine or the insoluble carrier on which the antibody fragment thereof is immobilized, is a sample and an antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or phosphocholine. It may be produced in a reaction solution for reaction with an antibody or an antibody fragment thereof that binds to phosphocholine. In this case, instead of the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide, or the insoluble carrier in which the antibody or antibody fragment that binds to phosphocholine is immobilized, one of a combination of affinity substances (A) is used. A bound antibody or antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody that binds to phosphocholine or the antibody fragment thereof and an insoluble carrier to which the other (a) of a combination of a pair of affinity substances is bound are used. Examples of the combination of a set of affinity substances, that is, the combination of A and a include the above-mentioned combinations and the like.

In the measurement kit of the present invention, a set of an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine, to which one (A) of a combination of a pair of affinity substances is bound. The insoluble carrier to which the other (a) of the combination of the affinity substances of the above is bound may be contained in the same reagent or may be contained in different reagents, but is preferably contained in different reagents. An antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine to which one (A) of a combination of a set of affinity substances is bound, or an antibody or an antibody fragment thereof that binds to phosphocholine and a combination of a set of affinity substances. When the insoluble carrier to which the other (a) is bound is contained in a separate reagent, the following kit is also included in the measurement kit of the present invention.

High-density lipoprotein, a first reagent containing an antibody that binds to phosphatidylcholine oxide or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof, to which one (A) of a combination of affinity substances is bound. A second reagent containing an antibody or an antibody fragment thereof, a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and a set. An oxidized high-density lipoprotein measuring kit containing a fourth reagent containing an insoluble carrier to which the other (a) of a combination of affinity substances is bound.

High-density lipoprotein, a first reagent containing an antibody that binds to phosphatidylcholine oxide or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment thereof, to which one (A) of a combination of affinity substances is bound. A second reagent containing an antibody or an antibody fragment thereof, a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and a set. At least one substance selected from the group consisting of a protein and polyethylene glycol comprises a fourth reagent containing an insoluble carrier to which the other (a) of the affinity substance combination is bound, and at least one of the first to fourth reagents is used. Oxidation high density lipoprotein measurement kit contained in one.

The method for measuring oxidized high-density lipoprotein when measuring by the measuring method 2 of the present invention using the measuring kit 3 of the present invention is a method including the following steps.
(1') A step of mixing the sample and the third reagent of the measurement kit 3 to prepare a sample diluent;
(2') The sample diluent prepared in the above step (1') is reacted with the second reagent of the measurement kit 3 in an aqueous medium to bind the oxidized high-density lipoprotein to the high-density lipoprotein. A step of producing an immune complex 3 consisting of the antibody fragment;
(3') The first reagent of the measurement kit 3 is reacted with the immune complex 3 produced in the step (2') in an aqueous medium to oxidize with an antibody or an antibody fragment thereof that binds to a high-density lipoprotein. A step of producing an immune complex 4 consisting of a high-density lipoprotein and an antibody or an antibody fragment thereof that binds to oxidized phosphatidylcholine, or an antibody or an antibody fragment thereof that binds to phosphocholine;
(4') Step of measuring the immune complex 4 produced in the step (3') In the above measuring method, the steps (1') and the step (2') may be performed sequentially or simultaneously. ..

In the measurement kit 3 of the present invention, it is preferable that the antibody or the antibody fragment thereof that binds to the high-density lipoprotein is immobilized on an insoluble carrier. Examples of the insoluble carrier include the above-mentioned insoluble carrier and the like.
Even if the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized is produced in the reaction solution of the reaction between the sample and the antibody that binds to the high-density lipoprotein or the antibody fragment. good. In this case, instead of the antibody that binds to the high-density lipoprotein or the insoluble carrier on which the antibody fragment is immobilized, one (B) of the combination of the affinity substances binds to the high-density lipoprotein. An insoluble carrier to which the antibody or the antibody fragment thereof is bound to the other (b) of a combination of a set of affinity substances is used. Examples of the combination of a set of affinity substances, that is, the combination of B and b include the above-mentioned combinations and the like.

In the measurement kit of the present invention, an antibody or an antibody fragment thereof that binds to a high-density lipoprotein to which one (B) of a combination of a set of affinity substances is bound, and the other (b) of a combination of a set of affinity substances. ) May be contained in the same reagent or in different reagents, but it is preferably contained in different reagents. An antibody or antibody fragment thereof bound to a high-density lipoprotein to which one (B) of a set of affinity substances is bound, and an insoluble carrier to which the other (b) of a combination of a pair of affinity substances is bound. The following kits are also included in the measurement kits of the invention when they are included in separate reagents.

A high-density lipoprotein to which one (B) of a combination of an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, an antibody that binds to phosphocholine or a first reagent containing the antibody fragment, and a set of affinity substances is bound. A second reagent containing an antibody or an antibody fragment thereof, a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and a set. An oxidized high-density lipoprotein measuring kit containing a fourth reagent containing an insoluble carrier to which the other (b) of the affinity substance combination is bound.

A high-density lipoprotein to which one (B) of a combination of an antibody or an antibody fragment thereof that binds to phosphatidylcholine oxide, an antibody that binds to phosphocholine or a first reagent containing the antibody fragment, and a set of affinity substances is bound. A second reagent containing an antibody or an antibody fragment thereof, a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, and a set. At least one substance selected from the group consisting of a protein and polyethylene glycol comprises a fourth reagent containing an insoluble carrier to which the other (b) of the affinity substance combination is bound, and at least one of the first to fourth reagents is used. Oxidation high density lipoprotein measurement kit contained in one.

To the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide or the antibody fragment thereof in the measurement kit of the present invention, or the antibody or antibody fragment thereof that binds to phosphatidylcholine oxide in the first reagent containing the antibody or antibody fragment that binds to phosphocholine. The content of the antibody or the antibody fragment to be bound is not particularly limited as long as it is a content that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned aqueous medium. It is 0.01 to 100 μg / mL in the state of being dissolved in, preferably 0.1 to 20 μg / mL. The content of the antibody that binds to the high-density lipoprotein or the antibody fragment in the second reagent containing the antibody that binds to the high-density lipoprotein of the measurement kit of the present invention or the antibody fragment thereof is the oxidized high-density lipoprotein of the present invention. The content is not particularly limited as long as it enables the measurement method, and is usually 0.01 to 100 μg / mL in the above-mentioned aqueous medium or in a state of being dissolved in the above-mentioned aqueous medium, preferably 0.1 to 20 μg / mL. ..

The constituent reagents of the measurement kit of the present invention may be freeze-dried or liquid.
When the constituent reagents of the freeze-dried measurement kit are used, they are dissolved in an aqueous medium and liquefied before measurement before measurement.
In the liquid measurement kit, an antibody or an antibody fragment thereof that binds to phosphatidyloxide oxide, an antibody or an antibody fragment thereof that binds to phosphocholine, an antibody or an antibody fragment thereof that binds to a high-density lipoprotein, and a polyoxyethylene-based surfactant. And at least one selected from the group consisting of cationic surfactants are in a state of being dissolved in an aqueous medium.
Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, protein stabilizers and the like. The liquid measurement kit contains an aqueous medium. Examples of the aqueous medium include the above-mentioned aqueous medium. The aqueous medium may contain the above-mentioned metal ions, sugars, preservatives, protein stabilizers and the like.

The content of at least one surfactant selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants in the constituent reagents of the measurement kit of the present invention is the content of the oxidized high-density lipoprotein of the present invention. The content is not particularly limited as long as it enables the measuring method, and is usually 0.0001 to 2.0% (w / v) in the above-mentioned aqueous medium or in the state of being dissolved in the above-mentioned aqueous medium. The content of 0.001 to 0.1% (w / v) is preferable, and the content of 0.005 to 0.05% (w / v) is particularly preferable.
In the measurement kit of the present invention, two or more kinds of surfactants selected from the group consisting of polyoxyethylene-based surfactants and cationic surfactants may be used in combination.

Examples of the polyethylene glycol in the measurement kit of the present invention include the above-mentioned polyethylene glycol. The content of polyethylene glycol in the constituent reagents of the measurement kit of the present invention is not particularly limited as long as it is a content that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually in the above-mentioned aqueous medium or in the above-mentioned aqueous medium. The content is 0.5 to 15% (w / v) when dissolved in the above-mentioned aqueous medium, and the content is preferably 1.0 to 10.0% (w / v).
Examples of the protein in the measurement kit of the present invention include the above-mentioned proteins. The protein content in the constituent reagents of the measurement kit of the present invention is not particularly limited as long as it is a content that enables the method for measuring the oxidized high-density lipoprotein of the present invention, and is usually in the above-mentioned aqueous medium or the above-mentioned. The content is 0.01 to 10.0% (w / v) when dissolved in an aqueous medium, and preferably 0.1 to 2.0% (w / v).

Hereinafter, the present invention will be described in more detail by way of examples, but these do not limit the scope of the present invention in any way. In this example, reagents from the following manufacturers were used.

Disodium hydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Ltd.), Sodium dihydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Ltd.), sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), Tween 20 (polyoxy) Ethylene sorbitan fatty acid ester; Sigma-Aldrich), Leodor TW-L106, Leodor TW-L120, Leodor TW-O120, Leodor TW-P120, Leodor TW-S120 (Polyoxyethylene sorbitan fatty acid ester; Kao) , Emanon 1112 (Polyoxyethylene fatty acid ester; manufactured by Kao), Triton X-100 (Polyoxyethylene alkyl phenyl ether; manufactured by Sigma-Aldrich), Nymeen L-207, Nymeen S-220 (all polyoxyethylene alkyl). Amin; Emalmin NL-70, Emalmin NL-90, Emalmin NL-110, Emalmin L-380 (above, polyoxyethylene alkyl ether; manufactured by Sanyo Kasei Kogyo Co., Ltd.), Neugen SD-150 (Polyoxy) Ethylenealkylamine; manufactured by Daiichi Kogyo Seiyaku Co., Ltd., BLAUNON EL-1521 (polyoxyalkyllauryl ether; manufactured by Aoki Oil & Fat Co., Ltd.), NIKKOL DLP-10 (polyoxyethylene alkyl ether phosphoric acid or a salt thereof; manufactured by Nikko Chemicals Co., Ltd.) , Newcol 704, Newcol 710, Newcol 714 (above, polyoxyethylene polycyclic phenyl ether; manufactured by Nippon Embroidery Co., Ltd.), Newcol 707-SF (polyoxyethylene polycyclic phenyl ether sulfate ester salt or salt thereof; Japan (Made by Embroidery Co., Ltd.), Unigli MK-230, Unigli MK-278 (all, polyoxyethylene fatty acid glyceryl; manufactured by Nichiyu Co., Ltd.), cation BB (quaternary ammonium salt).
BSA [Proliant], Polyethylene Glycol (PEG) 200 (Molecular Weight 200 PEG; Nichiyu), PEG600 (Molecular Weight 600 PEG; Nichiyu), PEG2000 (Molecular Weight 2,000 PEG; Nichiyu) PEG6000 (PEG with a molecular weight of 6,000; manufactured by Wako Pure Chemical Industries, Ltd.), PEG20000 (PEG with a molecular weight of 20,000; manufactured by Wako Pure Chemical Industries, Ltd.).

[Example 1]
Oxidized high-density lipoprotein measurement kits 1 to 4 consisting of the following reaction buffer, DLH3 antibody-immobilized microplate, and peroxidase (POD) -labeled anti-apoAI protein antibody solution were prepared.
As shown below, reaction buffers 1 to 4 constituting the oxidized high-density lipoprotein measurement kits 1 to 4 were prepared, respectively.

<Reaction buffer 1 (for measurement kit 1)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%

<Reaction buffer 2 (for measurement kit 2)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
BSA 1%

<Reaction buffer 3 (for measurement kit 3)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
PEG6000 4.8%

<Reaction buffer 4 (for measurement kit 4)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
BSA 1%
PEG6000 4.8%

In the measurement using each of the above measurement kits 1 to 4, the same DLH3 antibody-immobilized microplate and a peroxidase (POD) -labeled anti-apo AI protein antibody solution were used. The DLH3 antibody-immobilized microplate and the peroxidase (POD) -labeled anti-apoAI protein antibody solution were prepared as follows.

<DLH3 antibody solid-phase microplate>
In each well of a 96-well microplate (manufactured by Thermo Fisher Scientific Co., Ltd.), 100 μL of a 10 μg / mL Tris-HCl buffer (pH 8.0) solution of DLH3 antibody prepared by the method described in JP-A-7-238098 was added. Add to / well and incubate at 4 ° C for 16 hours, then remove the Tris-HCl buffer solution and add Tris-HCl buffer (pH 8.0) containing 1% (w / v) BSA. Blocking was performed by adding 350 μL to each well and incubating at 25 ° C for 2 hours, and then washing 4 times with PBS (pH 7.4) containing 0.05% (w / v) Tween 20 to immobilize the DLH3 antibody. Plates were prepared.

<POD-labeled anti-apo AI protein antibody solution>
Using the Peroxidase Labeling Kit-NH ₂ (manufactured by Dojin Kagaku Kenkyusho), label the anti-Apo AI protein monoclonal antibody (manufactured by Abnova) with POD according to the instruction manual of the kit, and POD-labeled anti-Apo A. -I protein monoclonal antibody was prepared. The obtained POD-labeled anti-apo AI protein monoclonal antibody was diluted 10000 times with a solution having the following composition to prepare a POD-labeled anti-apo AI protein monoclonal antibody solution.

Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
BSA 1%
Tween 20 0.05%

[Comparative Example 1]
Oxidized high-density lipoprotein measurement kits 5 to 8 composed of the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apoAI protein antibody solution were prepared.
As shown below, reaction buffers 5 to 8 constituting the oxidation high-density lipoprotein measurement kits 5 to 8 were prepared. In the measurement using each of the measurement kits 5 to 8, the same DLH3 antibody-immobilized microplate and a peroxidase (POD) -labeled anti-apo AI protein monoclonal antibody solution were used. The DLH3 antibody-immobilized microplate and the peroxidase (POD) -labeled anti-apo AI protein monoclonal antibody solution are the DLH3 antibody-immobilized microplate according to Example 1 and the peroxidase (POD) -labeled anti-apo. AI protein monoclonal antibody solutions were used respectively.

<Reaction buffer 5 (for measurement kit 5)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L

<Reaction buffer 6 (for measurement kit 6)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
BSA 1%

<Reaction buffer 7 (for measurement kit 7)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
PEG6000 4.8%

<Reaction buffer 8 (for measurement kit 8)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
BSA 1%
PEG6000 4.8%

[Example 2]
Using the measurement kits 1 to 8, serum (oxidized high-density lipoprotein concentration: 23.8 U / L) (hereinafter, sample A) collected from a healthy person was measured by the following method. The unit "U / L" here means 1 U / L oxidation of oxidized HDL obtained by copper oxidation of phospholipid (HDL-PL) in 1 mg / L high-density lipoprotein (HDL). It is defined as HDL.

To each well of the DLH3 antibody-immobilized plate prepared in Example 1, sample A diluted 1000-fold with the respective reaction buffer or 100 μL of the reaction buffer (0 U / L sample) was added, and the temperature was 37 ° C. The reaction was carried out for 2 hours (primary reaction). After washing each well of the plate after the primary reaction with a washing solution [0.05% Tween 20, 0.01 mol / L phosphate buffer containing 0.15 mol / L sodium chloride (pH 7.4)], POD was added to each well. 100 μL of the labeled anti-apo AI protein antibody solution 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 the above-mentioned washing solution, 100 μL of an aqueous solution of TMB (3,3', 5,5'-tetramethylbenzidine) was added to each well, and the reaction was carried out at 37 ° C. for 20 minutes. The reaction was stopped by adding 50 μL of 0.5 mol / L sulfuric acid (color development reaction). The absorbance (main wavelength 450 nm, sub-wavelength 650 nm) of the reaction solution obtained by the color development reaction was measured. Table 1 shows the absorbance obtained by subtracting the absorbance obtained when measuring a 0 U / L sample from the absorbance obtained when measuring sample A diluted 1000-fold.

As is clear from Table 1, the absorbance is 0.008 to 0.046 when measured using the measurement kits 5 to 8 containing the reaction buffer solution containing no Tween 20, that is, when Tween 20 is not present in the primary reaction. On the other hand, when measured using the measurement kits 1 to 4 containing the reaction buffer containing Tween 20, that is, when Tween 20 is present in the primary reaction, the absorbance is 0.112 to 0.714, and the measurement sensitivity is increased. It turned out to be.
Further, from the comparison between the measurement kit 1 and the measurement kits 2 to 4, it was found that the measurement sensitivity was further increased by the presence of BSA and / or PEG6000 together with Tween 20.

[Example 3]
Oxidized high-density lipoprotein measurement kits A to Z consisting of the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apoAI protein antibody solution were prepared.

<Reaction buffer solution (for measurement kits A to X)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Surfactants (surfactants and concentrations listed in Table 2)
BSA 1%
PEG6000 4.8%

<DLH3 antibody solid-phase microplate>
The DLH3 antibody-immobilized microplate described in Example 1 was used.

<POD-labeled anti-apo AI protein antibody solution>
The POD-labeled anti-apo AI protein antibody solution described in Example 1 was used.

[Comparative Example 2]
An oxidized high-density lipoprotein measurement kit Z consisting of the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apoAI protein antibody solution was prepared.

<Reaction buffer solution (for measurement kit Z)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L

<DLH3 antibody solid-phase microplate>
The DLH3 antibody-immobilized microplate described in Example 1 was prepared.

<POD-labeled anti-apo AI protein antibody solution>
The POD-labeled anti-apo AI protein antibody solution described in Example 1 was prepared.

[Example 4]
Using each of the measurement kits A to X of Example 3 and the measurement kit Z of Comparative Example 2, the sample A diluted 1000-fold with each reaction buffer in the same manner as in Example 2, and the sample A. , The absorbance obtained by measuring the reaction buffer (0 U / L sample) and measuring the 1000-fold diluted sample A minus the absorbance obtained when measuring the 0 U / L sample. Calculated. The results are shown in Table 2.

As is clear from Table 2, in addition to Tween 20 whose effect was shown in Example 2, in the presence of the polyoxyethylene-based surfactant and the cationic surfactant shown in Table 2. It was found that the measurement sensitivity was increased by performing the primary reaction.

[Example 5]
Oxidized high-density lipoprotein measurement kits a to o composed of the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apoAI protein antibody solution were prepared.

<Reaction buffer solution (for measurement kits a to o)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L
Tween20 0.01%
BSA 1%
PEG (PEG and concentration shown in Table 3)

<DLH3 antibody solid-phase microplate>
The DLH3 antibody-immobilized microplate described in Example 1 was prepared.

<POD-labeled anti-apo AI protein antibody solution>
The POD-labeled anti-apo AI protein antibody solution described in Example 1 was prepared.

[Comparative Example 3]
An oxidized high-density lipoprotein measurement kit x consisting of the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apoAI protein antibody solution was prepared.

<Reaction buffer solution (for measurement kit x)>
Phosphate buffer 0.01 mol / L (pH 7.4)
Sodium chloride 0.15 mol / L

<DLH3 antibody solid-phase microplate>
The DLH3 antibody-immobilized microplate described in Example 1 was prepared.

<POD-labeled anti-apo AI protein antibody solution>
The POD-labeled anti-apo AI protein antibody solution described in Example 1 was prepared.

[Example 6]
Using the measurement kits a to o of Example 5 and the measurement kit x of Comparative Example 3, sample A diluted 1000-fold with each reaction buffer in the same manner as in Example 2, and the reaction buffer. (0 U / L sample) was measured, and the absorbance obtained by measuring the sample A diluted 1000-fold was calculated by subtracting the absorbance obtained when the 0 U / L sample was measured. The results are shown in Table 3.

As is clear from Table 3, it was found that the measurement sensitivity was increased by using PEG having a molecular weight of 200 to 20000.

INDUSTRIAL APPLICABILITY The present invention provides a method for measuring oxidized high-density lipoprotein, a measuring reagent, and a measuring kit. The present invention is useful for diagnosing cardiovascular diseases such as arteriosclerosis.

Claims (25)

A method for measuring oxidized high-density lipoprotein in a sample, which comprises the following steps.
(1) A sample and an antibody that binds to phosphatidylcholine oxide or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody can be obtained from a polyoxyethylene fatty acid ester, a polyoxyethylene alkyl phenyl ether, or a polyoxyethylene alkyl. Amine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphate or its salt, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or its salt, or polyoxyethylene fatty acid glyceryl. An antibody or fragment of the antibody that binds to the oxidized high-density lipoprotein and phosphatidylcholine oxide by reacting in an aqueous medium containing at least one selected from the group consisting of an oxyethylene-based surfactant and a cationic surfactant. Or, a step of producing an immune complex 1 consisting of an antibody that binds to phosphocholine or a fragment of the antibody .
(2) An antibody that binds to a high-density lipoprotein or a fragment of the antibody is reacted with the immune complex 1 produced in the above step (1) in an aqueous medium to bind to the oxidized phosphatidylcholine or a fragment of the antibody. Or, a step of producing an immune complex 2 consisting of an antibody that binds to phosphocholine or a fragment of the antibody , an oxidized high-density lipoprotein, and an antibody that binds to the high-density lipoprotein or a fragment of the antibody;
(3) A step of measuring the immune complex 2 produced in the step (2). A method for measuring oxidized high-density lipoprotein in a sample, which comprises the following steps.
(1') A sample and an antibody that binds to a high-density lipoprotein or a fragment of the antibody are used as a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, a polyoxyethylene alkyl ether, or a polyoxy. Ethylene alkyl ether phosphate or its salt, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or its salt, or polyoxyethylene fatty acid glyceryl, a polyoxyethylene surfactant and cationic An immune complex 3 composed of an oxidized high-density lipoprotein and an antibody that binds to the high-density lipoprotein or a fragment of the antibody, which is reacted in an aqueous medium containing at least one selected from the group consisting of surfactants. Process to generate;
(2') An antibody that binds to oxidized phosphatidylcholine or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody is reacted with the immune complex 3 produced in the above step (1') in an aqueous medium. An antibody that binds to high-density lipoprotein or a fragment of the antibody, an antibody that binds to oxidized high-density lipoprotein and phosphatidylcholine oxide or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody . Step of producing an immune complex 4 consisting of;
(3') Step of measuring the immune complex 4 produced in the step (2'). The method of claim 1 or 2, wherein the aqueous medium of step (1) or step (1') further comprises at least one substance selected from the group consisting of protein and polyethylene glycol. The method according to claim 3, wherein the protein is albumin. The method according to claim 4, wherein the albumin is bovine serum albumin. The method according to any one of claims 1 to 5 , wherein the cationic surfactant is a quaternary ammonium salt. The method according to any one of claims 1 to 6 , wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein. The method according to claim 7 , wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein. The reagent used for the measurement method according to claim 1 or 2, which binds to an antibody that binds to oxidized phosphatidylcholine or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody, and a high-density lipoprotein. An antibody or a fragment of the antibody , a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, a polyoxyethylene alkyl ether, a polyoxyethylene alkyl ether phosphate or a salt thereof, a polyoxyethylene polycyclic phenyl Contains at least one selected from the group consisting of ether, polyoxyethylene polycyclic phenyl ether sulfate ester or a salt thereof, or a polyoxyethylene-based surfactant and a cationic surfactant which are polyoxyethylene fatty acid glyceryl. A reagent for measuring high-density oxidized lipoprotein. The reagent according to claim 9 , further comprising at least one substance selected from the group consisting of proteins and polyethylene glycols. The reagent according to claim 10 , wherein the protein is albumin. The reagent according to claim 11 , wherein the albumin is bovine serum albumin. The reagent according to any one of claims 9 to 12 , wherein the cationic surfactant is a quaternary ammonium salt. The reagent according to any one of claims 9 to 13 , wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein. The reagent according to claim 14 , wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein. An antibody that binds to phosphatidylcholine oxide or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody, and a polyoxyethylene fatty acid ester, a polyoxyethylene alkylphenyl ether, a polyoxyethylene alkylamine, or a polyoxyethylene alkyl ether. , Polyoxyethylene alkyl ether phosphate or its salt, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or its salt, or polyoxyethylene fatty acid glyceryl, a polyoxyethylene surfactant and High oxidation, including a first reagent containing at least one selected from the group consisting of cationic surfactants, and a second reagent containing an antibody that binds to a high density lipoprotein or a fragment of the antibody. Density lipoprotein measurement kit. An antibody that binds to a high-density lipoprotein or a fragment of the antibody, and a polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or a salt thereof. , Polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate ester or a salt thereof, or a polyoxyethylene-based surfactant and a cationic surfactant which are polyoxyethylene fatty acid glyceryl. High oxidation level, including a first reagent containing at least one of the following, and a second reagent containing an antibody that binds to phosphatidylcholine oxide or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody. Density lipoprotein measurement kit. The kit according to claim 16 or 17 , wherein at least one substance selected from the group consisting of protein and polyethylene glycol is contained in at least one reagent of the first reagent and the second reagent. A first reagent containing an antibody that binds to oxidized phosphatidylcholine or a fragment of the antibody, or an antibody that binds to phosphocholine or a fragment of the antibody, a second reagent that contains an antibody that binds to a high-density lipoprotein or a fragment of the antibody. , Polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene Contains a third reagent containing at least one selected from the group consisting of polycyclic phenyl ether sulfate ester or a salt thereof, or a polyoxyethylene-based surfactant and a cationic surfactant which are polyoxyethylene fatty acid glyceryl. , Oxidation high density lipoprotein measurement kit. The kit according to claim 19 , wherein at least one substance selected from the group consisting of protein and polyethylene glycol is contained in at least one reagent of the first to third reagents. The kit according to claim 18 or 20 , wherein the protein is albumin. The kit according to claim 21 , wherein the albumin is bovine serum albumin. The kit according to any one of claims 16 to 22 , wherein the cationic surfactant is a quaternary ammonium salt. The kit according to any one of claims 16 to 23 , wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apoA protein. The kit according to claim 24 , wherein the antibody that binds to the apoA protein is an antibody against the apoAI protein.