WO2018079658A1 - Method for measuring oxidized high-density lipoprotein - Google Patents

Abstract

Provided is a method for measuring an oxidized high-density lipoprotein in a sample, the method characterized by including the following: (1) a step for generating an immunocomplex 1 by reacting a sample with an antibody, or a fragment thereof, that binds to oxidized phosphatidylcholine, or with an antibody, or a fragment thereof, that binds to phosphocholine, such reaction carried out in an aqueous medium containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant; (2) a step for generating an immunocomplex 2 by reacting the immunocomplex 1 generated in step (1) with an antibody, or a fragment thereof, that binds to a high-density lipoprotein, such reaction carried out in an aqueous medium; and (3) a step for measuring the immunocomplex 2 generated in step (2).

Description

Method for measuring oxidized high density lipoprotein

The present invention relates to a method for measuring oxidized high density lipoprotein, a measuring reagent, and a measuring kit.
This application claims priority on the basis of Japanese Patent Application No. 2016-213873 for which it applied to Japan on October 31, 2016, and uses the content here.

Arteriosclerosis is a disease that occurs frequently in muscular arteries such as the aorta, coronary artery, cerebral artery, and carotid artery, and is a major cause of angina, myocardial infarction, cerebral infarction, and the like. Probable causes include increased serum cholesterol, platelet aggregation, and endothelial injury, but the cause has not been analyzed.

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

However, as a result of a comparison of the absolute amount of serum lipids in the above-mentioned disease patient group and the healthy subject group in a recent study, there was no significant difference between the two groups, but rather the denatured lipoprotein in the serum. It was reported that the abundance was clearly different between the two groups (see Non-Patent Document 1).

In recent years, 9F5-3a, an antibody that recognizes oxidized phospholipids (particularly lysophosphatidylcholine) of high-density lipoprotein, as a method for measuring oxidized high-density lipoprotein for the diagnosis of cardiovascular diseases including arteriosclerosis, 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 an apoprotein (see Patent Document 2), etc. Has been reported.

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

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

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

As a result of intensive studies to solve the above problems, the present inventors have determined that a sample and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, have a polyoxyethylene interface. It has been found that by reacting in an aqueous medium containing at least one selected from the group consisting of an active agent and a cationic surfactant, oxidized high density lipoprotein in a sample can be measured easily and with high sensitivity. 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, comprising the following steps.
(1) A sample and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine are selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant. An immune complex comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine Producing 1;
(2) an antibody or antibody fragment that binds to oxidized phosphatidylcholine by reacting an antibody that binds to high-density lipoprotein or the antibody fragment with the immune complex 1 produced in step (1) in an aqueous medium, or A step of generating an immune complex 2 comprising an antibody that binds to phosphocholine or the antibody fragment, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to the 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, comprising the following steps.
(1 ′) An aqueous medium containing at least one selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant, and a sample and an antibody or antibody fragment that binds to high-density lipoprotein Producing an immune complex 3 comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to the high-density lipoprotein;
(2 ′) reacting the antibody or antibody fragment that binds to oxidized phosphatidylcholine or the antibody or antibody fragment that binds to phosphocholine with the immune complex 3 produced in the step (1 ′) in an aqueous medium, An immune complex comprising an antibody that binds to high-density lipoprotein 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 Producing 4;
(3 ′) a step of measuring the immune complex 4 generated 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 a 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 surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether. In any one of [1] to [5], phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate 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 apo A protein.
[9] The method according to [8], wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein.

[10] A reagent used in the measurement method according to [1] or [2], wherein the antibody or antibody fragment binds to oxidized phosphatidylcholine, or the antibody or antibody fragment binds to phosphocholine, and a high-density lipoprotein A reagent for measuring oxidized high-density lipoprotein, comprising an antibody or an antibody fragment that binds to phospholipid, and at least one selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant.
[11] The reagent according to [10], further comprising at least one substance selected from the group consisting of a protein and polyethylene glycol.
[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 surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether. Any one of [10] to [13], which is phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof, or polyoxyethylene fatty acid glyceryl The reagent described.

[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 apo A protein.
[17] The reagent according to [16], wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein.

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

[21] An antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or a first reagent that contains an antibody that binds to phosphocholine or the antibody fragment, an antibody that binds to high-density lipoprotein, or a second reagent that contains the antibody fragment And a kit for measuring oxidized high density lipoprotein, comprising a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant.
[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 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 surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether. [18] to [24], which are phosphoric acid or a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof, or polyoxyethylene fatty acid glyceryl The described kit.
[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 apo A protein.
[28] The kit according to [27], wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein.

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

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

≪Process (1) ≫
In step (1), a sample and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine are composed of a polyoxyethylene-based surfactant and a cationic surfactant. A method of reacting in an aqueous medium containing at least one member selected from the group includes an oxidized high-density lipoprotein, 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 generation of an immune complex 1 comprising

The antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or the antibody that binds to phosphocholine or the antibody fragment may or may not be immobilized on an insoluble carrier, but may be immobilized. preferable.
An antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or an antibody fragment that binds to oxidized phosphatidylcholine, or an antibody fragment that binds to phosphocholine when the antibody fragment is immobilized on an insoluble carrier. The insoluble carrier on which the antibody or the antibody fragment is immobilized may be produced in the reaction solution of the antigen-antibody reaction. In this case, it binds to oxidized phosphatidylcholine to which one of the combination of affinity substances (A) is bound. The antibody or the antibody fragment that binds, or the antibody or the antibody fragment that binds to phosphocholine and the insoluble carrier to which the other (a) of the combination of a pair of affinity substances binds in the reaction solution of the antigen-antibody reaction An antibody or antibody fragment that binds to oxidized phosphatidylcholine, or Antibody or insoluble carrier antibody fragment is immobilized to bind to phosphocholine it can be produced in the reaction solution in the antigen-antibody reaction.

Examples of combinations of Aa include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-Combinations of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
An antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or a combination of an antibody that binds to phosphocholine or the 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 oxidized phosphatidylcholine or the antibody fragment, or an antibody that binds to phosphocholine or the antibody fragment, which can be used for the method of measuring oxidized high density lipoprotein of the present invention. For example, polystyrene plates such as microtiter plates, glass or synthetic resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, nitrocellulose membrane, etc. Various membranes, synthetic resin test tubes, and the like.

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

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

The linker is, for example, a molecule capable of covalently bonding both a functional group on the surface of an insoluble carrier and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or a functional group possessed by an antibody or antibody fragment that binds to phosphocholine. There is no particular limitation, for example, an antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or a first reactive group that can react with a functional group of the antibody or antibody fragment that binds to phosphocholine, and an insoluble carrier surface It is preferable that the first reactive group and the second reactive group are different groups having a second reactive group capable of reacting with the functional group. Examples of the functional group of the antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine, and the surface of the insoluble carrier on the surface thereof include, for example, a carboxyl group, an amino group, and glycidyl. Group, sulfhydryl group, hydroxyl group, amide group, imino group, N-hydroxysuccinimide group (NHS group), maleimide group and the like. Examples of the reactive group in the linker include aryl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, isothiocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen. And groups such as pyridyl disulfide and vinyl sulfone.

Alternatively, after the specimen and at least one selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant are mixed in advance, the resulting mixture may be subjected to an antigen-antibody reaction.

In the step (1), the reaction temperature of the reaction between the sample and the antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine is determined by the method for measuring oxidized high density lipoprotein of the present invention. The temperature is not particularly limited as long as the temperature can be adjusted, 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 oxidized high density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, 30 Particularly preferred are minutes to 2 hours. The concentration of the antibody or the antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or the antibody fragment that binds to phosphocholine in the reaction solution is particularly a concentration that enables the method for measuring oxidized high-density lipoprotein of the present invention. There is no limitation, and it is usually 0.01-100 μg / mL, preferably 0.1-20 μg / mL.

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

In the step (2), an antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or an antibody that binds to phosphocholine or the antibody fragment, an oxidized high-density lipoprotein, an antibody that binds to the high-density lipoprotein, or the antibody fragment The reaction temperature of the reaction for producing the immune complex 2 is not particularly limited as long as it enables the method for measuring oxidized high density lipoprotein of the present invention, and is usually 0 to 50 ° C., ~ 45 ° C is preferred, and 20-40 ° C is particularly preferred. The reaction time of the reaction is not particularly limited as long as it enables the method for measuring oxidized high density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, 30 Particularly preferred are minutes to 2 hours. The concentration of the antibody or antibody fragment that binds to the high-density lipoprotein in the reaction solution is not particularly limited as long as it enables the method for measuring oxidized high-density lipoprotein of the present invention, and is usually 0.01-100 μg / mL. 0.1 to 20 μg / mL is preferable.

Step (1) and step (2) may be performed sequentially or simultaneously.
Moreover, you may add the process of wash | cleaning the insoluble support | carrier after reaction of a process (1) as needed between a process (1) and a process (2). The washing liquid used for washing the insoluble carrier after the reaction in step (1) is not particularly limited as long as it is a washing liquid that enables the method for measuring oxidized high density lipoprotein of the present invention. Examples 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.

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

≪Process (3) ≫
In step (3), the concentration of the oxidized complex 2 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 high-density lipoprotein or the antibody fragment is not labeled An antibody that binds to high-density lipoprotein or an antibody that binds to the antibody fragment (hereinafter referred to as a third antibody) or the antibody A labeled third antibody having the label bound to the antibody fragment of the three antibody or the antibody fragment, the immune complex 2 (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 an antibody fragment that binds to a high-density lipoprotein in an oxidized complex comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to the high-density lipoprotein) and binds to oxidized phosphatidylcholine An antibody that binds to phosphocholine or an antibody fragment thereof, and an oxidized high-density lipoprotein Then, an immune complex 5 consisting of an antibody that binds to a high-density lipoprotein or the antibody fragment and a labeled third antibody or the antibody fragment is formed, and the label in the immune complex is measured by the method described later. Thereby, the quantity of the immune complex 2 produced | generated at the process (2) can be measured. 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, or the antibody fragment. Examples of the label include a label 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 oxidized high-density lipoprotein of the present invention. The temperature is not particularly limited as long as the temperature 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 oxidized high density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, 30 Particularly preferred are minutes to 2 hours. The concentration of the labeled third antibody or the antibody fragment in the reaction solution is not particularly limited as long as it enables the method for measuring oxidized high density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL, 0.1 to 20 μg / mL is preferred.

(Ii) When the antibody or antibody fragment that binds to high-density lipoprotein is labeled The amount of immune complex 2 produced in step (2) is measured by measuring the label in immune complex 2 can do.
Examples of the label include an enzyme, a fluorescent substance, a luminescent substance, a radioisotope, biotin, digoxigenin, a polypeptide containing a tag sequence, metal colloid particles, and colored latex particles.

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

An antibody that binds to oxidized phosphatidylcholine or the antibody fragment; or an antibody that binds to phosphocholine or the antibody fragment; an oxidized high-density lipoprotein; and a labeled antibody that binds to high-density lipoprotein or the antibody fragment; Measurement of the label in the immune complex consisting of, and the antibody or the antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or the antibody fragment that binds to phosphocholine, the oxidized high-density lipoprotein, and the high-density lipoprotein The measurement of the label in the immune complex comprising the antibody to be or the antibody fragment and the labeled third antibody or the antibody fragment can be appropriately selected depending on the label to be used.
When the label is a color developing substance, that is, a substance that absorbs light of a certain wavelength, the absorbance can be measured using a spectrophotometer, a multiwell plate reader, or the like.
When the label is a fluorescent substance, the fluorescence intensity can be measured using a fluorometer, a fluorescent multiwell plate reader, or the like.
When the label is a luminescent substance, the luminescence intensity can be measured using a luminescence photometer, a luminescence multiwell 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.
When the label is an enzyme, the amount of label can be quantified by measuring the enzyme activity. For example, the amount of labeling can be measured by reacting a substrate of the enzyme with the enzyme and measuring the produced substance.
When the enzyme is peroxidase, the peroxidase activity can be measured by, for example, an absorbance method, 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 and an oxidative coloring chromogen as a substrate, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. The method of measuring by etc. is mentioned. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

The leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active 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] An amine (BCMA) etc. are mentioned.

The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinder reagent), a combination of a coupler and a phenol.
Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.
Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), 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-Dimethylanily N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (3- Methylphenyl) -N'-succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -4 -Fluoro-3,5-dimethoxyaniline (F-DAOS) and the like.
Examples of 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 a fluorescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as a substrate, and the intensity of fluorescence generated by a fluorometer, a fluorescent multiwell plate reader or the like is measured. The measuring method etc. are mentioned. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

As a method for measuring peroxidase activity by a luminescence method, for example, a peroxidase is reacted with a combination of hydrogen peroxide and a luminescent substance as a substrate, and the intensity of luminescence generated by a luminescence intensity meter or a luminescence multiwell plate reader is measured. The measuring method etc. are mentioned. Examples of the luminescent substance include a luminol compound and a lucigenin compound.

When the enzyme is alkaline phosphatase, alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring alkaline phosphatase activity by a luminescence method include a method in which alkaline phosphatase and its substrate are reacted and the luminescence intensity of the produced luminescence is measured with a luminescence intensity meter, a luminescence multiwell plate reader, or the like.
Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro- 5- {4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.1 ^3.7 ] decane] -4-yl} phenyl phosphate disodium salt (CDP-Star ^TM ), 3- {4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.1 ^3.7 ] decane] -4'-yl} phenyl phosphate disodium salt (CSPD ^TM ), 9-[(Phenyloxy) (phosphoryloxy) methylidene] -10-methylacridane disodium, 9-[(4-chlorophenylthio) (phosphoryloxy) methylidene] -10-methylacridan disodium (Lumigen ^™ APS-5) and the like.

When the enzyme is β-D-galactosidase, β-D-galactosidase activity can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method. As a method of measuring β-D-galactosidase activity by absorbance method (colorimetric method), β-D-galactosidase and its substrate are reacted, and the absorbance of the reaction solution is measured with a spectrophotometer or a multiwell plate reader. And the like. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by an absorbance method (colorimetric method) include o-nitrofel-β-D-galactopyranoside. As a method for measuring β-D-galactosidase activity by a luminescence method, for example, a method in which β-D-galactosidase and its substrate are reacted and the luminescence intensity of the reaction solution is measured with a luminescence intensity meter, a luminescence multiwell plate reader, etc. Etc. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by a luminescence method include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) and its similar compounds. Can be mentioned. As a method of measuring β-D-galactosidase activity by a fluorescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescent multiwell plate reader, etc. Etc. Examples of the substrate for β-D-galactosidase in the method for measuring β-D-galactosidase activity by a 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 luciferase activity by the luminescence method include a method of reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of the luciferase substrate include luciferin and coelenterazine.

When the label is other than a fluorescent substance, a luminescent substance, a radioisotope, and an enzyme, a label that is specifically labeled with a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, etc. The fluorescent substance, the luminescent substance, the radioisotope, or the enzyme that binds the label in 2 or the label in the immune complex 5 and labels the substance that specifically binds to the label by the method described above. By measuring, the label can be measured. Examples of the substance that specifically binds to the label include an avidin and the like in addition to an antibody that specifically binds to the label and when the label is biotin.

By performing the following step (4) and step (5) after step (3), the concentration of 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 create a calibration curve representing the relationship between the oxidized high density lipoprotein concentration and the measured value of the label. Process;
(5) A step of determining the concentration of oxidized high density lipoprotein in the 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 a method characterized by including the following steps.
(1 ′) The sample and the antibody that binds to the high-density lipoprotein or the antibody fragment or the antibody fragment or the antibody fragment are selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant. Reacting in an aqueous medium containing at least one species to produce an immune complex 3 comprising oxidized high density lipoprotein and an antibody or antibody fragment that binds to the high density lipoprotein;
(2 ′) reacting the antibody or antibody fragment that binds to oxidized phosphatidylcholine or the antibody or antibody fragment that binds to phosphocholine with the immune complex 3 produced in the step (1 ′) in an aqueous medium, An immune complex comprising an antibody that binds to high-density lipoprotein 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 Producing 4;
(3 ′) a step of measuring the immune complex 4 generated in the step (2 ′)

≪Process (1 ') ≫
In the step (1 ′), a sample and an antibody that binds to a high-density lipoprotein or the antibody fragment or the antibody fragment or the antibody fragment are made of a polyoxyethylene surfactant and a cationic surfactant. The method of reacting in an aqueous medium containing at least one selected from the above enables the production of an immune complex 3 comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to the high-density lipoprotein. There is no particular limitation as long as it is a method.

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

Examples of the combination of Bb include the following combinations.
A combination of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
-Combinations 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 antibody fragment that binds to a high-density lipoprotein and enables the method for measuring oxidized high-density lipoprotein of the present invention. Examples include carriers.

The binding between the antibody that binds to the high-density lipoprotein or the antibody fragment and the insoluble carrier is not particularly limited as long as it enables the method for measuring the oxidized high-density lipoprotein of the present invention. A method is mentioned.
The antibody or antibody fragment that binds to high-density lipoprotein may be directly immobilized on an insoluble carrier or indirectly immobilized on an insoluble carrier using the physical adsorption and / or chemical bond described above. Good. Examples of the indirect immobilization method include the above-described method.
The antibody or antibody fragment that binds to the high-density lipoprotein may be immobilized on an insoluble carrier by covalent bond via a linker. The linker is not particularly limited as long as it is a molecule that can covalently bond both the functional group on the surface of the insoluble carrier and the functional group possessed by the antibody or antibody fragment that binds to the high-density lipoprotein. Can be mentioned.
Further, after the specimen and at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant are mixed in advance, the resulting mixture may be subjected to an antigen-antibody reaction.

In the step (1 ′), the reaction temperature between the sample and the antibody or antibody fragment that binds to the high-density lipoprotein is not particularly limited as long as it enables the method for measuring 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 oxidized high density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, 30 Particularly preferred are minutes to 2 hours. The concentration of the antibody or antibody fragment that binds to the high-density lipoprotein in the reaction solution is not particularly limited as long as it enables the method for measuring oxidized high-density lipoprotein of the present invention, and is usually 0.01-100 μg / mL. 0.1 to 20 μg / mL is preferable.

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

The step (1 ′) and the step (2 ′) may be performed sequentially, but may be performed simultaneously.
When the step (1 ′) and the step (2 ′) are performed simultaneously, the oxidized high-density lipoprotein in the sample is bound to the antibody or the antibody fragment that binds to the high-density lipoprotein, and the antibody that binds to oxidized phosphatidylcholine or The antibody fragment is reacted with 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 that binds to the high-density lipoprotein is not immobilized, even if it is immobilized on an insoluble carrier. Alternatively, the antibody or the antibody fragment that binds to the high-density lipoprotein can be immobilized on an insoluble carrier by the method described above.

Moreover, you may add the process of wash | cleaning the insoluble support | carrier after reaction of process (1 ') between process (1') and process (2 ') as needed. The washing liquid used for washing the insoluble carrier after the reaction in the step (1 ′) is not particularly limited as long as it is a washing liquid capable of measuring the oxidized high density lipoprotein of the present invention. Etc.
You may add the above-mentioned washing | cleaning process after a process (2 '). The washing solution used for washing the insoluble carrier after the step (2 ′) is not particularly limited as long as it is a washing solution that enables the method for measuring oxidized high density lipoprotein of the present invention, and examples thereof include the aforementioned washing solution. .

≪Process (3 ') ≫
In step (3 ′), the concentration of the oxidized complex 4 produced in step (2 ′) is measured using the following method to determine the oxidized high density lipoprotein concentration in the sample.

(I ′) An antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or an antibody that binds to phosphocholine or the antibody fragment that is not labeled An antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or binds to phosphocholine The antibody or the antibody that binds to the antibody fragment (hereinafter referred to as the fourth antibody) or the labeled fourth antibody or the antibody fragment bound to the antibody fragment is bound to the immune complex 4 (the high-density lipoprotein is bound). An antibody or an antibody fragment thereof, an oxidized high-density lipoprotein, an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody or antibody fragment that binds to phosphocholine) and binds to oxidized phosphatidylcholine That binds to the antibody or antibody fragment or phosphocholine An antibody or antibody fragment that binds to a high-density lipoprotein by reacting with 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 An immune complex 6 comprising the antibody fragment and a labeled fourth antibody or the antibody fragment is formed, and the label in the immune complex is measured by the method described later to produce in the step (2 ′) The amount of immune complex 4 thus obtained 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 determined according to the present invention. The temperature is not particularly limited as long as it enables a method for measuring oxidized high density lipoprotein, and is usually 0 to 50 ° C, preferably 4 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 oxidized high density lipoprotein of the present invention, and is usually 1 minute to 4 hours, preferably 10 minutes to 3 hours, 30 Particularly preferred are minutes to 2 hours. The concentration of the labeled fourth antibody or the antibody fragment in the reaction solution is not particularly limited as long as it enables the method for measuring oxidized high density lipoprotein of the present invention, and is usually 0.01 to 100 μg / mL, 0.1 to 20 μg / mL is preferred.

(Ii ′) When the antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine is labeled, by measuring the label in immune complex 4, step (2 ′ ) Can be measured.
Examples of the label include the aforementioned labeling substances, and examples of the measurement of the label include the aforementioned measurement method.
When the label is other than a fluorescent substance, a luminescent substance, a radioisotope, and an enzyme, a labeled substance in which a substance that specifically binds to the label is labeled with a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, etc., and an immune complex The fluorescent substance, the luminescent substance, the radioisotope or the enzyme labeled with the label in 4 or the label in the immune complex 6 and labeled with a substance that specifically binds to the label is obtained by the method described above. By measuring, the label can be measured. Examples of the substance that specifically binds to the label include an avidin and the like in addition to an antibody that specifically binds to the label and when the label is biotin.

By performing the following step (4 ′) and step (5 ′) after step (3 ′), the concentration of oxidized high-density lipoprotein in the sample can be determined.
(4 ′) A calibration curve representing the relationship between the oxidized high density lipoprotein concentration and the measured value of the label by performing steps (1 ′) to (3 ′) using a known concentration of oxidized high density lipoprotein as a sample. Creating a process;
(5 ′) determining the concentration of oxidized high density lipoprotein in the 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 refers to a high-density lipoprotein having apoA protein, which is an apoprotein constituting high-density lipoprotein, and exhibiting dysfunction due to oxidation of a lipid part constituting high-density lipoprotein. Say.

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

The antibody fragment of an antibody that binds to oxidized phosphatidylcholine or the antibody fragment of an antibody that binds to phosphocholine in the present invention binds to oxidized high-density lipoprotein, and enables the method for measuring oxidized high-density lipoprotein of the present invention There is no particular limitation as long as it is a fragment. For example, Fc portions such as Fab obtained by papain treatment of an antibody, F (ab ′) ₂ obtained by pepsin treatment, Fab ′ obtained by pepsin treatment-reduction treatment, and the like have been removed. Examples thereof include antibody fragments and antibody fragments from which the Fc portion has been removed by genetic engineering techniques.

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 oxidized high-density lipoprotein of the present invention. Either of these can be used.

In the present invention, the antibody that binds to the high-density lipoprotein is preferably an antibody that binds to the apoA protein, and more preferably an antibody against the apoA-I protein. Examples of such an antibody include anti-apo AI protein monoclonal antibody [manufactured by Abnova].
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 oxidized high-density lipoprotein of the present invention. And the aforementioned antibody fragment.

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

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. Examples of saturated or unsaturated fatty acids having 8 to 24 carbon atoms include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, Examples include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanic acid, docosahexaenoic acid, tetradocosanoic acid, tetracosapentaenoic acid, and the like. Examples of the saturated or unsaturated fatty acid 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, polyoxyethylene sorbitan monopalmitate, and the like. Examples of commercially available polyoxyethylene sorbitan fatty acid esters include Tween 20 (polyoxyethylene sorbitan monolaurate; manufactured by Sigma-Aldrich), Tween 40 (polyoxyethylene sorbitan monopalmitate; manufactured by Wako Pure Chemical Industries, Ltd.), Tween 60 (polyoxyethylene sorbitan monostearate; manufactured by Sigma-Aldrich), Tween 80 (polyoxyethylene sorbitan monooleate; manufactured by Wako Pure Chemical Industries, Ltd.), Rhedol TW-L106 (polyoxyethylene sorbitan monolaurate; Kao), Leodol TW-L120 (Polyoxyethylene sorbitan monolaurate; Kao), Leodol TW-O106V (Polyoxyethylene sorbitan monooleate; Kao), Leodol TW-O120V (Polyoxyethylene) Sorbitan monooleate; manufactured by Kao Corporation), Leodol TW-P120 (polyoxyethylene sorbitan monopalmitate; manufactured by Kao Corporation), Leodol TW-S106V (Polyoxyethylene sorbitan monostearate; manufactured by Kao Corporation), Leodol TW-S120V (Polyoxyethylene sorbitan monostearate; manufactured by Kao Corporation), BLAUNON OT-106 (polyoxyethylene sorbitan monooleate; manufactured by Aoki Yushi Co., Ltd.), BLAUNON OT-21 (polyoxyethylene sorbitan monooleate; manufactured by Aoki Yushi Co., Ltd.) ), New Coal 25 (Polyoxyethylene sorbitan laurate; manufactured by Nippon Emulsifier Co., Ltd.), New Coal 65 (Polyoxyethylene sorbitan stearate; manufactured by Nihon Emulsifier Co., Ltd.), New Coal 85 (Polyoxyethylene sorbitan oleate; Nihon Emulsifier Co., Ltd.) Etc.) .

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. Examples of saturated or unsaturated fatty acids having 8 to 24 carbon atoms include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, Examples include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanic acid, docosahexaenoic acid, tetradocosanoic acid, tetracosapentaenoic acid, and the like. Examples of the saturated or unsaturated fatty acid 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, polyoxyethylene distearate and the like. Examples of commercially available products of polyoxyethylene fatty acid esters include Emanon 1112 (polyoxyethylene monolaurate; manufactured by Kao), Emanon 4110 (polyoxyethylene monooleate; manufactured by Kao), Emanon 3199V (polyoxyethylene monostearate). Aleate; manufactured by Kao), Emanon 3299V (polyoxyethylene disteareate; manufactured by Kao), BLUINON L-400 (polyoxyethylene monolaurate; manufactured by Aoki Yushi Co., Ltd.), BLAUNON S-300A (polyoxyethylene mono) Stearate (manufactured by Aoki Yushi Co., Ltd.), BLAUNON® O-600SA® (polyoxyethylene monooleate; manufactured by Aoki Yushi Co., Ltd.), and the like.

Examples of the alkyl in the polyoxyethylene alkylphenyl ether include 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 octyl phenyl ether and polyoxyethylene nonyl phenyl ether. Commercially available products of polyoxyethylene alkylphenyl ether include, for example, Triton X-100 (polyoxyethylene octylphenyl ether; manufactured by Sigma-Aldrich), BLAUNON ポ リ NK-810 (polyoxyethylene octylphenyl ether; manufactured by Aoki Yushi) Is mentioned.

Examples of the alkyl in the polyoxyethylene alkylamine include alkyl having 8 to 24 carbon atoms, and alkyl having 10 to 20 carbon atoms is preferable. Examples of the alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl , Nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl and the like. Examples of the alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecyl, icosyl and the like. Can be mentioned. Examples of commercially available products of polyoxyethylene alkylamine include Nimin L-201 (oxyethylene dodecylamine; manufactured by NOF Corporation), Nimin L-202, Naimine L-207, and Naimine L-215 (above, polyoxyethylene dodecylamine). Nymeen S-202, Nymeen S-204, Nymeen S-210, Nymeen S-215, Nymeen S-220 (above, polyoxyethylene octadecylamine; made by NOF Corporation), Nymeen T2-202 Nymeen T2-210, Nymeen T2-230 [above, polyoxyethylene alkyl (tallow) amine; manufactured by NOF Corporation], Nymeen F-202, Nymeen F-203, Nymeen F-205, Nymeen F-210, Nymeen F -215 [above, polyoxyethylene Alkyl (coconut oil) amine; manufactured by NOF Corporation], BROWNON L-202, BROWNON L-205, BROWNON L-207, BROWNON L-210, BROWNON L-230 (above, polyoxyethylene dodecylamine; manufactured by Aoki Yushi Co., Ltd.) ), Brownon S-207, Brownon S-210, Brownon S-215, Brownon S-220, Brownon S-230 (above, polyoxyethylene octadecylamine; manufactured by Aoki Yushi), Brownon S-205T, Brownon S-208T BROWNON S-210T, BROWNON S-215T, BROWNON S-230T [above, polyoxyethylene alkyl (beef tallow) amine; manufactured by Aoki Yushi Co., Ltd.], New Coal OD420 (polyoxyethylene octadecylamine; manufactured by Nippon Emulsifier Co., Ltd.), pionine D3104 (Po Oxyethylene dodecylamine; manufactured by Takemoto Yushi Co., Ltd.), Pionein D3110 (polyoxyethylene dodecylamine; manufactured by Takemoto Yushi Co., Ltd.), Pionein D3605 [polyoxyethylene alkyl (soybean) amine; manufactured by Takemoto Yushi Co., Ltd.], Pionine D3615T [Polyoxyethylene Alkyl (beef tallow) amine; manufactured by Takemoto Yushi Co., Ltd.], BLAUNON O209 (polyoxyethylene oleylamine; manufactured by Aoki Yushi Co., Ltd.), 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 the alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl , Nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl and the like. Examples of the alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecyl, icosyl and the like. Can be mentioned. Examples of commercially available polyoxyethylene alkyl ethers include Nonion K-204, Nonion K-220 (above, polyoxyethylene lauryl ether; manufactured by NOF Corporation), Nonion E-205, Nonion E-215 (above, Polyoxy Ethylene oleyl ether (manufactured by NOF Corporation), Nonion S-215, Nonion S-225 (above, polyoxyethylene stearyl ether; manufactured by NOF Corporation), Emulgen 108, Emulgen 120 (above, polyoxyethylene lauryl ether; Kao Corporation Emulgen 220 (polyoxyethylene cetyl ether; manufactured by Kao Corporation), Emulgen 320P (polyoxyethylene stearyl ether; manufactured by Kao Corporation), Emulgen 420 (polyoxyethylene oleyl ether; manufactured by Kao Corporation), Adekatol LA-875, Adekato LA-975 (above, polyoxyethylene lauryl ether; manufactured by ADEKA), Adecatol OA-7 (polyoxyethylene oleyl ether; manufactured by ADEKA), Emalmin NL-70, Emalmin NL-90, Emalmin NL-100, Emalmin NL-110, Emalmin LS-80, Emalmin LS-90, Emalmin L-380 (above, polyoxyethylene lauryl ether; manufactured by Sanyo Chemical Industries), BLAUNON EL-1507, BLAUNON EL-1509, BLAUNON EL-1512P, BLAUNON EL-1515, BLAUNON EL-1521 (above, polyoxyethylene lauryl ether; manufactured by Aoki Yushi Co., Ltd.), BLAUNON CH-310, BLAUNON CH-310L, BLAUN N CH-313 (polyoxyethylene cetyl ether; manufactured by Aoki Yushi Co., Ltd.), BLAUNON SR-711 (polyoxyethylene stearyl ether; manufactured by Aoki Yushi Co., Ltd.), BLAUNON EN-1511 (polyoxyethylene oleyl ether; Aoki Yushi 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 polyoxyethylene alkyl ether phosphoric acid or a salt thereof include alkyl having 8 to 24 carbon atoms, preferably alkyl having 10 to 20 carbon atoms. Examples of the alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl , Nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl and the like. Examples of the alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecyl, icosyl and the like. 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 polyoxyethylene alkyl ether phosphoric acid or a salt thereof include polyoxyethylene lauryl ether phosphoric acid sodium salt. Examples of commercially available products of polyoxyethylene alkyl ether phosphoric acid or a salt thereof include Prisurf A212C, Prisurf A215C (above, polyoxyethylene tridecyl ether phosphate; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Prisurf A208F (Polyoxy A208F) Ethylene octyl ether phosphoric acid; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Prisurf A219B (polyoxyethylene lauryl ether phosphoric acid; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Plysurf DB-01 (polyoxyethylene lauryl ether phosphoric acid / monoethanol) Amine 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 acetate) Berlin sodium; manufactured by Nikko Chemicals Co.), NIKKOL DOP-6NV (sodium polyoxyethylene oleyl ether phosphate; manufactured by Nikko Chemicals Co., Ltd.).

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

The polyoxyethylene in the polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof is a phenyl group in which two or more groups (substituents) having one aromatic ring 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 and 1- (phenyl) ethyl. Examples of the group having two or more aromatic rings in the group include naphthyl. Examples of the salt include lithium salt, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, monoethanolamine salt and the like. Examples of commercially available products of polyoxyethylene polycyclic phenyl ether sulfate or salts thereof include, for example, New Coal 707-SF, New Coal 707-SFC, New Coal 707-SN, New Coal 714-SF, New Coal 714-SN (and above) , Manufactured by Nippon Emulsifier Co., Ltd.).

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. Examples of saturated or unsaturated fatty acids having 8 to 24 carbon atoms include octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, Examples include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanic acid, docosahexaenoic acid, tetradocosanoic acid, tetracosapentaenoic acid, and coconut oil fatty acid. Examples of the saturated or unsaturated fatty acid having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, coconut oil fatty acid and the like. Examples of the polyoxyethylene fatty acid glyceryl include polyoxyethylene coconut oil fatty acid glyceryl, polyoxyethylene lauric acid glyceryl, polyoxyethylene oleic acid glyceryl and the like. Commercially available products of polyoxyethylene fatty acid glyceryl include, for example, UNIGURI MK-207, UNIGURI MK-230, UNIGURI MK-278 (above, polyoxyethylene palm oil fatty acid glyceryl; manufactured by NOF Corporation), UNIGURI ML-212, UNIGLY ML -220 (Glyceryl polyoxyethylene laurate; manufactured by NOF Corporation), UNIGLY MO-220, UNIGLY MO-230 (above, glyceryl polyoxyethylene oleate; manufactured by NOF Corporation), and the like.

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

(Wherein 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 are the same or different and each represents 1 to 6 carbon atoms. And X represents an anion)

Examples of the alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl , Nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl and the like.

Examples of the alkyl having 1 to 24 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, isooctyl, nonyl, decyl, isodecyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl , Hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, 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 the anion 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 and iodine ion.
Examples of the anion derived from an inorganic acid include nitrate ion, sulfate ion, phosphate ion, carbonate ion, bicarbonate ion and the like.
Examples of the anions derived from organic acids include formate ions, acetate ions, lactate ions, citrate ions, carboxylate ions such as glutamate ions, and the like.

Examples of commercially available quaternary ammonium salts include cation AB (octadecyltrimethylammonium chloride; manufactured by NOF Corporation), cation BB (dodecyltrimethylammonium chloride; manufactured by NOF Corporation), and cation 2ABT (distearyldimethylammonium chloride; Japan). Oil), cation 2DB-500E (didecyldimethylammonium chloride; manufactured by NOF Corporation), cation 2-OLR (dioleoyldimethylammonium chloride; manufactured by NOF Corporation), and the like.

In the method for measuring oxidized high density lipoprotein of the present invention, a surfactant selected from the group consisting of a polyoxyethylene 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 oxidized high density lipoprotein of the present invention. For example, 0.0001 to 2.0% (w / v), 0.001 to 0.1% (w / v) is preferable, and 0.005 to 0.05% (w / v) is particularly preferable.
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 capable of measuring the oxidized high density lipoprotein of the present invention. For example, albumin, fetal bovine serum (FBS), casein, block ace (DS Pharma Biomedical) Etc.), and albumin is preferable. Examples of albumin include bovine serum albumin (BSA).
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 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 preferred.
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. ˜20,000 polyethylene glycols are 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 oxidized high density lipoprotein of the present invention, and is, for example, 0.5 to 15.0% (w / v), 1.0 to 10.0% (w / v) is preferable.

The aqueous medium in the present invention is not particularly limited as long as it is an aqueous medium that enables the method for measuring oxidized high density lipoprotein of the present invention, and examples thereof include deionized water, distilled water, and buffer solution. Is preferred. 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 set pH. The buffer used for preparing the buffer solution is not particularly limited as long as it has a buffering capacity. For example, a lactate buffer, a citrate buffer, an acetate buffer, a succinate buffer, a phthalate buffer, a phosphorus Acid buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, Good buffering agents and the like can be mentioned.

Examples of the good buffer include 2-morpholinoethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamido) iminodiacetic acid (ADA) buffer, piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamido) 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-morpholinopropanesulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxy Til) 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-hydroxyethyl) -N ′-(2-hydroxy-3-sulfopropyl) piperazine (HEPPSO) buffer, N- (2-hydroxyethyl) -N ′-(3-sulfopropyl) piperazine (EPPS) buffer, [N-tris (hydride (Xymethyl) methylglycine] (Tricine) buffer, [N, N-bis (2-hydroxyethyl) glycine] (Bicine) buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) Buffer, 2- (N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propane Examples include sulfonic acid (CAPS) buffer.

The hydrophobic 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 and sorbitol. Examples of the preservative include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Procrine 300, Proxel GXL, and the like. Examples of the protein stabilizer include peroxidase stabilization buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation).

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

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

When the measurement reagent of the present invention is used in the above-described measurement method 1, the antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine is immobilized on an insoluble carrier. Is preferred.
Examples of the insoluble carrier include the aforementioned insoluble carrier.
When the measurement reagent of the present invention is used in the above-described measurement method 1, an antibody or the antibody fragment that binds to oxidized phosphatidylcholine, or an insoluble carrier on which the antibody or the antibody fragment that binds to phosphocholine is immobilized May be produced in a reaction solution of a reaction between a sample and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine.

In this case, the measurement reagent of the present invention includes an oxidized phosphatidylcholine to which one of a pair of affinity substances (A) is bound, instead of the insoluble carrier on which the antibody or the antibody fragment bound to phosphocholine is immobilized. An antibody to be bound or the antibody fragment, or an antibody to bind to phosphocholine or the antibody fragment, and an insoluble carrier to which the other (a) of the combination of a pair of affinity substances is bound. Examples of the combination of one set of affinity substances, that is, the combination of A and a include the aforementioned combinations.

When the measurement reagent of the present invention is used in the above-described measurement method 2, the antibody or antibody fragment that binds to high-density lipoprotein is preferably immobilized on an insoluble carrier.
Examples of the insoluble carrier include the aforementioned insoluble carrier.
When the measurement reagent of the present invention is used in the above-described measurement method 2, an antibody or an insoluble carrier having the antibody fragment immobilized thereon is immobilized on the sample and the high-density lipoprotein. It may be produced in the reaction solution of the reaction with the antibody to be bound or the antibody fragment.

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

The measurement reagent of the present invention may be lyophilized or liquid. When a measurement reagent in a lyophilized state is used, it is dissolved in an aqueous medium before measurement and made into a liquid state for measurement.
In the liquid measurement reagent, an antibody or antibody fragment that binds to oxidized phosphatidylcholine, an antibody or antibody fragment that binds to phosphocholine, an antibody or antibody fragment that binds to high-density lipoprotein, and a polyoxyethylene interface At least one selected from the group consisting of an active agent and a cationic surfactant is dissolved in an aqueous medium.
As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, protein stabilizers, and the like.

Examples of the polyoxyethylene-based surfactant and the cationic surfactant in the measurement reagent of the present invention include the aforementioned polyoxyethylene-based surfactant and cationic surfactant.
The surfactant content of each of the polyoxyethylene-based surfactant and the cationic surfactant in the measurement reagent of the present invention should be a content that enables the method for measuring oxidized high-density lipoprotein of the present invention. There is no particular limitation, and the content is usually 0.0001 to 2.0% (w / v) in the above-mentioned aqueous medium or dissolved in the above-mentioned aqueous medium, and 0.001 to 0.1% (w / v) A content of 0.005 to 0.05% (w / v) is particularly preferable.

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

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

<Measurement kit 1>
The kit for measuring oxidized high density lipoprotein of the present invention comprises an antibody that binds to oxidized phosphatidylcholine or an antibody fragment thereof, or an antibody that binds to phosphocholine or the antibody fragment, a polyoxyethylene surfactant and a cationic surfactant. A kit comprising: a first reagent containing at least one selected from the group consisting of agents; and a second reagent containing an antibody that binds to a high-density lipoprotein or an antibody fragment thereof.
In the kit, at least one substance selected from the group consisting of protein and polyethylene glycol may be contained in at least one 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, and the oxidized high-density lipoprotein, the antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or the antibody that binds to phosphocholine or the antibody Generating an immune complex 1 comprising the fragments;
(2) The second reagent of the measurement kit 1 is reacted with the immune complex 1 generated in the step (1) in an aqueous medium, and bound to the antibody or the antibody fragment or oxidized phosphocholine that binds to oxidized phosphatidylcholine. Producing an immune complex 2 comprising an antibody or antibody fragment, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to the high-density lipoprotein;
(3) The step of measuring the immune complex 2 generated in the step (2) In the measurement kit 1 of the present invention, the antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or the antibody that binds to phosphocholine or the antibody fragment It is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier.

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

In the measurement kit of the present invention, a set of an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, to which one (A) of a set of affinity substances is bound The insoluble carrier to which the other (a) of the combination of affinity substances is bound may be contained in the same reagent or in separate reagents, but is preferably contained in separate reagents. An antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, and one set of affinity substance combinations, to which one of the combination of affinity substances (A) is bound On the other hand, when the insoluble carrier to which (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 antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, to which one of a pair of affinity substances (A) is bound, and a polyoxyethylene surfactant and a positive chain A first reagent containing at least one selected from the group consisting of ionic surfactants, an antibody that binds to high-density lipoprotein or a second reagent containing the antibody fragment, and a set of affinity substances A kit for measuring an oxidized high-density lipoprotein comprising a third reagent containing an insoluble carrier to which the other (a) of the combination is bound.

An antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, to which one of a pair of affinity substances (A) is bound, and a polyoxyethylene surfactant and a positive chain A first reagent containing at least one selected from the group consisting of ionic surfactants, an antibody that binds to high-density lipoprotein 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 (a) of the combination is bound, and at least one substance selected from the group consisting of protein and polyethylene glycol is present in at least one of the first to third reagents. A kit for measuring oxidized high density lipoprotein.

<Measurement kit 2>
In addition, the oxidized high density lipoprotein measurement kit of the present invention is at least selected from the group consisting of an antibody that binds to high density lipoprotein or the antibody fragment thereof, a polyoxyethylene-based surfactant, and a cationic surfactant. A kit comprising: a first reagent comprising one species; and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or a second reagent that comprises an antibody or antibody fragment 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 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 comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to the high-density lipoprotein by reacting the sample with the first reagent of the measurement kit 2 in an aqueous medium. Producing
(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, and the antibody or the antibody fragment that binds to high-density lipoprotein is oxidized. Generating an immune complex 4 comprising a high-density lipoprotein and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment 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 antibody fragment that binds to the high-density lipoprotein is immobilized on an insoluble carrier. Preferably it is. Examples of the insoluble carrier include the aforementioned insoluble carrier.

The insoluble carrier on which the antibody or antibody fragment that binds to the high-density lipoprotein is immobilized may be produced in the reaction solution of the reaction between the sample and the antibody or antibody fragment that binds to the high-density lipoprotein. Good. In this case, instead of the insoluble carrier on which the antibody or antibody fragment that binds to the high-density lipoprotein is immobilized, it binds to the high-density lipoprotein to which one of the combination of affinity substances (B) is bound. An antibody or the antibody fragment and an insoluble carrier to which the other (b) of the combination of a pair of affinity substances is bound are used. Examples of the combination of a pair of affinity substances, that is, the combination of B and b include, for example, the combinations described above.

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

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

<Measurement kit 3>
Furthermore, the kit for measuring oxidized high-density lipoprotein of the present invention binds to an antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or a first reagent that contains an antibody that binds to phosphocholine or the antibody fragment, or a high-density lipoprotein. And a second reagent containing the antibody fragment, and a third reagent containing at least one selected from the group consisting of a polyoxyethylene 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 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 measurement method of oxidized high density lipoprotein in the case of measuring by the measurement method 1 of the present invention using the measurement 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 diluted solution prepared in the above step (1) and the first reagent of the measurement kit 3 are reacted in an aqueous medium, and the antibody or the antibody fragment that binds to oxidized high density lipoprotein and oxidized phosphatidylcholine, Or a step of generating an immune complex 1 comprising an antibody that binds to phosphocholine or the antibody fragment;
(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, and the antibody or the antibody fragment that binds to oxidized phosphatidylcholine, or the phosphocholine. Producing an immune complex 2 comprising an antibody or antibody fragment, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to the high-density lipoprotein;
(4) The process of measuring the immune complex 2 produced | generated at the said process (3) In the said measuring method, a process (1) and a process (2) may be performed sequentially or may be performed simultaneously.

In the measurement kit 3 of the present invention, the antibody or antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or antibody fragment that binds to phosphocholine is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier.
The antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or the antibody that binds to phosphocholine or the insoluble carrier on which the antibody fragment is immobilized comprises a sample, an antibody that binds to oxidized phosphatidylcholine, the antibody fragment, or phosphocholine. It may be produced in a reaction solution of a reaction with an antibody that binds to the antibody or the antibody fragment. In this case, instead of an antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or an insoluble carrier on which an antibody that binds to phosphocholine or the antibody fragment is immobilized, one of the combinations of affinity substances (A) is The bound antibody or the antibody fragment that binds to oxidized phosphatidylcholine, or the antibody or the antibody fragment that binds to phosphocholine, and the insoluble carrier to which the other (a) of a pair of affinity substances is bound. Examples of the combination of one set of affinity substances, that is, the combination of A and a include the aforementioned combinations.

In the measurement kit of the present invention, a set of an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, to which one (A) of a set of affinity substances is bound The insoluble carrier to which the other (a) of the combination of affinity substances is bound may be contained in the same reagent or in separate reagents, but is preferably contained in separate reagents. An antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine, and one set of affinity substance combinations, to which one of the combination of affinity substances (A) is bound On the other hand, when the insoluble carrier to which (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 antibody fragment that binds to oxidized phosphatidylcholine, or a first reagent containing the antibody or antibody fragment that binds to phosphocholine, to which one of the combinations of affinity substances (A) is bound, high-density lipoprotein A third reagent containing at least one selected from the group consisting of a second reagent containing an antibody that binds to the antibody or the antibody fragment, a polyoxyethylene surfactant and a cationic surfactant, and a set of A kit for measuring oxidized high density lipoprotein, comprising a fourth reagent containing an insoluble carrier to which the other (a) of the combination of affinity substances is bound.

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

The method for measuring oxidized high density lipoprotein in the case of 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 diluted solution prepared in the step (1 ′) and the second reagent of the measurement kit 3 are reacted in an aqueous medium, and the oxidized high-density lipoprotein and the antibody that binds to the high-density lipoprotein or Generating an immune complex 3 comprising 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, and the antibody or the antibody fragment that binds to the high-density lipoprotein is oxidized. Generating an immune complex 4 comprising a high-density lipoprotein and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine;
(4 ′) A step of measuring the immune complex 4 generated in the step (3 ′) In the measurement method, the step (1 ′) and the step (2 ′) may be performed sequentially or simultaneously. .

In the measurement kit 3 of the present invention, the antibody that binds to the high-density lipoprotein or the antibody fragment is preferably immobilized on an insoluble carrier. Examples of the insoluble carrier include the aforementioned insoluble carrier.
The insoluble carrier on which the antibody or antibody fragment that binds to the high-density lipoprotein is immobilized may be produced in the reaction solution of the reaction between the sample and the antibody or antibody fragment that binds to the high-density lipoprotein. Good. In this case, instead of the insoluble carrier on which the antibody or antibody fragment that binds to the high-density lipoprotein is immobilized, it binds to the high-density lipoprotein to which one of the combination of affinity substances (B) is bound. An antibody or the antibody fragment and an insoluble carrier to which the other (b) of the combination of a pair of affinity substances is bound are used. Examples of the combination of a pair of affinity substances, that is, the combination of B and b include, for example, the combinations described above.

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

High density lipoprotein to which antibody (or antibody fragment) that binds to oxidized phosphatidylcholine, or antibody (1) containing phosphocholine or the first reagent containing the antibody fragment, or one of a pair of affinity substances (B) binds A third reagent containing at least one selected from the group consisting of a second reagent containing an antibody that binds to the antibody or the antibody fragment, a polyoxyethylene surfactant and a cationic surfactant, and a set of A kit for measuring an oxidized high density lipoprotein comprising a fourth reagent containing an insoluble carrier to which the other (b) of the combination of affinity substances is bound.

High density lipoprotein to which antibody (or antibody fragment) that binds to oxidized phosphatidylcholine, or antibody (1) containing phosphocholine or the first reagent containing the antibody fragment, or one of a pair of affinity substances (B) binds A third reagent containing at least one selected from the group consisting of a second reagent containing an antibody that binds to the antibody or the antibody fragment, a polyoxyethylene surfactant and a cationic surfactant, and a set of A fourth reagent containing an insoluble carrier to which the other (b) of the combination of affinity substances is bound, and at least one substance selected from the group consisting of protein and polyethylene glycol is at least one of the first to fourth reagents. A kit for measuring oxidized high density lipoprotein contained in one.

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

The constituent reagent of the measurement kit of the present invention may be lyophilized or liquid.
When using a constituent reagent of a measurement kit in a lyophilized state, the reagent is dissolved in an aqueous medium and measured before use for measurement.
In a liquid measurement kit, an antibody or antibody fragment that binds to oxidized phosphatidylcholine, an antibody or antibody fragment that binds to phosphocholine, an antibody or antibody fragment that binds to high-density lipoprotein, and a polyoxyethylene surfactant And at least one selected from the group consisting of cationic surfactants is in a state dissolved in an aqueous medium.
As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, protein stabilizers, and the like. The liquid measurement kit includes an aqueous medium. As an aqueous medium, the above-mentioned aqueous medium is mentioned, for example. The aqueous medium may contain the aforementioned metal ions, sugars, preservatives, protein stabilizers, and the like.

The content of at least one surfactant selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant in the constituent reagent 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 measurement method, and is usually a content that becomes 0.0001 to 2.0% (w / v) in the above-mentioned aqueous medium or in a state dissolved in the above-mentioned aqueous medium, A content of 0.001 to 0.1% (w / v) is preferable, and a 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 a polyoxyethylene surfactant and a cationic surfactant may be used in combination.

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

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

Disodium hydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Inc.), sodium dihydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Inc.), sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), Tween 20 (polyoxy Ethylene sorbitan fatty acid ester; Sigma-Aldrich), Rheodor TW-L106, Rhedol TW-L120, Rhedol TW-O120, Rhedol TW-P120, Rhedol TW-S120 (above, polyoxyethylene sorbitan fatty acid ester; Kao Corporation) Emanon 1112 (polyoxyethylene fatty acid ester; manufactured by Kao Corporation), Triton X-100 (polyoxyethylene alkylphenyl ether; manufactured by Sigma-Aldrich), Naimine L-207, Naimine S-220 (above, polyoxyethylene alkyl) Amine; NOF Corporation , Emalmin NL-70, Emalmin NL-90, Emalmin NL-110, Emalmin L-380 (above, polyoxyethylene alkyl ether; manufactured by Sanyo Chemical Industries), Neugen SD-150 (Polyoxyethylene alkylamine; Daiichi Kogyo) (Manufactured by Pharmaceutical Co., Ltd.), BLAUNON EL-1521 (polyoxyalkyl lauryl ether; manufactured by Aoki Yushi Co., Ltd.), NIKKOL DLP-10 (polyoxyethylene alkyl ether phosphoric acid or salt thereof; manufactured by Nikko Chemicals), New Coal 704, New Coal 710, New Coal 714 (polyoxyethylene polycyclic phenyl ether; manufactured by Nippon Emulsifier Co., Ltd.), New Coal 707-SF (Polyoxyethylene polycyclic phenyl ether sulfate or salt thereof; manufactured by Nihon Emulsifier Co., Ltd.), Unigri MK 230, Yuniguri MK-278 (above, polyoxyethylene fatty acid glyceryl; manufactured by NOF Corporation), cationic BB (quaternary ammonium salt).
BSA (manufactured by Proliant), polyethylene glycol (PEG) 200 (PEG having a molecular weight of 200; manufactured by NOF Corporation), PEG600 (PEG having a molecular weight of 600; manufactured by NOF Corporation), PEG2000 (PEG having a molecular weight of 2,000; NOF Corporation) PEG6000 (molecular weight 6,000; Wako Pure Chemical Industries, Ltd.), PEG 20000 (molecular weight 20,000 PEG; Wako Pure Chemical Industries, Ltd.).

[Example 1]
Oxidized high density lipoprotein measurement kits 1 to 4 comprising the following reaction buffer, DLH3 antibody-immobilized microplate, and peroxidase (POD) labeled anti-apo AI 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.

<Reaction buffer 1 (for measurement kit 1)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%

<Reaction buffer 2 (for measurement kit 2)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
BSA 1%

<Reaction buffer 3 (for measurement kit 3)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
PEG6000 4.8%

<Reaction buffer 4 (for measurement kit 4)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Tween 20 0.01%
BSA 1%
PEG6000 4.8%

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

<DLH3 antibody immobilized microplate>
100 μL of 10 μg / mL Tris-HCl buffer solution (pH 8.0) of DLH3 antibody prepared by the method described in JP-A-7-238098 is added to each well of a 96-well microplate (Thermo Fisher Scientific). Incubate for 16 hours at 4 ° C, 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. Prepared plates.

<POD-labeled anti-apo AI protein antibody solution>
Using Peroxidase Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories), anti-apoA-I protein monoclonal antibody (Abnova) is labeled with POD according to the instruction manual of the kit, and POD-labeled anti-apoA -I protein monoclonal antibody was prepared. The obtained POD-labeled anti-apo AI protein monoclonal antibody was diluted 10,000 times with a solution having the following composition to prepare a POD-labeled anti-apo AI protein monoclonal antibody solution.

Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
BSA 1%
Tween 20 0.05%

[Comparative Example 1]
Oxidized high density lipoprotein assay kits 5 to 8 comprising the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apo AI protein antibody solution were prepared.
As shown below, reaction buffer solutions 5 to 8 constituting oxidized 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 the 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 and the peroxidase (POD) -labeled anti-apoposome described in Example 1. Each of the AI protein monoclonal antibody solutions was used.

<Reaction buffer 5 (for measurement kit 5)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L

<Reaction buffer 6 (for measurement kit 6)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
BSA 1%

<Reaction buffer 7 (for measurement kit 7)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
PEG6000 4.8%

<Reaction buffer 8 (for measurement kit 8)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
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 referred to as sample A) collected from healthy individuals was measured by the following method. The unit “U / L” here refers to 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 reaction buffer (0 U / L sample) was added at 37 ° C. The reaction was carried out for 2 hours (primary reaction). Each well of the plate after the primary reaction was washed with a wash solution [0.01% mol / L phosphate buffer (pH 7.4) containing 0.05% Tween20, 0.15% mol / L sodium chloride], and each well was subjected to POD. 100 μL of the labeled anti-apo AI protein antibody solution was added, and the reaction was performed at 37 ° C. for 1 hour (secondary reaction). After washing each well of the plate after the secondary reaction with the above washing solution, 100 μL of TMB (3,3 ′, 5,5′-tetramethylbenzidine) aqueous solution was added to each well and reacted at 37 ° C. for 20 minutes. The reaction was stopped by adding 50 μL of 0.5 mol / L sulfuric acid (coloring reaction). The absorbance (main wavelength: 450 nm, sub wavelength: 650 nm) of the reaction solution obtained by the color reaction was measured. Table 1 shows the absorbance obtained by subtracting the absorbance obtained when the 0 U / L sample was measured from the absorbance obtained when the sample A diluted 1000 times was measured.

As is apparent from Table 1, the absorbance when measured using measurement kits 5 to 8 containing a reaction buffer not containing Tween 20, that is, the absence of Tween 20 in the primary reaction is 0.008 to 0.046. On the other hand, when measurement was performed using measurement kits 1 to 4 containing a reaction buffer containing Tween 20, that is, when Tween 20 was present in the primary reaction, the absorbance was 0.112 to 0.714, and the measurement sensitivity increased. Turned out to be.
Further, it was found from the comparison between the measurement kit 1 and the measurement kits 2 to 4 that the measurement sensitivity is 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 comprising the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apo AI protein antibody solution were prepared.

<Reaction buffer (for measurement kits A to X)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Surfactant (surfactants and concentrations listed in Table 2)
BSA 1%
PEG6000 4.8%

<DLH3 antibody immobilized 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 assay kit Z comprising the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apo AI protein antibody solution was prepared.

<Reaction buffer (for measurement kit Z)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L

<DLH3 antibody immobilized 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]
Sample A diluted 1000-fold with each reaction buffer in the same manner as in Example 2, using each of measurement kits A to X of Example 3 and measurement kit Z of Comparative Example 2, and Measure the reaction buffer solution (0 U / L sample) and subtract the absorbance obtained when measuring 0 U / L sample from the absorbance obtained when measuring sample A diluted 1000 times. Calculated. The results are shown in Table 2.

As is apparent from Table 2, in addition to the Tween 20 that showed the effect 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 is increased by performing the primary reaction.

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

<Reaction buffer (for measurement kits a to o)>
Phosphate buffer solution 0.01 mol / L (pH7.4)
Sodium chloride 0.15 mol / L
Tween20 0.01%
BSA 1%
PEG (PEGs and concentrations listed in Table 3)

<DLH3 antibody immobilized 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 assay kit x comprising the following reaction buffer, DLH3 antibody-immobilized microplate, and POD-labeled anti-apo AI protein antibody solution was prepared.

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

<DLH3 antibody immobilized 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]
Sample A diluted 1000-fold with each reaction buffer and reaction buffer using the measurement kits a to o of Example 5 and the measurement kit x of Comparative Example 3 in the same manner as in Example 2. (0 U / L sample) was measured, and an absorbance obtained by subtracting the absorbance obtained when measuring the 0 U / L sample from the absorbance obtained when measuring sample A diluted 1000 times was calculated. The results are shown in Table 3.

As is apparent from Table 3, it was found that the use of PEG having a molecular weight of 200-20000 increases the measurement sensitivity.

According to the present invention, a method for measuring oxidized high density lipoprotein, a measuring reagent, and a measuring kit are provided. The present invention is useful for the diagnosis of cardiovascular diseases such as arteriosclerosis.

Claims (28)

A method for measuring oxidized high density lipoprotein in a sample, comprising the following steps.
(1) A sample and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine are selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant. An immune complex comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to oxidized phosphatidylcholine, or an antibody or antibody fragment that binds to phosphocholine Producing 1;
(2) an antibody or antibody fragment that binds to oxidized phosphatidylcholine by reacting an antibody that binds to high-density lipoprotein or the antibody fragment with the immune complex 1 produced in step (1) in an aqueous medium, or A step of generating an immune complex 2 comprising an antibody that binds to phosphocholine or the antibody fragment, an oxidized high-density lipoprotein, and an antibody or antibody fragment that binds to the high-density lipoprotein;
(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, comprising the following steps.
(1 ′) An aqueous medium containing at least one selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant, and a sample and an antibody or antibody fragment that binds to high-density lipoprotein Producing an immune complex 3 comprising an oxidized high-density lipoprotein and an antibody or antibody fragment that binds to the high-density lipoprotein;
(2 ′) reacting the antibody or antibody fragment that binds to oxidized phosphatidylcholine or the antibody or antibody fragment that binds to phosphocholine with the immune complex 3 produced in the step (1 ′) in an aqueous medium, An immune complex comprising an antibody that binds to high-density lipoprotein 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 Producing 4;
(3 ′) a step of measuring the immune complex 4 generated in the step (2 ′) The method according to claim 1 or 2, wherein the aqueous medium in the step (1) or the step (1 ') further contains at least one substance selected from the group consisting of a 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 polyoxyethylene surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or The method according to any one of claims 1 to 5, which is a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof, or polyoxyethylene fatty acid glyceryl. The method according to any one of claims 1 to 6, wherein the cationic surfactant is a quaternary ammonium salt. The method according to any one of claims 1 to 7, wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apo A protein. The method according to claim 8, wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein. A reagent used in the measurement method according to claim 1 or 2, wherein the antibody or antibody fragment binds to oxidized phosphatidylcholine, the antibody or antibody fragment that binds to phosphocholine, and the antibody that binds to high-density lipoprotein or A reagent for measuring oxidized high density lipoprotein, comprising the antibody fragment and at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant. The reagent according to claim 10, further comprising at least one substance selected from the group consisting of a protein and polyethylene glycol. The reagent according to claim 11, wherein the protein is albumin. The reagent according to claim 12, wherein the albumin is bovine serum albumin. The polyoxyethylene surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or The reagent according to any one of claims 10 to 13, which is a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof, or polyoxyethylene fatty acid glyceryl. The reagent according to any one of claims 10 to 14, wherein the cationic surfactant is a quaternary ammonium salt. The reagent according to any one of claims 10 to 15, wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apo A protein. The reagent according to claim 16, wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein. An antibody that binds to oxidized phosphatidylcholine or the antibody fragment, or an antibody that binds to phosphocholine or the antibody fragment, and at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant, A kit for measuring oxidized high-density lipoprotein, comprising: a first reagent to be contained; and a second reagent containing an antibody or antibody fragment that binds to the high-density lipoprotein. A first reagent comprising an antibody or antibody fragment that binds to high-density lipoprotein, and at least one selected from the group consisting of a polyoxyethylene surfactant and a cationic surfactant, and oxidized phosphatidylcholine A kit for measuring oxidized high-density lipoprotein, comprising an antibody that binds to or an antibody fragment thereof, or an antibody that binds to phosphocholine or a second reagent containing the antibody fragment. Furthermore, the kit according to claim 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. An antibody that binds to oxidized phosphatidylcholine or the antibody fragment; or an antibody that binds to phosphocholine or a first reagent containing the antibody fragment; an antibody that binds to high-density lipoprotein; or a second reagent containing the antibody fragment; and A kit for measuring oxidized high density lipoprotein comprising a third reagent containing at least one selected from the group consisting of a polyoxyethylene-based surfactant and a cationic surfactant. The kit according to claim 21, wherein at least one substance selected from the group consisting of protein and polyethylene glycol is contained in at least one of the first to third reagents. The kit according to claim 20 or 22, wherein the protein is albumin. The kit according to claim 23, wherein the albumin is bovine serum albumin. The polyoxyethylene surfactant is polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkylphenyl ether, polyoxyethylene alkylamine, polyoxyethylene alkyl ether, polyoxyethylene alkyl ether phosphoric acid or The kit according to any one of claims 18 to 24, which is a salt thereof, polyoxyethylene polycyclic phenyl ether, polyoxyethylene polycyclic phenyl ether sulfate or a salt thereof, or polyoxyethylene fatty acid glyceryl. The kit according to any one of claims 18 to 25, wherein the cationic surfactant is a quaternary ammonium salt. The kit according to any one of claims 18 to 26, wherein the antibody that specifically binds to the high-density lipoprotein is an antibody that binds to the apo A protein. 28. The kit according to claim 27, wherein the antibody that binds to the apoA protein is an antibody against the apoA-I protein.