Method for measuring sterols in lipoproteins, reagents and reagent kits for measuring sterols in lipoproteins

Abstract

To provide a novel method for measuring sterol in lipoprotein, a reagent, and a reagent kit.SOLUTION: There is formed a composite body including lipoprotein including a tag-added sterol and a first capturing body, by bringing lipoprotein in a sample obtained from a subject, a tag-added sterol obtained by adding a tag to the C3 position of a sterol skelton, and a first capturing body specifically binding to the tag and having a label material in contact with one another. The above object is achieved by detecting a signal generated by the label material in the composite body.SELECTED DRAWING: Figure 3B

Description

Technical Field

[0001] The present invention relates to a method for measuring sterols in lipoproteins. The present invention relates to a reagent for measuring sterols in lipoproteins. The present invention relates to a reagent kit for measuring sterols in lipoproteins.

Background Art

[0002] In recent years, indices reflecting the functions of lipoproteins have attracted attention. As a method for examining the functions of lipoproteins, for example, the methods described in Patent Documents 1 and 2 are known. In this method, the cholesterol uptake ability, which is a qualitative activity of lipoproteins, is measured. These documents disclose that the cholesterol uptake ability of lipoproteins was measured by contacting lipoproteins in a sample with tagged cholesterol and forming lipoproteins that incorporated tagged cholesterol esterified by lecithin-cholesterol acyltransferase (LCAT) in the sample, and detecting a signal derived from the incorporated tagged cholesterol.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the tagged cholesterol described in Patent Documents 1 and 2, the tag is attached via the hydrocarbon chain at the C17 position of the sterol skeleton, and the hydroxyl group at the C3 position can be esterified by LCAT. The methods described in Patent Documents 1 and 2 do not use cholesterol in which the tag is attached to other parts of the sterol skeleton. As a method for functional analysis of lipoproteins, there are still few methods for measuring sterols in lipoproteins, and further development of measurement methods is needed. [Means for solving the problem]

[0005] The present invention provides a method for measuring sterols in a lipoprotein, wherein the tag is attached to the C3 position of the sterol backbone, in the case of a tagged sterol. The method comprises the steps of: contacting a lipoprotein in a sample with a tagged sterol and a first capture body that specifically binds to the tag and has a labeling substance, to form a complex containing a lipoprotein containing a tagged sterol and a first capture body; and detecting a signal generated by the labeling substance contained in the complex.

[0006] The present invention provides a reagent for measuring sterols in lipoproteins, which is used in the above-mentioned method, comprising a tagged sterol, wherein the tag is attached to the C3 position of the sterol skeleton.

[0007] The present invention provides a reagent kit for measuring sterols in lipoproteins, comprising a first reagent containing a tagged sterol and a second reagent containing a first capture body that specifically binds to the tag and has a labeling substance, wherein the tagged sterol has the tag attached to the C3 position of the sterol backbone. [Effects of the Invention]

[0008] The present invention provides a method for measuring sterols in lipoproteins, as well as reagents and reagent kits that can be used in this method. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows an example of a reagent for measuring sterols in lipoproteins according to this embodiment. [Figure 2A] This figure shows an example of a reagent kit for measuring sterols in lipoproteins according to this embodiment. [Figure 2B] This figure shows an example of a reagent kit for measuring sterols in lipoproteins according to this embodiment. [Figure 2C] This figure shows an example of a reagent kit for measuring sterols in lipoproteins according to this embodiment. [Figure 2D] This figure shows an example of a reagent kit for measuring sterols in lipoproteins according to this embodiment. [Figure 3A] This graph shows the results of measuring high-density lipoprotein (HDL) fractions (C0-C5) at different concentrations using tagged sterols (C5-Amide). [Figure 3B] This graph shows the results of measuring C0-C5 using tagged sterols (PEG3). [Figure 3C] This graph shows the results of measuring C0-C5 using tagged sterols (PEG7). [Figure 3D] This graph shows the results of measuring C0-C5 using tagged sterols (PEG11). [Figure 3E] This graph shows the results of measuring C0-C5 using tagged sterols (PEG23). [Figure 4A] This graph shows the results of measuring samples (sample H and sample L) containing HDL with different cholesterol uptake capabilities using tagged sterols (C5-Amide). [Figure 4B] This graph shows the results of measuring samples H and L using tagged sterols (PEG3). [Figure 4C] This graph shows the results of measuring samples H and L using tagged sterols (PEG7). [Figure 4D]This graph shows the results of measuring samples H and L using tagged sterols (PEG11). [Figure 4E] This graph shows the results of measuring samples H and L using tagged sterols (PEG23). [Figure 5A] This graph shows the results of measuring tagged sterol (C2-Amide) using sample H and sample L. [Figure 5B] This graph shows the results of measuring samples H and L using tagged sterols (PEG3). [Figure 5C] This graph shows the results of measuring samples H and L using tagged sterols (PEG7). [Figure 5D] This graph shows the results of measuring samples H and L using tagged sterols (PEG11). [Figure 5E] This graph shows the results of measuring samples H and L using tagged sterols (PEG23). [Figure 6A] This graph shows the results of measuring C0-C5 using tagged sterols (PEG1). [Figure 6B] This graph shows the results of measuring C0-C5 using tagged sterols (PEG2). [Figure 7A] This graph shows the results of measuring samples H and L using tagged sterols (PEG1). [Figure 7B] This graph shows the results of measuring samples H and L using tagged sterols (PEG2). [Figure 8A] This graph shows the results of measuring C0-C5 using tagged sterols (PEG7 (ether)). [Figure 8B] This graph shows the results of measuring samples H and L using tagged sterols (PEG7 (ether)). [Figure 9A] This graph shows the results of measuring LCAT-added samples (rLCAT+) and LCAT-unadded samples (rLCAT-) using a tagged sterol (C2-Amide). [Figure 9B] This graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG3). [Figure 9C] This graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG7). [Figure 9D] This graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG11). [Figure 9E] This graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG23). [Figure 10] This graph shows the results of measuring samples with and without N-ethylmaleimide (NEM) added using tagged sterols (PEG3). [Figure 11] This graph shows the results of measuring samples oxidized with hydrogen peroxide and samples without hydrogen peroxide using tagged sterols (PEG3). [Modes for carrying out the invention]

[0010] Naturally occurring cholesterol, upon contact with lipoproteins in the body, is incorporated into those lipoproteins. Free cholesterol in the blood initially binds to the surface (phospholipid membrane) of lipoproteins. Here, when the hydroxyl group at the C3 position of cholesterol is esterified by LCAT, its lipophilicity improves, and the cholesterol moves from the surface to the center of the lipoprotein particle. On the other hand, tagged sterols used in the method for measuring sterols in lipoproteins of this embodiment (hereinafter also referred to as "the measurement method of this embodiment") have a tag attached to the C3 position of the sterol skeleton, so esterification by LCAT does not occur. In this specification, "sterol skeleton" refers to the skeleton represented by the following formula.

[0011] [ka] (In the formula, the numbers represent the positions of carbon atoms in the sterol skeleton.)

[0012] The surface layer of lipoproteins in samples obtained from living organisms contains endogenous cholesterol derived from the organism. The samples also contain endogenous LCAT derived from the organism. The endogenous cholesterol on the surface of the lipoprotein is esterified by LCAT to produce cholesterol esters. These cholesterol esters then migrate into the interior of the lipoprotein. This migration of endogenous cholesterol from the surface to the interior creates space for tagged cholesterol to bind to the surface. While tagged cholesterol can come into contact with LCAT, as mentioned above, tagged sterols are not considered to be esterified by LCAT. Therefore, tagged sterols are thought to remain on the surface of the lipoprotein. If a lipoprotein can move more endogenous cholesterol from the surface to the interior, it is thought that more tagged sterols can bind to the surface. When more tagged sterols bind to the surface of a lipoprotein, a stronger signal will be detected from that lipoprotein. Therefore, the signal obtained by the measurement method of this embodiment can serve as an indicator of the function of the lipoprotein.

[0013] When a sample comes into contact with a tagged sterol, the tagged sterol is thought to bind to the surface of the lipoprotein, and the tag portion is thought to be exposed on the outer surface of the lipoprotein. Here, "outer surface of the lipoprotein" refers to the outer surface of the lipoprotein particle. "Exposed on the outer surface" means both being on the outer surface of the lipoprotein and protruding from the outer surface of the lipoprotein. The tagged sterol in the lipoprotein is detected by binding the tag exposed on the outer surface of the lipoprotein containing the tagged sterol to a first capture body that specifically binds to the tag. The steps in the measurement method of this embodiment are described below.

[0014] In the measurement method of this embodiment, a complex is formed by bringing a lipoprotein containing a tagged sterol, a tagged sterol, and a first capture body that specifically binds to the tag and has a labeling substance, thereby forming a complex containing a lipoprotein containing a tagged sterol and the first capture body.

[0015] The sample is not particularly limited as long as it contains lipoproteins. Examples of such samples include blood samples. Examples of blood samples include blood (whole blood), plasma, serum, etc. A sample containing lipoproteins may be separated or fractionated by known methods such as ultracentrifugation or polyethylene glycol (PEG) precipitation to obtain a fraction containing a predetermined lipoprotein. The fraction containing the predetermined lipoprotein obtained in this way may be used as the lipoprotein-containing sample.

[0016] The lipoprotein may be any of the following: high-density lipoprotein (HDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), or chylomicron (CM). HDL is a lipoprotein with a density of 1.063 g / mL or higher. LDL is a lipoprotein with a density of 1.019 g / mL or higher and less than 1.063 g / mL. IDL is a lipoprotein with a density of 1.006 g / mL or higher and less than 1.019 g / mL. VLDL is a lipoprotein with a density of 0.95 g / mL or higher and less than 1.006 g / mL. CM is a lipoprotein with a density of less than 0.95 g / mL. The preferred lipoprotein is HDL.

[0017] Samples containing lipoproteins may be diluted. For example, to adjust the lipoprotein concentration, a solution obtained by diluting the above-mentioned blood sample or a fraction containing a specific lipoprotein in an aqueous medium may be used as the sample. Examples of aqueous mediums include water, physiological saline, and buffer solutions. Examples of buffer solutions include phosphate-buffered saline (PBS), Tris-HCl, and Good's buffer.

[0018] The concentration of apolipoproteins, which are components of lipoproteins, serves as an indicator of the lipoprotein concentration in the sample. Based on the apolipoprotein concentration, the lipoprotein concentration in the sample may be adjusted by diluting the lipoprotein-containing sample. The apolipoprotein concentration can be measured by known immunological assay methods (e.g., immunoturbidimetry). ApoAI or ApoE are preferred as the apolipoprotein.

[0019] Blocking agents may be added to the sample as needed. Examples of blocking agents include casein, bovine serum albumin (BSA), cyclic oligosaccharides (e.g., cyclodextrin, hydroxypropyl cyclodextrin, etc.), and 2-methacryloyloxyethyl phosphorylcholine polymers (e.g., the Lipidure® series from NOF Corporation, particularly Lipidure-BL203). In addition, fatty acids or compositions containing them (e.g., liposomes) necessary for the esterification reaction of cholesterol by lipoproteins may be added to the sample. Liposomes can be prepared, for example, by mixing dimyristoyl phosphatidylglycerol, cholesterol, and hydrogenated soybean phosphatidylcholine.

[0020] Tagged sterols are sterols having a sterol skeleton represented by the above formula, in which a tag is directly or indirectly attached to the carbon atom at the C3 position. Here, "indirectly attached to the carbon atom at the C3 position" includes the attachment of a tag to the carbon atom at the C3 position of the sterol via a linker, the attachment of a tag via a substituent if one is bonded to the carbon atom at the C3 position of the sterol, and the attachment of a tag via a linker to the substituent at the C3 position as described above. Preferably, tagged sterols have a hydrocarbon chain at the C17 position of the sterol skeleton represented by the above formula, which may have a substituent.

[0021] The tagged sterol can be prepared by adding a tag to the C3 position of the sterol. The tag may be added directly to the carbon atom at the C3 position of the sterol or via a linker. When a substituent is bonded to the carbon atom at the C3 position of the sterol, the tag may be added to this substituent. Alternatively, the tag may be added to the substituent at the C3 position of the sterol via a linker. For example, when using a sterol having a hydroxy group at the C3 position such as cholesterol, the tag may be added directly to the hydroxy group at the C3 position or via a linker. The sterol used for the preparation of the tagged sterol is preferably bonded to the surface of the lipoprotein. Examples of the sterol include cholesterol and its analogs. Examples of cholesterol analogs include epicholesterol, allocholesterol, cholestanol, coprostanol, 7-dehydrocholesterol, stigmasterol, sitosterol, campesterol, α-spinasterol, brassicasterol, 24-methylene sterol, and the like.

[0022] Examples of the tagged sterol include, for example, a compound represented by the following formula (I) (hereinafter, also referred to as "tagged sterol of formula (I)").

[0023] [Chemical formula] (In the formula, the double line of the solid line and the broken line each independently represents a single bond or a double bond, R 1 is an alkyl group having 1 to 6 carbon atoms which may have a substituent, or an alkenyl group having 2 to 6 carbon atoms which may have a substituent, X and Y are the same or different and are -R 2 -NH-, -NH-R 2 -, -R 2 -(C=O)-NH-, -(C=O)-NH-R 2 -, -R 2 -NH-(C=O)-, -NH-(C=O)-R 2 -, -R 2 -(C=O)-, -(C=O)-R 2-, -R 2 -(C=O)-O-, -(C=O)-OR 2 -, -R 2 -O-(C=O)-, -O-(C=O)-R 2 -, -R 2 -(C=S)-NH-, -(C=S)-NH-R 2 -, -R 2 -NH-(C=S)-, -NH-(C=S)-R 2 -, -R 2 -O-, -OR 2 -, -R 2 -S-, or -SR 2 - is represented by, where R 2 Each of these is independently a bond, an alkylene group having 1 to 10 carbon atoms which may have substituents, an arylene group or heteroarylene group having 6 to 12 carbon atoms which may have substituents, or a cycloalkylene group or heterocycloalkylene group having 3 to 8 carbon atoms which may have substituents. L is -(CH2) d -[R 3 -(CH2) e ] f -, or -[(CH2) e -R 3 ] f -(CH2) d - is represented by, where R 3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH- or a bond, Z is a tag, a and c are either the same or different integers between 0 and 6, and are between 0 and 6. b is either 0 or 1. d and e are integers, either identical or distinct, between 0 and 12. f is an integer between 0 and 24 (inclusive).

[0024] In equation (I), "-[X] a -[L] b -[Y] cThe part represented by "-" corresponds to a linker that connects the tag and the sterol moiety. In formula (I), when a, b, and c are all 0, the tagged sterol represented by this formula does not have a linker. That is, the tag is bonded to the oxygen atom at the C3 position of the sterol moiety (hereinafter also referred to as "C3 O"). In formula (I), when any of a, b, and c is not 0, the tagged sterol of formula (I) has a linker between the tag and the sterol moiety. It is thought that the linker makes it easier for the tag exposed on the outer surface of the lipoprotein to bind to the first capture body. The substituents of formula (I) are described below.

[0025] Preferably, in formula (I), either the bond between C5 and C6 or the bond between C7 and C8 is a double bond, or both the bond between C5 and C6 and the bond between C7 and C8 are single bonds. Particularly preferably, in formula (I), the bond between C5 and C6 is a double bond, and the bond between C7 and C8 is a single bond.

[0026] R 1 The main chain consists of an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms, and may have substituents at any of these positions. 1 When the compound has substituents, the carbon number mentioned above does not include the carbon number of the substituents. Examples of alkyl groups with 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, and hexyl groups. Examples of alkenyl groups with 2 to 6 carbon atoms include vinyl, propenyl, butenyl, pentenyl, and hexenyl groups.

[0027] R 1 Examples of substituents in include methyl, ethyl, phenyl, naphthyl, benzyl, alkoxy, nitro, halogen, haloalkyl, and thioether groups. Halogen represents fluorine, chlorine, bromine, or iodine. Alkoxy represents an -O-alkyl group, which is a linear or branched saturated aliphatic hydrocarbon group having 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.1 It may have multiple substituents. 1 A methyl group is particularly preferred as the substituent in this. When the substituent is a methyl group, preferred R 1 This is a 1,5-dimethylhexyl group. This is the same as the alkyl chain at positions C20 to C27 of naturally occurring cholesterol.

[0028] When a is an integer greater than or equal to 1, [X] a O, who is in third place in C, and L, [Y] c Alternatively, it corresponds to the connection part with Z (tag). L corresponds to a spacer and has a linear structure that gives a predetermined length to the linker. When c is an integer of 1 or more, [Y] c These are Z (tag), L, and [X] a Alternatively, it corresponds to the linkage with O at the C3 position. X and Y are determined according to the type of reaction that binds the sterol moiety to the linker and the reaction that binds the linker to the tag.

[0029] R 2 and R 3 In relation to this, a bonding bond refers to a direct bond that does not involve other atoms in between.

[0030] R 2 However, when the alkylene group has 1 to 10 carbon atoms, examples of such alkylene groups include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene, 2-ethylhexylene, nonylene, and desilene. Among these, alkylene groups with 1 to 4 carbon atoms are preferred. 2 However, when it is an alkylene group with substituents, the above carbon number does not include the carbon atoms of the substituents.

[0031] R 2However, when it is an arylene group or a heteroarylene group, such a group may be an aromatic ring with 6 to 12 carbon atoms, which may contain one or more heteroatoms selected from N, S, O, and P. Examples include groups such as phenylene, naphthylene, biphenylene, furanylene, pyrlorene, thiophenylene, triazolene, oxadiazolene, pyridylene, and pyrimidylene. 2 However, when it is an arylene group or heteroarylene group having a substituent, the above carbon number does not include the carbon number of the substituent.

[0032] R 2 However, when it is a cycloalkylene group or a heterocycloalkylene group, such a group may be a non-aromatic ring having 3 to 8 carbon atoms, which may contain one or more heteroatoms selected from N, S, O, and P. Examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, pyrrolidinylene, piperidinylene, and morpholinylene. 2 However, when it is a substituted cycloalkylene group or heterocycloalkylene group, the above carbon number does not include the carbon number of the substituent.

[0033] R 2 Examples of substituents in R2 include hydroxy, cyano, alkoxy, nitro, =O, =S, -SH, halogen, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether groups. R2 may have multiple substituents. Here, halogen represents fluorine, chlorine, bromine, or iodine. Alkoxy represents an -O-alkyl group, which is a linear or branched saturated aliphatic hydrocarbon group having 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.

[0034] It is preferable that L has a structure that does not inhibit the binding of lipoproteins and cholesterol, and whose linker portion is not easily incorporated into lipoproteins. Examples of such structures include structures containing hydrophilic polymers. For example, in formula (I), b is 1 and R3 It is preferable that is an oxygen atom. In this case, L is -(CH2) d -[O-(CH2) e ] f -or-[(CH2) e -O] f -(CH2) d It is represented by -. Here, d is an integer between 0 and 12, e is an integer between 1 and 6, and f is an integer between 1 and 24. Preferably, d and e are the same or different integers between 1 and 4. More preferably, d and e are 2. f is preferably an integer between 1 and 23, more preferably an integer between 3 and 23, and particularly preferably an integer between 3 and 11.

[0035] In a preferred embodiment, in formula (I), a is 0 or 1, b and c are 1, and X is -NH-(C=O)-R 2 Represented by -, Y is -R 2 It is represented as -(C=O)-NH-, R 2 Each of these is an unsubstituted alkylene group having 1 to 6 carbon atoms, and L is -[(CH2)2-O] f -(CH2) d It is represented by -, where d is an integer between 1 and 6 (inclusive), and f is an integer between 0 and 24 (inclusive). Furthermore, R 1 It is more preferable that the group is a 1,5-dimethylhexyl group.

[0036] For example, in equation (I), a, b, and c are 1, and X is -NH-(C=O)-(CH2) n It is represented by - (where n is an integer between 1 and 6, preferably 2, 3, or 4), Y is represented by -(CH2)4-(C=O)-NH-, and L is -[(CH2)2-O] f It is represented as -(CH2)2-, where f is an integer between 1 and 23 (inclusive). Alternatively, in equation (I), a, b, and c are 1, and X is -NH-(C=O)-(CH2) n It is represented as - (where n is an integer between 1 and 6, preferably 2, 3, or 4), Y is represented as -(CH2)4-(C=O)-NH-, and L is -(CH2) fIt is represented by -, where f is an integer between 1 and 6, preferably between 2 and 5. Alternatively, in equation (I), a is 0, b and c are 1, Y is represented by -(CH2)4-(C=O)-NH-, and L is -[(CH2)2-O] f It is represented as -(CH2)2-, and f is an integer between 1 and 23. In either case, R 1 It is more preferable that the group is a 1,5-dimethylhexyl group.

[0037] The tag is not particularly limited, as long as a substance capable of specifically binding to it exists or can be obtained. The tag may be either a naturally occurring or synthetic substance, such as a compound, peptide, protein, nucleic acid, or complex thereof. Examples of combinations of a tag and a substance capable of specifically binding to it include an antigen and an antibody that recognizes the antigen, a hapten and an anti-hapten antibody, a peptide or protein and an aptamer that recognizes them, a ligand and its receptor, an oligonucleotide and an oligonucleotide having a complementary chain, biotins and avidins, a histidine tag (a peptide containing 6-10 histidine residues) and Ni-NTA (nitrilotriacetate chelated with nickel ions), glutathione-S-transferase (GST) and glutathione. The antigen used as the tag may be a peptide tag or protein tag known in this technology, such as FLAG®, hemagglutinin (HA), Myc protein, or green fluorescent protein (GFP). Examples of haptens used as tags include the 2,4-dinitrofer (DNP) group. Anti-DNP antibodies are suitable as capture agents that specifically bind to DNP.

[0038] In this specification, "biotins" includes biotin and its analogues. Examples of biotin analogues include desthiobiotin and biocitin. In this specification, "avidins" includes avidin and its analogues. Examples of avidin analogues include streptavidin, avidin-like protein derived from Tamogitake mushroom (Tamavidin®), bladavidin, and rizavidin.

[0039] The compound used as a tag may be, for example, a labeling compound known in the art. Examples of such compounds include biotin groups and dye compounds. In this specification, "biotin group" refers to the heterocyclic portion of the chemical structure of biotins that includes at least an imidazolidine ring. The preferred biotin group is the biotin group of biotin. Suitable capture agents that specifically bind to the biotin group include avidins. An example of a dye compound is borondipyrometen (BODIPY®). A commercially available capture agent that binds to BODIPY is an anti-BODIPY antibody (BODIPY FL Rabbit IgG Fraction, A-5770, Lifetechnologies). Among these, the biotin group is preferred as a tag.

[0040] The tagged sterol is preferably tagged cholesterol. Examples of tagged cholesterol include biotin-added cholesterol represented by the following formulas (II), (III), or (IV).

[0041] [ka] (In the formula, n is an integer between 1 and 23, inclusive.)

[0042] [ka] (In the formula, n is an integer between 1 and 7, inclusive.)

[0043] [ka] (In the formula, n is an integer between 2 and 5, inclusive.)

[0044] In tagged sterols, the mode of bonding between the sterol moiety and the tag is not particularly limited, but covalent bonding is preferred. For example, the tag may be covalently bonded to the C3 position of the sterol moiety, or the C3 position of the sterol moiety and the tag may be covalently bonded via a linker. The bonding method is not particularly limited, but crosslinking using a functional group is simple and preferred. The functional group is not particularly limited, but for example, amino groups, carboxyl groups, and sulfhydryl groups are preferred because commercially available crosslinkers can be used.

[0045] When cholesterol is used as a sterol, the hydroxyl group at the C3 position is unreactive as a functional group. Therefore, as shown in the first step of the figure below, for example, the hydroxyl group at the C3 position may be reacted with tosyl chloride to produce tosylate (in the figure, R-OH is cholesterol). Then, as shown in the second step of the figure below, the tosylate is reacted with a tagged alcohol to remove the tosyl group and obtain tagged cholesterol (in the figure, R'-OH is a tagged alcohol).

[0046] [ka]

[0047] Alternatively, as shown in the first step of the figure below, an ether may be produced by reacting the hydroxyl group at the C3 position of cholesterol with an alkylating agent such as ethyl bromo, ethyl bromopropionate, or ethyl bromobutyrate (in the figure, R-OH is cholesterol, R' is, for example, a methylene group, an ethylene group, or a propylene group, and Et is an ethyl group). Then, as shown in the second step of the figure below, a carboxyl group may be conferred to the C3 position of cholesterol by hydrolysis of the product with an alkali such as potassium hydroxide. Tag-added cholesterol can be obtained by attaching a tag to this carboxyl group at the C3 position by a cross-linking reaction.

[0048] [ka]

[0049] The functional groups present in a tag vary depending on the type of tag. For example, when using a peptide or protein as a tag, amino groups, carboxyl groups, and sulfhydryl groups (SH groups) can be used. When using biotin as a tag, the carboxyl groups of the biotin side chain can be used. The linker is preferably a compound with a chain structure having functional groups at both ends (e.g., a polymer compound). When adding biotin as a tag, commercially available biotin labeling reagents may be used. These reagents contain biotin to which spacer arms of various lengths (e.g., PEG chains) having functional groups at the ends are attached.

[0050] The following describes a typical crosslinking reaction of functional groups. A compound having a carboxyl group as a functional group can be bonded to a compound having an amino group as a reactant through the three steps shown in the figure below. First, as shown in the first step of the figure below, the compound having a carboxyl group is reacted with a compound having a carbodiimide group (-N=C=N-) (in the figure below, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC)). Next, as shown in the second step of the figure below, the product from the first step is reacted with NHS to form an unstable NHS ester. Then, as shown in the third step of the figure below, the product from the second step is reacted with the compound having an amino group to crosslink the two. For example, when linking a sterol or linker having a carboxyl group to a tag having an amino group, crosslinking can be performed in this way. Alternatively, when crosslinking a sterol or linker having a carboxyl group to a tag having a carboxyl group, a crosslinking reagent having amino groups at both ends may be used.

[0051] [ka]

[0052] Compounds having an amino group as a functional group can be crosslinked with compounds having an N-hydroxysuccinimide (NHS) ester or an isothiocyano group as a reactive group, as shown in the figure below. For example, when crosslinking a linker having an amino group with a tag having an amino group, a crosslinking reagent having NHS esters at both ends may be used.

[0053] [ka]

[0054] Compounds having a sulfhydryl group as a functional group can be crosslinked with compounds having a maleimide group or a bromo(or iod)acetamide group as a reactive group, as shown in the figure below. For example, when crosslinking a linker having a sulfhydryl group with a tag having a sulfhydryl group, a crosslinking reagent having maleimide at both ends may be used.

[0055] [ka]

[0056] When covalently bonding a compound having a carboxyl group as a functional group with a compound having an amino group as a reactive group, an amidation reaction using a known condensing agent may be used. Examples of such condensing agents include O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphonate (HATU), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride, 2-chloro-1,3-dimethylimidazolinium, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, diphenyl phosphoryl azide, chlorotripyrrolidinophosphonium hexafluorophosphate, and N,N'-diisopropylcarbodiimide.

[0057] The above cross-linking and amidation reactions can be carried out at room temperature and atmospheric pressure. The solvent used in the reaction is not particularly limited, as long as it is inert to the reaction and can dissolve or disperse each compound involved in the reaction. Examples of such solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; ethers such as tetrahydrofuran (THF), diethyl ether, ethylene glycol dimethyl ether, and 1,4-dioxane; amides such as N,N-dimethylformamide (DMF); sulfoxides such as dimethyl sulfoxide; and halogenated hydrocarbons such as dichloromethane and chloroform. These solvents can be used individually or as mixtures.

[0058] The first capture body, which specifically binds to the tag and has a labeling substance, is a capture body that is labeled with a labeling substance and specifically binds to the tag. The capture body that specifically binds to the tag can be appropriately determined depending on the type of tag. For example, by referring to the above-mentioned combination of the tag and a substance that can specifically bind to the tag, it can be selected from antibodies, aptamers, ligand receptors, oligonucleotides, biotins, avidins, histidine tags, Ni-NTA, GST, glutathione, etc. Among these, avidins or antibodies are preferred. As avidins, avidin or streptavidin is preferred.

[0059] In this specification, the term "antibody" also includes antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, domain antibodies (dAb), reduced IgG (rIgG), diabodies, triabodies, and so on. The antibody may be either a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited and may be derived from any mammal such as mouse, rat, hamster, rabbit, goat, horse, or camel. The antibody isotype may be any of IgG, IgM, IgE, or IgA, but IgG is preferred. The antibody that specifically binds to the tag may be a commercially available antibody or an antibody produced by a method known in the art.

[0060] The labeling substance is not particularly limited and can be, for example, a substance that generates a signal itself (hereinafter also called a "signal-generating substance") or a substance that generates a signal by catalyzing the reaction of other substances. Examples of signal-generating substances include fluorescent substances and radioactive isotopes. Examples of substances that generate a detectable signal by catalyzing the reaction of other substances include enzymes. Enzymes generate signals such as light and color by reacting with appropriate substrates. Examples of enzymes include alkaline phosphatase (ALP), peroxidase (POD), β-galactosidase, and luciferase. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor®, and fluorescent proteins such as GFP. Examples of radioactive isotopes include, for example 125 I, 14 C, 32 Examples include P. As labeling substances, enzymes are preferred, and ALP and POD are particularly preferred.

[0061] Labeling of a capture entity with a labeling substance that specifically binds to a tag can be performed by directly or indirectly binding the labeling substance to the capture entity. A capture entity to which the labeling substance is directly bound is, for example, a capture entity to which the labeling substance is covalently bound. This can be obtained, for example, by covalently bonding the labeling substance and the capture entity using a commercially available labeling kit or crosslinker. If both the capture entity and the labeling substance are proteins, the capture entity to which the labeling substance is directly bound may be a fusion protein of the capture entity and the labeling substance. This can be produced by genetic engineering techniques known in the art.

[0062] When a labeling substance is indirectly bound to a capture agent, a substance to which the labeling substance is covalently bound and which specifically binds to the capture agent can be used. For example, if the capture agent that specifically binds to the tag is an avidin, a biotin to which the labeling substance is covalently bound (labeled biotin) can be used. Avidins usually form tetramers and can bind to four molecules of biotin. By mixing avidins and labeled biotin in a ratio such that one or two molecules of labeled biotin bind to each tetrameric avidin, avidins bound with labeled biotin can be obtained. If the capture agent that specifically binds to the tag is an antibody, an antibody to which the labeling substance is covalently bound and which specifically binds to the antibody (labeled secondary antibody) can be used.

[0063] In a preferred embodiment, the first capture agent is a labeled antibody, labeled avidin, or labeled streptavidin that specifically binds to the tag. As the labeled antibody, an antibody to which a labeling substance is covalently attached or an antibody to which a labeling substance is fused is preferred. As the labeled avidin and labeled streptavidin, avidin and streptavidin to which a labeling substance is covalently attached, avidin and streptavidin to which a labeling substance is fused, and avidin and streptavidin to which one or two molecules of labeled biotin are bound are preferred.

[0064] Contact between the lipoprotein in the sample, the tagged sterol, and the first capture agent can be achieved by mixing the sample, the tagged sterol, and the first capture agent. This mixing is thought to cause the tagged sterol to bind to the lipoprotein, and the first capture agent to bind to the tag exposed on the outer surface of the lipoprotein. As a result, a complex is formed containing the lipoprotein containing the tagged sterol and the first capture agent. The order of mixing is not particularly limited; the sample, the tagged sterol, and the first capture agent may be mixed substantially simultaneously or sequentially.

[0065] The amount of tagged sterol added is not particularly limited. For example, tagged sterol can be added to the sample to a final concentration of 10 nM to 10 μM, preferably 20 nM to 5 μM. The amount of the first capture agent added is not particularly limited and can be appropriately set depending on the type of capture agent and labeling substance.

[0066] In a preferred embodiment, the lipoprotein in the sample is first brought into contact with the tagged sterol. This causes the tagged sterol to bind to the lipoprotein. Subsequently, the lipoprotein containing the tagged sterol is brought into contact with the first capture body. This causes the first capture body to bind to the tag exposed on the outer surface of the lipoprotein, forming a complex containing the lipoprotein containing the tagged sterol and the first capture body. The temperature and time conditions for mixing the sample and the tagged sterol are not particularly limited. For example, the mixture of the sample and the tagged sterol may be incubated at 20 to 48°C, preferably 25 to 42°C, for 1 minute to 24 hours, preferably 10 minutes to 2 hours. During incubation, the mixture may be left to stand, stirred, or shaken. The temperature and time conditions for mixing the lipoprotein containing the tagged sterol with the first capture body are also not particularly limited and can be appropriately determined from the above range.

[0067] In a preferred embodiment, a second capture agent that specifically binds to lipoproteins is used for contact between the lipoprotein, the tagged sterol, and the first capture agent. Specifically, the sample, the tagged sterol, the first capture agent, and the second capture agent are mixed. The order of mixing is not particularly limited. Furthermore, the amount of the second capture agent added is not particularly limited and can be appropriately set depending on the type of capture agent, etc.

[0068] The second capture agent is not particularly limited as long as it is a substance that can specifically bind to a portion of the surface of the lipoprotein. Examples of such substances include antibodies and aptamers. As the second capture agent, an antibody that specifically binds to lipoproteins is preferred, and an antibody that can specifically bind to apolipoproteins, which are components of lipoproteins, is more preferred. Examples of such antibodies include anti-ApoA antibodies (anti-ApoAI antibodies and anti-ApoAII antibodies), anti-ApoB antibodies and anti-ApoE antibodies (anti-ApoE2 antibodies, anti-ApoE3 antibodies and anti-ApoE4 antibodies). Among these, anti-ApoAI antibodies are particularly preferred. Commercially available anti-lipoprotein antibodies or anti-ApoAI antibodies may also be used.

[0069] Upon contact between the lipoprotein, the tagged sterol, and the first capture, further contact with the second capture is made, thereby forming a complex of the first capture, the lipoprotein to which the tagged sterol is bound, and the second capture. In this complex, the first capture binds to the tag exposed on the outer surface of the lipoprotein, and the second capture binds to the surface of the lipoprotein. That is, the lipoprotein containing the tagged sterol is sandwiched between the first and second captures. In this embodiment, the complex of the first capture, the lipoprotein containing the tagged sterol, and the second capture is also referred to below as the "sandwich complex".

[0070] The preferred sequence of contacts in the formation of the sandwich complex is as follows: First, the lipoprotein in the sample is brought into contact with the tagged sterol. This causes the tagged sterol to bind to the lipoprotein. Next, the lipoprotein containing the tagged sterol is brought into contact with the second capture. This causes the second capture to bind to the surface of the lipoprotein containing the tagged sterol, forming a complex of the lipoprotein containing the tagged sterol and the second capture. Subsequently, the complex of the lipoprotein containing the tagged sterol and the second capture is brought into contact with the first capture. This causes the first capture to bind to the tag exposed on the outer surface of the lipoprotein, forming a sandwich complex.

[0071] The temperature and time conditions for mixing the lipoprotein containing tagged sterols with the second capture agent are not particularly limited. For example, the mixture of the lipoprotein containing tagged sterols and the second capture agent may be incubated at 20-48°C, preferably 25-42°C, for 1 minute to 24 hours, preferably 10 minutes to 2 hours. During incubation, the mixture may be left to stand, stirred, or shaken.

[0072] The complex of the lipoprotein containing the tagged sterol and the second capture agent may be formed on a solid phase. For example, the complex can be formed on a solid phase by bringing the lipoprotein in the sample, the tagged sterol, the second capture agent, and the solid phase into contact. The order of contact is not particularly limited, but for example, the lipoprotein in the sample may be brought into contact with the tagged sterol first, and then the lipoprotein containing the tagged sterol, the second capture agent, and the solid phase may be brought into contact. Alternatively, the lipoprotein containing the tagged sterol may be brought into contact with the second capture agent first, and then the complex of the lipoprotein containing the tagged sterol and the second capture agent may be brought into contact with the solid phase.

[0073] The second capture body may be pre-immobilized on a solid phase. For example, after contacting the lipoprotein in the sample with the tagged sterol, the lipoprotein containing the tagged sterol may be contacted with the solid phase on which the second capture body is immobilized. Alternatively, the lipoprotein containing the tagged sterol may be contacted with the solid phase on which the second capture body is immobilized. The temperature and time conditions when using a solid phase are not particularly limited. For example, the same conditions as those for mixing the lipoprotein containing the tagged sterol with the second capture body may be used.

[0074] The solid phase can be any insoluble carrier capable of immobilizing the second capture. For example, the capture can be immobilized on the solid phase by direct or indirect bonding between the solid phase and the second capture. Examples of direct bonding between the solid phase and the second capture include adsorption to the solid phase surface via hydrophobic interactions or covalent bonding. For example, if the second capture is an antibody and the solid phase is an ELISA microplate, the antibody can be immobilized in the plate wells by adsorption. Also, if the second capture is an antibody and the solid phase has functional groups on its surface, the antibody can be immobilized on the solid phase surface by covalent bonding using the functional groups. For example, if the solid phase is a particle having a carboxyl group, the crosslink reaction of the carboxyl group compound described above can be used. Specifically, the carboxyl group on the particle surface is activated with WSC, and then reacted with NHS to form an NHS ester. When the particle having the NHS ester is brought into contact with the antibody, the NHS ester reacts with the amino group of the antibody, and the antibody is covalently immobilized on the particle surface.

[0075] One method of indirect binding between a solid phase and a second capture is binding via a molecule that specifically binds to the second capture. By pre-immobilizing such a molecule on the surface of the solid phase, the second capture can be immobilized on the solid phase. For example, when the second capture is an antibody, molecules that specifically bind to the second capture include protein A, protein G, and antibodies that specifically recognize antibodies (secondary antibodies). Alternatively, the two can be bound using a combination of substances that interpose between the second capture and the solid phase. Examples of such combinations include biotins and avidins, and haptens and anti-hapten antibodies. For example, if the second capture is pre-modified with DNP, the second capture can be immobilized on the solid phase by a solid phase immobilized with an anti-DNP antibody.

[0076] The solid-phase material can be selected from organic polymers, inorganic compounds, biopolymers, etc. Examples of organic polymers include latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and polyvinyl acetate acrylate. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, cobalt, and ferrite, etc.), silica, alumina, and glass. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these may be used in combination.

[0077] The shape of the solid phase is not particularly limited and examples include particles, microplates, microtubes, and test tubes. Among these, particles and microplates are preferred, and magnetic particles are particularly preferred. When the solid phase is in the form of particles, a suspension of particles can be used as the solid phase for the formation of the composite. When the solid phase is in the form of a container such as a microplate, the formation of the composite can be carried out within the container as the solid phase. When a second capture body immobilized on magnetic particles is used, the measurement may be performed using a commercially available fully automated immunoassay analyzer such as the HISCL® series (Sysmex Corporation).

[0078] Bound / Free (B / F) separation may be performed between the contact of the lipoprotein containing tagged sterols with the second capture body and the contact of the formed complex with the first capture body to remove unreacted free components. Unreacted free components are those that do not constitute the complex between the lipoprotein containing tagged sterols and the second capture body. Examples include free tagged sterols that did not bind to the lipoprotein, free second capture bodies that did not bind to the lipoprotein, and impurities in the sample. The means of B / F separation are not particularly limited, but for example, the complex can be separated from the unreacted free components by recovering only the complex using ultracentrifugation. If the complex is formed on a solid phase, and the solid phase is particles, the particles can be recovered by centrifugation or magnetic separation and the supernatant can be removed to separate the complex from the unreacted free components. If the solid phase is a container such as a microplate or microtube, the complex can be separated from the unreacted free components by removing the liquid containing the unreacted free components.

[0079] After removing unreacted free components, the recovered complex or the solid phase on which the complex is immobilized can be washed with a suitable aqueous medium. Examples of such aqueous mediums include water, physiological saline, PBS and Tris-HCl, and Good's buffer. A surfactant may be added to the aqueous medium as needed. The surfactant is not particularly limited and can be appropriately selected from surfactants used as washing buffers in the fields of biochemistry and molecular biology, for example.

[0080] In this embodiment, the measurement method detects a signal generated by a labeling substance. This labeling substance is a labeling substance possessed by a first capture body bound to a tag exposed on the outer surface of the lipoprotein. Therefore, the signal generated by this labeling substance reflects the amount of tagged sterols in the lipoprotein. In other words, the detection result of this signal serves as an indicator of the lipoprotein's sterol uptake ability.

[0081] In this specification, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantitatively detecting the intensity of a signal, and semi-quantitatively detecting the intensity of a signal. "Semi-quantitatively detecting the intensity of a signal" means detecting the signal intensity in multiple stages, such as "no signal," "weak," and "strong." Preferably, the intensity of the signal generated by the labeling substance contained in the above complex is quantified and a measurement value is obtained. If necessary, a value obtained by subtracting the background value from the measured signal intensity may be obtained. The background value may be, for example, a measured signal intensity obtained by measurement without using the sample, tagged sterol, the first capturer, or the second capturer.

[0082] The method for detecting the signal is known in the art. In this embodiment, an appropriate measurement method can be selected depending on the type of signal derived from the labeled substance. For example, if the labeled substance is an enzyme, the signal, such as light or color, generated by the reaction between the enzyme and its substrate can be measured using a known device. Examples of such measuring devices include spectrophotometers and luminometers.

[0083] The substrate of the enzyme can be appropriately selected from known substrates depending on the type of enzyme. For example, when using peroxidase as the enzyme, examples of substrates include chemiluminescent substrates such as luminol and its derivatives, and chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB). Also, when using alkaline phosphatase as the enzyme, examples of substrates include CDP-Star(registered trademark)(4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)trixyl[3.3.1.1 3, 7Examples of chemiluminescent substrates include 1-(3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. In preferred embodiments, the signal is a chemiluminescent signal produced by contacting the enzyme with the substrate.

[0084] If the labeling material is a radioactive isotope, the radiation signal can be measured using a known device such as a scintillation counter. If the labeling material is a fluorescent substance, the fluorescence signal can be measured using a known device such as a fluorescence microplate reader. The excitation wavelength and fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

[0085] Before detecting the signal, B / F separation may be performed to remove unreacted free components. Unreacted free components include, for example, the first free capture organism that did not bind to the tag. The specific method and washing solution for B / F separation are the same as those used in the washing step described above.

[0086] Each of the above steps is performed in vitro. Furthermore, each of the above steps is performed in a substantially cell-free system. A substantially cell-free system means that cells are not actively added for the purpose of measuring sterols in lipoproteins. For example, conventional methods for measuring cholesterol efflux function use cells that accumulate cholesterol, such as macrophages. However, in the measurement method of this embodiment, tagged sterols bind directly to lipoproteins in the sample, so there is no need to use such cells. Even if the sample contains cells of biological origin, it is considered that the cells themselves have little effect on the binding of lipoproteins and tagged sterols, and therefore the measurement method is considered a cell-free system.

[0087] A further embodiment of the present invention relates to a reagent for measuring sterols in lipoproteins (hereinafter also referred to as "the reagent of this embodiment"). The reagent of this embodiment includes tagged sterols and is used in the measurement method of this embodiment described above. Details of the tagged sterols are the same as those described in the measurement method of this embodiment.

[0088] The reagent of this embodiment may be provided to the user in a container containing tagged sterols. An example of the reagent of this embodiment is shown in Figure 1. Referring to Figure 1, 10 shows the reagent of this embodiment contained in a container. The tagged sterols in the reagent may be solid (e.g., powder, crystals, lyophilized product, etc.) or liquid (e.g., solution, suspension, emulsion, etc.). When the tagged sterols are included in the reagent in liquid form, the solvent may be, for example, the aqueous medium mentioned above. If necessary, stabilizers such as casein and BSA may be added to the aqueous medium.

[0089] Further embodiments of the present invention relate to the use of tagged sterols for the production of reagents for measuring sterols in lipoproteins, wherein the tagged sterol is attached to the C3 position of the sterol skeleton. Details of the tagged sterol are the same as those described in the measurement method of this embodiment.

[0090] Further embodiments of the present invention relate to a reagent kit for measuring sterols in lipoproteins (hereinafter also referred to as "the reagent kit of this embodiment"), comprising a reagent containing tagged sterols. The reagent kit of this embodiment is used in the measurement method of this embodiment described above. Details of tagged sterols are the same as those described in the measurement method of this embodiment. For example, a container containing the reagent containing tagged sterols may be packaged in a box and provided to the user as the reagent kit of this embodiment. An accompanying document may be included in the box. The accompanying document may describe the composition of the reagent, the structure of the tagged sterols, how to use the reagent, how to store the reagent, etc. An example of the reagent kit of this embodiment is shown in Figure 2A. Referring to Figure 2A, 11 shows the reagent kit of this embodiment, 12 shows a container containing the reagent containing tagged sterols, 13 shows a packaging box, and 14 shows an accompanying document.

[0091] The reagent kit of this embodiment may comprise a first reagent containing a tag-added sterol and a second reagent containing a first capture body that specifically binds to the tag and has a labeling substance. The reagent kit of this embodiment may further comprise a third reagent containing a second capture body that specifically binds to lipoproteins. Details of the first and second capture bodies are the same as those described in the measurement method of this embodiment.

[0092] The reagent kit of this embodiment may be provided to the user in a box containing containers for each reagent. An accompanying document may be included in the box. The accompanying document may describe the composition of each reagent, the structure of the tagged sterol, how to use each reagent, how to store each reagent, etc. An example of the reagent kit of this embodiment is shown in Figure 2B. Referring to Figure 2B, 21 shows the reagent kit of this embodiment, 22 shows the first container containing the first reagent which contains the tagged sterol, 23 shows the second container which contains the second reagent which contains the first capture body, 24 shows the packaging box, and 25 shows the accompanying document.

[0093] The reagent kit of this embodiment may include a third reagent containing a second capture agent in addition to the first and second reagents described above. Referring to Figure 2C, 31 shows the reagent kit of this embodiment, 32 shows a first container containing the first reagent containing the tagged sterol, 33 shows a second container containing the second reagent containing the first capture agent, 34 shows a third container containing the third reagent containing the second capture agent, 35 shows the packaging box, and 36 shows the accompanying documentation.

[0094] The tagged sterol, the first capturer, and the second capturer in the reagent may each be in solid form (e.g., powder, crystal, lyophilized product, etc.) or liquid form (e.g., solution, suspension, emulsion, etc.). When the tagged sterol is included in the reagent in liquid form, the solvent may be, for example, the aqueous medium mentioned above. If necessary, stabilizers such as casein or BSA may be added to the aqueous medium.

[0095] In the third reagent, the second capture body may be pre-immobilized on a solid phase. In this case, the third reagent includes the second capture body immobilized on the solid phase. Details of the solid phase are the same as those described in the measurement method of this embodiment. When the solid phase is particles, in Figure 2C, 34 indicates a container containing the reagent including the second capture body immobilized on particles. An example of the reagent kit of this embodiment, where the solid phase is a microplate and the third reagent is a microplate on which the second capture body is immobilized, is shown in Figure 2D. Referring to Figure 2D, 41 indicates the reagent kit of this embodiment, 42 indicates a first container containing the first reagent containing tagged sterols, 43 indicates a second container containing the second reagent containing the first capture body, 44 indicates a microplate on which the second capture body is immobilized, 45 indicates a packaging box, and 46 indicates an accompanying document. In Figure 2D, the microplate is a 96-well plate.

[0096] The reagent kit of this embodiment may further include a calibrator. The calibrator may include, for example, a lipoprotein-free buffer (negative control) and a buffer containing lipoprotein of known concentration. The reagent kit of this embodiment may further include a washing solution. Details of the washing solution and the aqueous medium and surfactant contained therein are the same as those described in the measurement method of this embodiment.

[0097] Further embodiments of the present invention relate to the use of a tagged sterol, a first capture body having a labeling substance that specifically binds to the tag, and a second capture body that specifically binds to the lipoprotein, for the production of a reagent kit for measuring sterols in lipoproteins, wherein the tagged sterol is attached to the C3 position of the sterol skeleton. Details of the tagged sterol, the first capture body, and the second capture body are the same as those described in the measurement method of this embodiment.

[0098] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0099] Example 1: Preparation of tagged sterols Biotin-PEG as a tagged sterol. n -Cholesterol, Biotin- (CH2) n -Cholesterol and biotin-PEG7(ether)-cholesterol were prepared. In both cases, the biotin group in these biotin-added cholesterols was the biotin group of D-biotin. Their structural formulas are shown below.

[0100] [ka] (In the formula, n is 1, 2, 3, 7, 11, or 23.)

[0101] [ka] (In the formula, n is either 2 or 5.)

[0102] [ka]

[0103] (1) Biotin-PEG n - Cholesterol preparation Biotin-PEG n -The cholesterol synthesis scheme is shown below.

[0104] [ka]

[0105] Referring to Step 1, cholesterol (3 mmol) (Tokyo Chemical Industries, Ltd. (hereinafter also referred to as TCI)), ethyl bromo (6 mmol), and sodium hydride (9 mmol) were dissolved in DMF (15 mL), stirred at room temperature under an argon atmosphere for 18 hours, and then purified by silica gel column chromatography. Referring to Step 2, the compound obtained in Step 1 (0.11 mmol) was dissolved in a mixture of 2.5 mL of THF and 0.5 mL of water. Subsequently, ground potassium hydroxide (0.22 mmol) was added, and the mixture was stirred at room temperature under an argon atmosphere for 1 hour. The solution of the starting material (cholesterol), the reaction solution obtained in Step 1, and the reaction solution obtained in Step 2 were spotted onto silica gel plates and developed with the developing solvent (hexane:ethyl acetate = 10:1). The Rf value of the starting material was 0, the Rf value of the product from Step 1 was 0.3, and the Rf value of the product from Step 2 was 0.

[0106] Refer to step 3, the compound obtained in step 2 (0.03 mmol), Biotin-PEG n-Amine (0.03 mmol, n is 1, 2, 3, 7, 11 or 23) (BroadPharm), HATU (0.05 mmol), and triethylamine (TEA) (8.3 μL) were dissolved in DMF (1 mL) and stirred at room temperature under an argon atmosphere for 18 hours. The reaction mixture obtained in step 2, the reaction mixture obtained in step 3, and their mixtures were spotted onto silica gel plates and developed with the developing solvent (dichloromethane:methanol = 10:1). The product of step 3 (Biotin-PEG) was developed. n The Rf value of -cholesterol was 0.1-0.2. By HPLC preparative method, biotin-PEG was identified as tagged cholesterol. n -Cholesterol was recovered. Below, the specified biotin-PEG was used. n -When referring to cholesterol, it is called "PEG1," "PEG2," "PEG3," "PEG7," "PEG11," or "PEG23" depending on the length of the PEG chain (the number of n).

[0107] (2) Biotin-(CH2) n - Cholesterol preparation In the third step of (1) above, Biotin-PEG n Except for using N-(2-aminoethyl)biotinamide (0.03 mmol) (Cat No. A3131: TCI) or biotin-C5-amine (0.03 mmol) (Cat No. A3155: TCI) instead of -Amine, the procedure is the same as in (1) above, but with the addition of biotin-(CH2) n -Cholesterol was prepared. Below, the specified biotin-(CH2) n -When referring to cholesterol, it is called "C2-Amide" or "C5-Amide" depending on the number of carbon atoms (n) between the nitrogen atoms of the two amide groups.

[0108] (3) Preparation of biotin-PEG7(ether)-cholesterol The synthesis scheme for biotin-PEG7(ether)-cholesterol is shown below.

[0109] [ka]

[0110] Cholesterol (1.94 mmol) (TCI) was dissolved in pyridine (6 mL), and the resulting solution was cooled to 0°C. Tosyl chloride (3.87 mmol) was dissolved in pyridine (1.2 mL). Referring to step 1, the tosyl chloride solution was added to the cholesterol solution and stirred overnight at room temperature. The reaction mixture obtained in step 1 was concentrated using a rotary evaporator, and the resulting solid was recovered by dissolving it in 1.2 mL of chloroform. Methanol (15 mL) was added to obtain a precipitate of cholest-5-ene-3β-tosylate. The precipitate was obtained by filtration, washed with methanol (15 mL) and acetonitrile (6 mL), and vacuum dried. Referring to step 2, the cholest-5-ene-3β-tosylate (0.02 mmol) obtained in step 1 and Biotin-PEG7-alchol (BroadPharm) were dissolved in 0.2 mL of 1,4-dioxane and heated under reflux at 110°C for 18 hours. Biotin-PEG7(ether)-cholesterol (hereinafter also referred to as "PEG7(ether)") was recovered as tagged cholesterol by silica gel column chromatography. As shown in the synthesis scheme above, PEG7(ether) undergoes a nucleophilic reaction with Biotin-PEG7-alchol, resulting in the elimination of the tosyl group and the addition of a tag to the oxygen atom at position 3 of the sterol skeleton, thus inverting the stereochemistry of the oxygen atom at position 3.

[0111] Example 2: Measurement of sterols in lipoproteins (1) Of the biotin-modified cholesterol prepared in Example 1, C5-Amide, PEG3, PEG7, PEG11, and PEG23 were used. Each biotin-modified cholesterol was mixed with lipoprotein-containing samples at various concentrations, and the biotin-modified cholesterol in the lipoproteins was measured to examine its quantitative properties. In addition, two lipoproteins with different activities were measured in the same manner to investigate whether the results reflected the qualitative activity of the lipoproteins. The measurements were performed using a fully automated, high-sensitivity immunoassay analyzer HI-1000 (Sysmex Corporation).

[0112] (1) Sample preparation (1.1) Preparation of samples with different lipoprotein concentrations Equal volumes of 22% polyethylene glycol 4000 (Nacalai Tesque Co., Ltd.) were mixed with pooled serum from healthy individuals and allowed to stand at room temperature for 20 minutes. The mixture was then centrifuged at 860 xg for 15 minutes at room temperature. The resulting supernatant was collected as the HDL fraction. Five samples with different HDL concentrations were prepared by progressively diluting the HDL fraction with PBS. These samples, in descending order of HDL concentration, are referred to as "C1," "C2," "C3," "C4," and "C5." PBS was used as the calibrator "C0," which does not contain the HDL fraction.

[0113] (1.2) Preparation of samples containing HDL with different cholesterol uptake capabilities Two types of pooled serum, each known to have different HDL cholesterol uptake capabilities, were processed in the same manner as described in (1.1) above, and the HDL fractions were recovered. Each HDL fraction was diluted with PBS at the same dilution ratio. Hereinafter, the HDL dilution fraction derived from serum containing HDL with high uptake capability will be referred to as "Sample H," and the HDL dilution fraction derived from serum containing HDL with low uptake capability will be referred to as "Sample L." The cholesterol uptake capability of HDL in the above-mentioned pooled serum was determined by the method described in U.S. Patent Application Publication No. 2017 / 0315112.

[0114] (2) Preparation of reagents (2.1) R1 reagent (reagent containing tagged sterols) As the R1 reagent, a buffer solution was prepared containing each biotin-modified cholesterol and 0.3% sodium caseinate. The concentration of biotin-modified cholesterol in each R1 reagent was 0.5 μM.

[0115] (2.2) R2 reagent (a reagent containing a solid phase on which the second capture body is immobilized) As the R2 reagent, magnetic particles immobilized with anti-ApoAI antibody were prepared. Specifically, they were prepared as follows: WSC (Dojin Chemical Laboratories Co., Ltd.) and NHS (Kishida Chemical Co., Ltd.) were added to the magnetic particles, followed by the addition of anti-ApoA1 antibody, thereby binding the anti-ApoA1 antibody to the carboxyl groups on the surface of the magnetic particles. This resulted in obtaining a suspension containing magnetic particles immobilized with anti-ApoAI antibody as the R2 reagent.

[0116] (2.3) R3 reagent (reagent containing the first capture agent) As the R3 reagent, a buffer solution containing alkaline phosphatase (ALP)-labeled streptavidin (Promega) was used.

[0117] (2.4) R4 reagent (measurement buffer) and R5 reagent (substrate solution) As reagents R4 and R5, the HISCL® luminescent substrate set (Sysmex Corporation), which contains a measurement buffer and an ALP chemiluminescent substrate solution, was used.

[0118] (2.5) Cleaning solution As a washing solution, a buffer containing 0.1% (w / v) Kolliphor P188 was prepared by dissolving the surfactant Kolliphor® P188 (Sigma Aldrich) in a buffer solution.

[0119] (3) Measurement (3.1) Contact between HDL and tagged sterols Reagents R1 to R5 were set in the HI-1000 (Sysmex Corporation). Calibrators C0 to C5, sample H, and sample L were used as samples. Each sample (30 μL) was added to reagent R1 (90 μL) and mixed, and allowed to stand at 42°C for 3 minutes. This brought HDL and biotin-modified cholesterol into contact and bound them together.

[0120] (3.2) Formation of a complex between HDL and anti-ApoA1 antibody on magnetic particles A cuvette containing a mixture of reagent R1 and the sample (120 μL) was mixed with reagent R2 (30 μL) and allowed to stand at 42°C for 2 minutes. This allowed the HDL to react with the anti-ApoA1 antibody. Subsequently, the magnetic particles in the mixture were collected and the supernatant was removed, and the magnetic particles were washed with a washing solution. After collecting the magnetic particles and removing the supernatant, reagent R3 (100 μL) was added to the magnetic particles immobilized with a complex of biotinylated cholesterol-bound HDL and anti-ApoA1 antibody, and allowed to stand at 42°C for 3 minutes. This allowed the HDL-bound biotinylated cholesterol to come into contact with ALP-labeled streptavidin. The magnetic particles in the mixture were collected and the supernatant was removed, and the magnetic particles were washed with a washing solution.

[0121] (3.3) Measurement of tagged sterols in HDL The washed magnetic particles were magnetized and the supernatant was removed. R4 reagent (50 μL) and R5 reagent (100 μL) were added to the cuvette containing the magnetic particles, and the mixture was reacted at 37°C for 5 minutes. After the reaction, the chemiluminescence intensity (Count) was measured.

[0122] (4) Results The measurement results for the calibrators are shown in Figures 3A to 3E. The measurement results for samples H and L are shown in Figures 4A to 4E. Referring to Figures 3A to 3E, the measured values ​​for C1 to C5 were higher than the measured values ​​for CO without HDL, regardless of which tagged sterol was used. This indicates that the signal originating from tagged sterols bound to HDL could be detected. Therefore, it was shown that tagged sterols in HDL can be measured regardless of which tagged sterol is used. Furthermore, it was shown that the higher the HDL concentration in the calibrator, the higher the measured value. Since C1 to C5 were prepared from the same pooled serum, it is thought that calibrators with higher HDL concentrations contain more tagged sterols in HDL. Therefore, it was suggested that quantitative measurement of tagged sterols in HDL is possible.

[0123] Comparing the results in Figures 3A to 3E, it was found that using tagged sterols with longer linkers resulted in a stronger detected signal intensity. Furthermore, Figure 3A demonstrates that tagged sterols in HDL can be measured even if the spacer portion in the linker is a hydrocarbon chain instead of PEG.

[0124] Referring to Figures 4A to 4E, the measured values ​​for sample H were higher than those for sample L in all cases where tagged sterols were used. Since samples L and H are thought to contain HDL fractions of approximately the same concentration, the difference in measured values ​​between sample L and sample H is suggested to correspond to the difference in the cholesterol uptake capacity of HDL. Therefore, it is suggested that measuring tagged sterols in HDL yields results that reflect the qualitative activity of lipoproteins.

[0125] Example 3: Measurement of sterols in lipoproteins (2) Of the biotin-modified cholesterol prepared in Example 1, C2-Amide, PEG3, PEG7, PEG11, and PEG23 were used. PEG7, PEG11, and PEG23 were used at different concentrations than in Example 2. The results reflecting lipoprotein activity were investigated using each biotin-modified cholesterol and samples L and H prepared in Example 2. Measurements were performed in the same manner as in Example 2.

[0126] (1) Reagents, samples and measurements As the R1 reagent, a buffer solution containing each biotin-modified cholesterol and 0.3% sodium caseinate was prepared. The concentrations of biotin-modified cholesterol in each R1 reagent were 6.2 μM for C2-Amide, 0.5 μM for PEG3, 0.2 μM for PEG7, 0.1 μM for PEG11, and 0.05 μM for PEG23. The same reagents as in Example 2 were used for reagents R2 to R5. Samples L and H prepared in Example 2 were used as samples. The measurements were performed in the same manner as in Example 2, except that the above-mentioned R1 reagent was used.

[0127] (2) Results The measurement results are shown in Figures 5A to 5E. From Figures 5A to 5E, it can be seen that, as with other biotin-modified cholesterols, the measured value for sample H was higher than that for sample L when using C2-Amide. Therefore, it is suggested that even if the spacer portion in the linker is a short hydrocarbon chain, it is possible to measure tag-modified sterols in HDL, and results that reflect the qualitative activity of lipoproteins can be obtained. Referring to Figures 5C to 5E, the chemiluminescence intensity (count) decreased when the concentrations of PEG7, PEG11, and PEG23 in reagent R1 were reduced, but the results were the same as in Example 2.

[0128] Example 4: Measurement of sterols in lipoproteins (3) Of the biotin-modified cholesterol prepared in Example 1, PEG1 and PEG2 were used. The biotin-modified cholesterol in lipoproteins was measured using each biotin-modified cholesterol, the calibrator prepared in Example 2, sample L, and sample H. The measurement was performed in the same manner as in Example 2.

[0129] (1) Reagents, samples and measurements As reagent R1, a buffer solution containing each biotin-modified cholesterol and 0.3% sodium caseinate was prepared. The concentration of biotin-modified cholesterol in each R1 reagent was 0.5 μM. The same reagents as in Example 2 were used for reagents R2 to R5. Calibrators C0 to C5, sample L, and sample H prepared in Example 2 were used as samples. The measurements were performed in the same manner as in Example 2, except that the above-mentioned R1 reagent was used.

[0130] (2) Results The measurement results for the calibrator are shown in Figures 6A and 6B. The measurement results for sample H and sample L are shown in Figures 7A and 7B. Referring to Figures 6A and 6B, in both cases using PEG1 and PEG2, the measured values ​​for C1-C5 were higher than the measured values ​​for CO without HDL. Furthermore, it was shown that the higher the HDL concentration in the calibrator, the higher the measured values. Referring to Figures 7A and 7B, in both cases using PEG1 and PEG2, the measured values ​​for sample H were higher than the measured values ​​for sample L. Therefore, it is suggested that quantitative measurement of tagged sterols in HDL is possible even when using tagged sterols, which have short PEG chains in the spacer portion of the linker, and that results reflecting the qualitative activity of lipoproteins can be obtained.

[0131] Example 5: Measurement of sterols in lipoproteins (4) Of the biotinylated cholesterol prepared in Example 1, PEG7 (ether) was used. Biotinylated cholesterol in lipoproteins was measured using PEG7 (ether), the calibrator prepared in Example 2, sample L, and sample H. The measurement was performed in the same manner as in Example 2.

[0132] (1) Reagents, samples and measurements A buffer solution containing 1 μM PEG7 (ether) and 0.3% sodium caseinate was prepared as reagent R1. The same reagents as in Example 2 were used for reagents R2 to R5. Calibrators C0 to C5, sample L, and sample H prepared in Example 2 were used as samples. The measurement was performed in the same manner as in Example 2, except for the use of the above-mentioned reagent R1.

[0133] (2) Results The measurement results for the calibrator are shown in Figure 8A. The measurement results for sample H and sample L are shown in Figure 8B. Referring to Figure 8A, the measured values ​​for C1-C5 were higher than those for CO without HDL. It was also shown that the higher the HDL concentration in the calibrator, the higher the measured value. Referring to Figure 8B, the measured value for sample H was higher than that for sample L. As described above, unlike other tagged sterols, PEG7 (ether) has an inverted stereochemistry at the C3 position where the tag is attached, but this was found not to affect the measurement. Therefore, it was suggested that even when using tagged sterols in which the linkage between the C3 position of the sterol skeleton and the linker is an ether linkage, quantitative measurement of tagged sterols in HDL is possible, and results reflecting the qualitative activity of lipoproteins can be obtained.

[0134] Example 6: Investigation of the effect of LCAT on sterol measurement (1) Cholesterol is incorporated into the central part of lipoproteins after its hydroxyl group at the C3 position is esterified by LCAT. However, the tagged sterols in this embodiment do not have this hydroxyl group. We investigated whether the measurement of tagged sterols in lipoproteins is affected by LCAT using recombinant LCAT.

[0135] (1) Sample preparation Equal volumes of 22% polyethylene glycol 4000 (Nacalai Tesque Co., Ltd.) were mixed with pooled serum from healthy individuals and allowed to stand at room temperature for 20 minutes. The mixture was then centrifuged at 860 xg for 15 minutes at room temperature. The resulting supernatant was collected as the HDL fraction. A portion of the HDL fraction was taken, and the ApoAI concentration was measured using the ApoAI measurement kit N-Assay TIA ApoAI-H (Nitto Boseki Medical Co., Ltd.). The specific procedure for concentration measurement was followed according to the manual included with the kit. Recombinant LCAT (Sino Biological Co., Ltd.) was mixed with the HDL fraction at a weight ratio of 1:10 to ApoA1 contained in the HDL fraction to prepare an LCAT-added sample. For comparison, an HDL fraction without LCAT was used as the LCAT-free sample for measurement.

[0136] (2) Reagents and measurements As the R1 reagent, a buffer solution was prepared containing biotin-modified cholesterols C2-Amide, PEG3, PEG7, PEG11, and PEG23, along with 0.3% sodium caseinate. The concentrations of biotin-modified cholesterol in each R1 reagent were 6.2 μM for C2-Amide, 0.5 μM for PEG3, 0.2 μM for PEG7, 0.1 μM for PEG11, and 0.05 μM for PEG23. The same reagents as in Example 2 were used for reagents R2 to R5. Measurements were performed in the same manner as in Example 2, except for the use of the above samples and R1 reagent. In addition, PBS was measured in the same manner as for each sample to measure the background.

[0137] (3) Results The measurement results are shown in Figures 9A to 9E. In the figures, "Net count" refers to the value obtained by subtracting the PBS measurement value from the measurement value of each sample, "rLCAT+" refers to the LCAT-added sample, and "rLCAT-" refers to the LCAT-unadded sample. Referring to Figures 9A to 9E, the rLCAT+ measurement value was higher than the rLCAT- measurement value in all cases where tagged sterols were used. This indicates that the addition of recombinant LCAT increased the signal derived from tagged sterols bound to HDL. In the presence of recombinant LCAT, it is thought that the esterification of endogenous cholesterol present near the surface of HDL and its movement into the HDL interior were promoted, increasing the amount of tagged sterols bound to the surface of the HDL. Therefore, it is suggested that the measurement of tagged sterols in HDL is indirectly affected by LCAT.

[0138] Example 7: Investigation of the effect of LCAT on sterol measurement (2) We investigated whether the measurement of tagged sterols in lipoproteins is affected by LCAT using LCAT inhibitors.

[0139] (1) Sample preparation N-ethylmaleimide (NEM) was added to pooled serum from healthy individuals to a final concentration of 10 mM as an LCAT inhibitor, and the mixture was incubated at 37°C for 45 minutes. The pooled serum was then diluted 1600-fold with PBS to prepare the NEM-added sample. For comparison, diluted pooled serum without NEM was used as the NEM-free sample for measurement.

[0140] (2) Reagents and measurements A buffer solution containing 0.5 μM PEG3 and 0.3% sodium caseinate was prepared as reagent R1. The same reagents as in Example 2 were used for reagents R2 to R5. The measurements were performed in the same manner as in Example 2, except that the above samples and reagent R1 were used. The measurements were performed independently twice.

[0141] (3) Results The measurement results are shown in Figure 10. In the figure, "% of control" indicates the ratio of the measured values ​​of the NEM-added sample to the measured values ​​of the NEM-free sample, which are set to 100%. Referring to Figure 10, the measured values ​​of the NEM-added sample were lower than those of the NEM-free sample. This indicates that the addition of NEM reduced the signal derived from tagged sterols bound to HDL. It is thought that the inhibition of endogenous LCAT activity by NEM suppressed the esterification of endogenous cholesterol by HDL and its movement into HDL, thereby reducing the amount of tagged sterols bound to the HDL. Considering the results of Example 6 as well, it is suggested that the method of this embodiment can evaluate the qualitative activity of lipoproteins that reflects the activity of LCAT.

[0142] Example 8: Evaluation of the qualitative activity of lipoproteins by sterol measurement We investigated whether the qualitative activity of lipoproteins, whose function had been reduced by oxidation, could be evaluated using tagged sterols in samples containing such lipoproteins.

[0143] (1) Sample preparation Serum from healthy individuals was diluted 100-fold with PBS. Diethylenetriaminepentaacetic acid (final concentration 100 μM: Tokyo Chemical Industry Co., Ltd.), hydrogen peroxide (final concentrations 20, 40, or 60 μM: Fujifilm Wako Pure Chemical Industries, Ltd.), recombinant myeloperoxidase (final concentration 10 nM: R&D Systems, Inc.), and sodium nitrite (200 μM: Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the diluted serum, and the mixture was incubated at 37°C for 1 hour. Subsequently, L-methionine was added to a final concentration of 2 mM to stop the oxidation reaction. The resulting reaction solution was diluted 15-fold with PBS to prepare the oxidized sample. For comparison, a sample prepared in the same manner as above, except that hydrogen peroxide was not added, was also measured.

[0144] (2) Reagents and measurements A buffer solution containing 0.5 μM PEG3 and 0.3% sodium caseinate was prepared as reagent R1. The same reagents as in Example 2 were used for reagents R2 to R5. The measurement was performed in the same manner as in Example 2, except that the above-mentioned samples and reagent R1 were used.

[0145] (3) Results The measurement results are shown in Figure 11. In the figure, "% of 0 μM" indicates the percentage of the measured values ​​for each oxidized sample, with the measured value for the sample without added hydrogen peroxide set to 100%. Referring to Figure 11, the measured values ​​for the oxidized samples were lower than those for the sample without added hydrogen peroxide. Furthermore, it was shown that the higher the concentration of added hydrogen peroxide, the lower the measured value. It is thought that the oxidation treatment reduced the activity of HDL, thereby suppressing the uptake of endogenous cholesterol by HDL, and as a result, the amount of tagged sterols bound to the HDL decreased. This suggests that the method of this embodiment can evaluate the qualitative activity of lipoproteins. [Explanation of Symbols]

[0146] 10: Reagent for measuring sterols in lipoproteins 11, 21, 31, 41: Reagent kit for measuring sterols in lipoproteins 12: Container 22, 32, 42: 1st container 23, 33, 43: 2nd container 34: Third container 44: Microplate with the second capture body immobilized 13, 24, 35, 45: Packaging box 14, 25, 36, 46: Attached document

Claims

1. A step of forming a complex comprising the lipoprotein containing the tagged sterol and the first capture body by contacting the lipoprotein in the sample with the tagged sterol and a first capture body having a labeling substance that specifically binds to the tag, A step of detecting a signal generated by the labeling substance contained in the composite. Includes, The aforementioned tag-added sterol is given by the following formula (I): 【Chemistry 1】 (In the formula, the solid and dashed lines represent, independently, single or double bonds.) R1 is an alkyl group having 1 to 6 carbon atoms, which may have substituents, or an alkenyl group having 2 to 6 carbon atoms, which may have substituents. X and Y are the same or different; -(C=O)-O-, -(C=O)-OR 2 -, -R 2 -O-(C=O)-, -O-(C=O)-R 2 -, -R 2 -(C=S)-NH-, -(C=S)-NH-R 2 -, -R 2 -NH-(C=S)-, -NH-(C=S)-R 2 -, -R 2 -O-, -OR 2 -, -R 2 -S-, or -SR 2 Represented by -, where R2 is independently a bond, an alkylene group having 1 to 10 carbon atoms which may have substituents, an arylene group or heteroarylene group having 6 to 12 carbon atoms which may have substituents, or a cycloalkylene group or heterocycloalkylene group having 3 to 8 carbon atoms which may have substituents. L is represented as -(CH2)d-[R3-(CH2)e]f- or -[(CH2)e-R3]f-(CH2)d-, where R3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH- or a bond. Z is a tag, a and c are either the same or different integers between 0 and 6, and are between 0 and 6. b is either 0 or 1. d and e are integers between 0 and 12, and are either the same or different. f is an integer between 0 and 24 (inclusive). A method for measuring sterols in lipoproteins, represented by [the formula shown].

2. In equation (I) above, a is 0 or 1, b and c are 1, and X is -NH-(C=O)-R 2 Represented by -, Y is -R 2 It is represented as -(C=O)-NH-, R 2 Each of these is an unsubstituted alkylene group having 1 to 6 carbon atoms, and L is -[(CH 2 ) 2 -O] f -(CH 2 ) d The method according to claim 1, wherein the expression is represented by -, where d is an integer between 1 and 6, and f is an integer between 0 and 24.

3. The method according to claim 1 or 2, wherein the tag is a biotin group.

4. The method according to any one of claims 1 to 3, wherein the tagged sterol is tagged cholesterol.

5. The aforementioned tag-added sterol is given by the following formula (II): 【Chemistry 2】 (In the formula, n is an integer between 1 and 23, inclusive.) Alternatively, use the following formula (III): 【Transformation 3】 (In the formula, n is an integer between 1 and 7, inclusive.) Or the following formula (IV): 【Chemistry 4】 (In the formula, n is an integer between 2 and 5, inclusive.) The method according to any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein the first capture body is a labeled antibody, labeled avidin, or labeled streptavidin that specifically binds to the tag.

7. The method according to any one of claims 1 to 6, wherein in the forming step, the second capture body that specifically binds to the lipoprotein is used in contact between the lipoprotein, the tag-added sterol, and the first capture body.

8. The method according to claim 7, wherein in the step of forming, the lipoprotein containing the tagged sterol is brought into contact with the second capture body, and then the complex of the lipoprotein containing the tagged sterol and the second capture body is brought into contact with the first capture body.

9. The method according to claim 8, wherein, in the step of forming, B / F separation is performed to remove unreacted free components between the contact between the lipoprotein containing the tagged sterol and the second capture body and the contact between the complex and the first capture body.

10. The method according to any one of claims 7 to 9, wherein the second capture body is immobilized on a solid phase, and a complex of the lipoprotein containing the tagged sterol and the second capture body is formed on the solid phase.

11. The method according to any one of claims 7 to 10, wherein the second capture body is an antibody that specifically binds to the lipoprotein.

12. The method according to claim 11, wherein the antibody that specifically binds to the lipoprotein is an anti-ApoAI antibody.

13. The method according to any one of claims 1 to 12, wherein B / F separation is performed to remove unreacted free components between the forming step and the detection step.

14. The method according to any one of claims 1 to 13, wherein the labeling substance is an enzyme, and the signal is a chemiluminescent signal produced by contacting the enzyme with a substrate.

15. The method according to claim 14, wherein the enzyme is alkaline phosphatase or peroxidase.

16. The method according to any one of claims 1 to 15, wherein the sample is blood, serum, or plasma.

17. The method according to any one of claims 1 to 16, wherein the lipoprotein is a high-density lipoprotein.

18. A reagent containing a tagged sterol, wherein the tagged sterol is of the following formula (I): 【Transformation 5】 (In the formula, the solid and dashed lines represent, independently, single or double bonds.) R1 is an alkyl group having 1 to 6 carbon atoms, which may have substituents, or an alkenyl group having 2 to 6 carbon atoms, which may have substituents. X and Y are the same or different; -(C=O)-O-, -(C=O)-OR 2 -, -R 2 -O-(C=O)-, -O-(C=O)-R 2 -, -R 2 -(C=S)-NH-, -(C=S)-NH-R 2 -, -R 2 -NH-(C=S)-, -NH-(C=S)-R 2 -, -R 2 -O-, -OR 2 -, -R 2 -S-, or -SR 2 Represented by -, where R2 is independently a bond, an alkylene group having 1 to 10 carbon atoms which may have substituents, an arylene group or heteroarylene group having 6 to 12 carbon atoms which may have substituents, or a cycloalkylene group or heterocycloalkylene group having 3 to 8 carbon atoms which may have substituents. L is represented as -(CH2)d-[R3-(CH2)e]f- or -[(CH2)e-R3]f-(CH2)d-, where R3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH- or a bond. Z is a tag, a and c are either the same or different integers between 0 and 6, and are between 0 and 6. b is either 0 or 1. d and e are integers between 0 and 12, and are either the same or different. f is an integer between 0 and 24 (inclusive). A reagent for measuring sterols in lipoproteins, used in the method according to any one of claims 1 to 17, as represented by the formula.

19. A reagent kit comprising a first reagent containing a tag-adding sterol and a second reagent containing a first capture body that specifically binds to the tag and has a labeling substance, wherein the tag-adding sterol has the following formula (I): 【Transformation 6】 (In the formula, the solid and dashed lines represent, independently, single or double bonds.) R1 is an alkyl group having 1 to 6 carbon atoms, which may have substituents, or an alkenyl group having 2 to 6 carbon atoms, which may have substituents. X and Y are the same or different; -(C=O)-O-, -(C=O)-OR 2 -, -R 2 -O-(C=O)-, -O-(C=O)-R 2 -, -R 2 -(C=S)-NH-, -(C=S)-NH-R 2 -, -R 2 -NH-(C=S)-, -NH-(C=S)-R 2 -, -R 2 -O-, -OR 2 -, -R 2 -S-, or -SR 2 Represented by -, where R2 is independently a bond, an alkylene group having 1 to 10 carbon atoms which may have substituents, an arylene group or heteroarylene group having 6 to 12 carbon atoms which may have substituents, or a cycloalkylene group or heterocycloalkylene group having 3 to 8 carbon atoms which may have substituents. L is represented as -(CH2)d-[R3-(CH2)e]f- or -[(CH2)e-R3]f-(CH2)d-, where R3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH- or a bond. Z is a tag, a and c are either the same or different integers between 0 and 6, and are between 0 and 6. b is either 0 or 1. d and e are integers between 0 and 12, and are either the same or different. f is an integer between 0 and 24 (inclusive). A reagent kit for measuring sterols in lipoproteins, represented as shown.

20. The reagent kit according to claim 19, comprising a third reagent containing a second capture agent that specifically binds to lipoproteins.

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