Method for measuring sterol in lipoprotein, reagent and kit

Abstract

The present application aims to provide a new method, reagent and kit for measuring sterol in lipoprotein. By contacting lipoprotein in a test sample obtained from a subject, a labeled sterol having a label attached to the C3 position of the sterol skeleton, and a first capture body that specifically binds to the label and has a labeling substance, by forming a complex containing the lipoprotein containing the labeled sterol and the first capture body, detecting a signal generated from the labeling substance contained in the complex, the problem is solved.

Description

[Technical Field]

[0001] This invention relates to a method for determining sterols in lipoproteins. This invention relates to reagents for determining sterols in lipoproteins. This invention relates to a kit for determining sterols in lipoproteins. [Background Technology]

[0002] In recent years, attention has been paid to indicators reflecting the function of lipoproteins. As a method for studying the function of lipoproteins, for example, the methods described in Patent Documents 1 and 2 are known. In these methods, the cholesterol harvesting energy, which is a qualitative activity of the lipoprotein, is measured. These documents disclose that by contacting a lipoprotein in a sample with tagged cholesterol to form a lipoprotein that harvests tagged cholesterol esterified by lecithin-cholesterol acyltransferase (LCAT) in the sample, the signal from the harvested tagged cholesterol is detected, and the cholesterol harvesting energy of the lipoprotein is measured.

[0003] [Existing Technical Documents]

[0004] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 2016 / 0109469

[0006] [Patent Document 2] U.S. Patent Application Publication No. 2017 / 0315112

[0007] [Summary of the Invention]

[0008] [The problem the invention aims to solve]

[0009] In the tagged cholesterol described in Patent Documents 1 and 2, the tag is attached to the hydrocarbon chain at the C17 position of the sterol backbone, and the hydroxyl group at the C3 position is esterified by LCAT. In the methods described in Patent Documents 1 and 2, cholesterol tagged at other sites on the sterol backbone is not used. As a functional analysis method for lipoproteins, there are few methods for determining sterols in lipoproteins, and further development of such methods is needed.

[0010] [Methods used to solve problems]

[0011] The present invention provides a method for determining sterols in a lipoprotein tagged at the C3 position of the sterol backbone by contacting a lipoprotein, a tagged sterol, and a first trap having a label specifically bound to the label in a sample to form a complex containing a lipoprotein containing a tagged sterol and the first trap; detecting a signal generated by the label contained in the complex; and determining sterols in a lipoprotein tagged at the C3 position of the sterol backbone.

[0012] The present invention provides a reagent containing a tagged sterol, wherein the tagged sterol is a reagent for the determination of sterols in lipoproteins used in the above-described method, wherein the tagged sterol is attached at the C3 position of the sterol backbone.

[0013] The present invention provides a kit comprising a first reagent containing a tagged sterol and a second reagent containing a first trap containing a labeling substance specifically bound to the label, wherein the kit is for the determination of sterols in lipoproteins tagged at the C3 position of the sterol backbone.

[0014] [The effects of the invention]

[0015] The present invention provides a method for determining sterols in lipoproteins, as well as reagents and kits that can be used in the method.

[0016] [Brief explanation of the attached image]

[0017]

【 Figure 1 [Image 1] is an example of a reagent for determining sterols in lipoproteins according to this embodiment.

[0018]

【 Figure 2A The figure shown is an example of a kit for determining sterols in lipoproteins according to this embodiment.

[0019]

【 Figure 2B The figure shown is an example of a kit for determining sterols in lipoproteins according to this embodiment.

[0020]

【 Figure 2C The figure shown is an example of a kit for determining sterols in lipoproteins according to this embodiment.

[0021]

【 Figure 2D The figure shown is an example of a kit for determining sterols in lipoproteins according to this embodiment.

[0022]

【 Figure 3A The graph shows the results of measuring high-density lipoprotein (HDL) fractions (C0–C5) at different concentrations using tagged sterols (C5-amides).

[0023]

【 Figure 3B The graph shows the results of C0–C5 determinations using labeled sterols (PEG3).

[0024]

【 Figure 3C The graph shows the results of C0–C5 determinations using tagged sterols (PEG7).

[0025]

【 Figure 3DThe graph shows the results of C0–C5 determinations using labeled sterols (PEG11).

[0026]

【 Figure 3E The graph shows the results of C0–C5 determinations using tagged sterols (PEG23).

[0027]

【 Figure 4A The graph is a coordinate graph showing the results of measuring samples (sample H and sample L) containing different cholesterol collection energies using labeled sterols (C5-amides).

[0028]

【 Figure 4B The graph shows the results of measurements of samples H and L using labeled sterol (PEG3).

[0029]

【 Figure 4C The graph shows the results of measurements of samples H and L using tagged sterols (PEG7).

[0030]

【 Figure 4D The graph shows the results of measurements of samples H and L using a labeled sterol (PEG11).

[0031]

【 Figure 4E The graph shows the results of measuring samples H and L using a labeled sterol (PEG23).

[0032]

【 Figure 5A The graph shows the results of measurements of samples H and L using labeled sterols (C2-amides).

[0033]

【 Figure 5B The graph shows the results of measurements of samples H and L using labeled sterol (PEG3).

[0034]

【 Figure 5C The graph shows the results of measurements of samples H and L using tagged sterols (PEG7).

[0035]

【 Figure 5D The graph shows the results of measurements of samples H and L using a labeled sterol (PEG11).

[0036]

【 Figure 5E The graph shows the results of measuring samples H and L using a labeled sterol (PEG23).

[0037]

【 Figure 6AThe graph shows the results of C0–C5 determinations using labeled sterols (PEG1).

[0038]

【 Figure 6B The graph shows the results of C0–C5 determinations using labeled sterols (PEG2).

[0039]

【 Figure 7A The graph shows the results of measurements of samples H and L using tagged sterols (PEG1).

[0040]

【 Figure 7B The graph shows the results of measuring samples H and L using pebbly sterol (PEG2) with an added label.

[0041]

【 Figure 8A The graph shows the results of C0–C5 determinations using labeled sterols (PEG7 (ether)).

[0042]

【 Figure 8B The graph shows the results of measurements of samples H and L using a labeled sterol (PEG7 (ether)).

[0043]

【 Figure 9A The graph shows the results of measurements using labeled sterols (C2-amides) on LCAT-added samples (rLCAT+) and LCAT-unadded samples (rLCAT-).

[0044]

【 Figure 9B The graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG3).

[0045]

【 Figure 9C The graph shows the results of measuring rLCAT+ and rLCAT- using tagged sterols (PEG7).

[0046]

【 Figure 9D The graph shows the results of measuring rLCAT+ and rLCAT- using a tagged sterol (PEG11).

[0047]

【 Figure 9E The graph shows the results of measuring rLCAT+ and rLCAT- using a tagged sterol (PEG23).

[0048]

【 Figure 10 The graph shows the results of measurements on N-ethylmaleimide (NEM) samples with and without NEM, using a sterol (PEG3) with an added label.

[0049]

【 Figure 11 The graph shows the results of measurements using labeled sterols (PEG3) on samples treated with hydrogen peroxide and samples without added hydrogen peroxide.

Detailed Implementation Methods

[0050] When naturally occurring cholesterol comes into contact with lipoproteins in vivo, it is collected by the lipoproteins. Free cholesterol in the blood begins to bind to the surface (phospholipid membrane) of the lipoprotein. Specifically, when the hydroxyl group at the C3 position of cholesterol is esterified by LCAT, its lipophilicity increases, and cholesterol migrates from the surface of the lipoprotein particle towards its center. On the other hand, the tagged sterols used in the method for determining sterols in lipoproteins according to this embodiment (hereinafter also referred to as "the determination method of this embodiment") are not esterified by LCAT because the sterol backbone is tagged at the C3 position. In this specification, "sterol backbone" refers to a backbone represented by the following formula.

[0051]

Chemistry 1

[0052]

[0053] (In the formula, the numbers represent the positions of carbons in the sterol skeleton.)

[0054] The lipoprotein in the sample obtained from a living organism contains endogenous cholesterol derived from the organism in its surface layer. Additionally, the sample also contains endogenous LCAT derived from the organism. The endogenous cholesterol in the lipoprotein surface layer is esterified by LCAT to form cholesterol esters. Furthermore, the cholesterol esters migrate into the interior of the lipoprotein. Due to the migration of endogenous cholesterol from the surface to the interior, there is room for tagged cholesterol to bind on the surface. Tagged cholesterol can contact LCAT, and as described above, it is assumed that tagged sterols are not esterified by LCAT. Therefore, tagged sterols are considered to terminate on the surface of the lipoprotein. When more endogenous cholesterol in the lipoprotein migrates from the surface to the interior, it is assumed that more tagged sterols can bind to the surface. With more tagged sterols binding to the surface of the lipoprotein, a stronger signal is detected from the lipoprotein. Therefore, the signal obtained by the measurement method of this embodiment can serve as an indicator of the function of the lipoprotein.

[0055] When the sample comes into contact with the tagged sterol, the tagged sterol is believed to bind to the surface of the lipoprotein, and the tag portion is believed to be exposed on the outer surface of the lipoprotein. Here, "outer surface of the lipoprotein" refers to the outer side of the lipoprotein particle. "Exposed on the outer surface" means present on the outer surface of the lipoprotein, and protruding from both sides of 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 trap that specifically binds to the tag. Next, each step in the determination method of this embodiment will be described.

[0056] In the determination method of this embodiment, a complex containing a lipoprotein containing a tagged sterol and a first trapping body having a labeling substance that specifically binds to the label is formed by contacting the lipoprotein in the sample.

[0057] The sample is not particularly limited as long as it contains lipoproteins. Examples of such samples include blood samples. Examples of blood samples include whole blood, plasma, and serum. The lipoprotein-containing sample can also be separated or fractionated using known methods such as ultracentrifugation or polyethylene glycol (PEG) precipitation to obtain a fraction containing the specified lipoproteins. This fraction containing the specified lipoproteins can also be used as a lipoprotein-containing sample.

[0058] Lipoproteins can be any of high-density lipoprotein (HDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), or chylomicrons (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 but less than 1.063 g / mL. IDL is a lipoprotein with a density of 1.006 g / mL or higher but less than 1.019 g / mL. VLDL is a lipoprotein with a density of 0.95 g / mL or higher but less than 1.006 g / mL. CM is a lipoprotein with a density of less than 0.95 g / mL. HDL is the preferred lipoprotein.

[0059] Samples containing lipoproteins may also be diluted. For example, to adjust the concentration of lipoproteins, a solution obtained by diluting the blood sample or a fraction containing a specified lipoprotein with an aqueous medium may be used as a sample. Examples of aqueous media include water, physiological saline, and buffer solutions. Examples of buffer solutions include phosphate-buffered saline (PBS), Tris-HCl, and Good buffer.

[0060] The concentration of apolipoproteins, which are components of lipoproteins, serves as an indicator of the lipoprotein concentration in a sample. The lipoprotein concentration in a sample can also be adjusted by diluting the sample containing lipoproteins based on the concentration of the apolipoproteins. The concentration of apolipoproteins can be determined by known immunological assays (e.g., immunoturbidimetry). ApoAI or ApoE are preferred as apolipoproteins.

[0061] A blocking agent may also 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 Nippon Oil Co., Ltd., specifically Lipidure-BL203). Additionally, fatty acids or compositions containing them (e.g., liposomes) that are essential for the esterification reaction of cholesterol from lipoproteins may be added to the sample. Liposomes, for example, can be prepared by mixing dimyristoylphosphatidylglycerol, cholesterol, and hydroaddition soybean phosphatidylcholine.

[0062] Tagned sterols refer to sterols having a sterol skeleton represented by the above formula, with a tag directly or indirectly attached to the carbon atom at the C3 position. "Indirect tagging at the C3 position" includes tagging the carbon atom at the C3 position of the sterol via a linker, tagging via the substituent when a substituent is attached to the carbon atom at the C3 position of the sterol, and tagging via a linker to the substituent at the C3 position. Preferably, the tagged sterol has a hydrocarbon chain optionally having a substituent at the C17 position of the sterol skeleton represented by the above formula.

[0063] Tag-added sterols can be modulated by attaching a tag to the C3 position of the sterol. The tag can also be attached directly or via a linker to the C3 carbon atom of the sterol. When a substituent is attached to the C3 carbon atom of the sterol, the tag can also be attached to that substituent. Alternatively, the tag can be attached to the substituent at the C3 position of the sterol via a linker. For example, when using sterols with a hydroxyl group at the C3 position, such as cholesterol, the tag can also be attached directly or via a linker to the hydroxyl group at the C3 position. The sterol used in the modulation of tagged sterols is preferably bound to the surface of lipoproteins. Examples of sterols include, for example, cholesterol and its analogues. Examples of cholesterol analogues include, for example, epicholesterol, allocholesterol, cholesterol, coprosterol, 7-dehydrocholesterol, stigmasterol, sitosterol, campesterol, α-spinasterol, brassosterol, 24-methylenesterol, etc.

[0064] Examples of labeled sterols include, for example, compounds represented by the following formula (I) (hereinafter also referred to as "labeled sterols of formula (I)").

[0065]

Chemistry 2

[0066]

[0067] In the formula, the solid and dashed double lines each independently represent a single bond or a double bond.

[0068] R 1 It can be an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms.

[0069] Whether X and Y are the same or different, determined by -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 -express,

[0070] Among them, R 2 Each of the following is independently a linking bond: an alkylene group having 1 to 10 carbon atoms with a substituent; an aryl or heteroaryl group having 6 to 12 carbon atoms with a substituent; or a cycloalkyl or heterocycloalkyl group having 3 to 8 carbon atoms with a substituent.

[0071] L is derived from -(CH2) d -[R 3 -(CH2) e ] f -、or-[(CH2)] e -R 3 ] f -(CH2) d -express,

[0072] in,

[0073] R 3 It is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH-, or a connecting bond.

[0074] Z is a label.

[0075] Whether a and c are the same or different, they are integers greater than 0 and less than 6.

[0076] b is 0 or 1.

[0077] d and e may be the same or different, and are integers greater than 0 and less than 12.

[0078] f is an integer greater than 0 and less than 24.

[0079] In equation (I), "-[X]" a -[L] b -[Y] c The part indicated by the "-" corresponds to a linker connecting 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 "O at the C3 position"). 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. Due to the linker, the tag exposed to the outer surface of the lipoprotein and the first trap are considered to become more likely to bind. Next, the substituents of formula (I) will be explained.

[0080] Preferably, in formula (I), either the combination between C5 and C6 or the combination between C7 and C8 is a double bond, or both the combination between C5 and C6 and the combination between C7 and C8 are single bonds. Particularly preferably, in formula (I), the combination between C5 and C6 is a double bond, and the combination between C7 and C8 is a single bond.

[0081] R 1 The main chain is composed of an alkyl group with 1 to 6 carbon atoms or an alkenyl group with 2 to 6 carbon atoms, and substituents are optionally present at any position. R 1 When a substituent is present, the number of carbon atoms in the aforementioned carbon number does not include those without the substituent. 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.

[0082] As R 1Examples of substituents in R include methyl, ethyl, phenyl, naphthyl, benzyl, alkoxy, nitro, halogen, haloalkyl, and thioether groups. Halogen refers to fluorine, chlorine, bromine, or iodine. Alkoxy refers to -O-alkyl, which is a straight-chain or branched saturated aliphatic hydrocarbon group with 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms. 1 It may optionally have multiple substituents. As R 1 The substituent in the [substituent name] is particularly preferred to be methyl. When the substituent is methyl, R is preferred. 1 It is 1,5-dimethylhexyl. This is the same as the alkyl chain at the C20 to C27 positions of naturally occurring cholesterol.

[0083] When a is an integer greater than or equal to 1, [X] a Equivalent to O and L at C3 bit, [Y] c Or the connecting part of Z (label). L is equivalent to a spacer, having a straight chain-like structure that gives the joint a specified length. When c is an integer greater than 1, [Y] c Equivalent to Z (label) and L, [X] a Or the O-connection portion at the C3 position. X and Y correspond to the types of reactions that bind the sterol moiety and the linker, and the types of reactions that bind the linker and the tag.

[0084] Regarding R 2 and R 3 A connecting bond is a direct bond formed without the presence of other atoms.

[0085] R 2 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 decylene. Among these, alkylene groups having 1 to 4 carbon atoms are preferred. 2 When it is an alkylene group with substituents, the number of carbon atoms without substituents in the above-mentioned number of carbon atoms.

[0086] R 2 When the group is arylene or heteroarylene, it can be any aromatic ring containing 6 to 12 carbon atoms, chosen from N, S, O, and P. Examples include phenylene, naphthylene, biphenylene, furanylene, pyrrolylene, phenylthionylene, triazolylene, oxadiazolylene, pyridinylene, and pyrimidinylene. 2 When it is a substituent aryl or heteroaryl group, the number of carbon atoms in the above-mentioned number of carbon atoms does not contain substituents.

[0087] R 2When the group is a cycloalkylene or heteroalkylene, it can be any non-aromatic ring containing 3 to 8 carbon atoms, chosen from N, S, O, and P. Examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, pyrrolidineene, piperidinylene, and morpholinylene. 2 When it is a cyclic alkylene or heterocyclic alkylene with substituents, the number of carbon atoms without substituents in the above-mentioned number of carbon atoms.

[0088] As R 2 Examples of substituents in R2 include hydroxyl, cyano, alkoxy, nitro, =O, =S, -SH, halogen, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether groups. R2 may optionally have multiple substituents. Halogen represents fluorine, chlorine, bromine, or iodine. Alkoxy represents -O-alkyl, which is a straight-chain or branched saturated aliphatic hydrocarbon group with 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.

[0089] L is preferably a structure that does not inhibit the binding of lipoproteins and cholesterol, and the linker portion has a structure that makes it difficult to collect lipoproteins. As such a structure, a structure containing a hydrophilic polymer can be cited. For example, it is preferable that in formula (I), b is 1, R... 3 It is an oxygen atom. At this point, L is composed of -(CH2). d -[O-(CH2) e ] f -or-[(CH2)] e -O] f -(CH2) d - indicates. Wherein, d is an integer from 0 to 12, e is an integer from 1 to 6, and f is an integer from 1 to 24. Preferably, d and e are the same or different, and are integers from 1 to 4. More preferably, d and e are 2. f is preferably an integer from 1 to 23, more preferably an integer from 3 to 23, and particularly preferably an integer from 3 to 11.

[0090] In a preferred embodiment, in formula (I), a is 0 or 1, b and c are 1, and X is derived from -NH-(C=O)-R 2 - indicates that Y is derived from -R 2 -(C=O)-NH- indicates that R 2 Each is an alkylene group with 1 to 6 carbon atoms that is not substituted, and L is -[(CH2)2-O]. f -(CH2) d - indicates that d is an integer greater than 1 and less than 6, and f is an integer greater than 0 and less than 24. Furthermore, R... 1 More preferably, it is 1,5-dimethylhexyl.

[0091] For example, in equation (I), a, b, and c are 1, and X is derived from -NH-(C=O)-(CH2). n - represents (however, 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 represented by -[(CH2)2-O]. f -(CH2)2- indicates that f is an integer greater than 1 and less than 23. Alternatively, in equation (I), a, b, and c are 1, and X is derived from -NH-(C=O)-(CH2). n - represents (however, 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 represented by -(CH2)4-(C=O)-NH-. f - indicates that f is an integer greater than or equal to 1 and less than or equal to 6, preferably an integer greater than or equal to 2 and less than or equal to 5. Alternatively, in formula (I), a is 0, b and c are 1, Y is represented by -(CH2)4-(C=O)-NH-, and L is represented by -[(CH2)2-O]. f -(CH2)2- indicates that f is an integer greater than 1 and less than 23. In any case, R 1 More preferably, it is 1,5-dimethylhexyl.

[0092] The tag is not particularly limited as long as a substance that can specifically bind to it exists or is available. The tag can be any substance of natural origin or synthetic origin, such as compounds, peptides, proteins, nucleic acids, and complexes thereof. Examples of combinations of a tag and a substance that can specifically bind to it include, for example, antigens and antibodies that recognize the antigen, haptens and anti-hapten antibodies, peptides or proteins and their aptamers, ligands and receptors, oligonucleotides and oligonucleotides having their complementary chains, biotinylate and avidin analogs, histidine tags (peptides containing 6-10 histidine residues) and Ni-NTA (nitroglycerin triacetic acid that forms a chelate with nickel ions), glutathione S-transferase (GST), and glutathione. The antigen used as the tag can be peptide tags and protein tags known in the art, such as FLAG (registered trademark), hemagglutinin (HA), Myc protein, and green fluorescent protein (GFP). Examples of haptens that can be used as tags include the 2,4-dinitrophenol (DNP) group. Anti-DNP antibodies are suitable as traps that specifically bind to DNP.

[0093] In this specification, "biotin class" includes biotin and its analogues. Examples of biotin analogues include, for example, desulfobiotin and biocytidine. In this specification, "avidin class" includes avidin and its analogues. Examples of avidin analogues include, for example, streptavidin, avidin-like protein derived from *Trichoderma oleracea* (Tamavidin (registered trademark)), bradavidin, and rhizavidin.

[0094] The compound used as a label can also be, for example, a labeling compound known in the art. Examples of such compounds include biotinyl groups and dye compounds. In this specification, "biotinyl group" refers to a heterocyclic moiety in the chemical structure of a biotinylate class containing at least an imidazole and lysine ring. A preferred biotinyl group is the biotinyl group of biotin. Avidin-like compounds are suitable as traps that specifically bind to the biotinyl group. Examples of dye compounds include boron dipyrrole methylene (BODIPY (registered trademark)). Anti-BODIPY antibodies (BODIPY FL Rabbit IgG Fraction, A-5770, Lifetechnologies) are commercially available as traps that bind to BODIPY. Among these, the biotinyl group is also preferred as a label.

[0095] The labeled sterol is preferably a labeled cholesterol. Examples of labeled cholesterol include, for example, biotin-added cholesterol represented by formulas (II), (III) or (IV) below.

[0096]

Transformation 3

[0097]

[0098] (In the formula, n is an integer between 1 and 23.)

[0099]

Chemistry 4

[0100]

[0101] (In the formula, n is an integer between 1 and 7.)

[0102]

Transformation 5

[0103]

[0104] (In the formula, n is an integer between 2 and 5.)

[0105] In tagged sterols, the bonding pattern between the sterol moiety and the tag is not particularly limited, but covalent bonding is preferred. For example, the tag can be covalently bonded to the C3 position of the sterol moiety, or the C3 position of the sterol moiety and the tag can be covalently bonded via a linker. The linking segment is not particularly limited; for example, cross-linking using functional groups is simple and preferred. The functional groups are not particularly limited; for example, amino, carboxyl, and thiol groups can be used, but commercially available cross-linking agents are preferred.

[0106] When cholesterol is used as a sterol, the hydroxyl group at the C3 position lacks reactivity as a functional group. Therefore, as shown in step 1 of the diagram below, the hydroxyl group at the C3 position can be reacted with toluenesulfonyl chloride to generate toluenesulfonic acid (in the diagram, R-OH is cholesterol). Furthermore, as shown in step 2 of the diagram below, by reacting toluenesulfonic acid with a labeled alcohol, the toluenesulfonyl group is deactivated to obtain labeled cholesterol (in the diagram, R'-OH is the labeled alcohol).

[0107]

Transformation 6

[0108] (Step 1)

[0109]

[0110] (Step 2)

[0111]

[0112] Alternatively, as shown in step 1 of the diagram below, the hydroxyl group at the C3 position of cholesterol can be reacted with an alkylating agent such as ethyl bromoacetate, ethyl bromopropionate, or ethyl bromobutyrate to generate an ether (in the diagram, R-OH is cholesterol, R' is, for example, methylene, ethylene, or propylene, and Et is ethyl). Furthermore, as shown in step 2 of the diagram below, a carboxyl group can be imparted to the C3 position of cholesterol by alkaline hydrolysis of the product with potassium hydroxide or the like. This C3 carboxyl group can then be tagged by a cross-linking reaction to obtain tagged cholesterol.

[0113]

Transformation 7

[0114] (Step 1)

[0115] R-OH+Br-R′-(C=O)-O-Et→ROR′-(C=O)-O-Et

[0116] (Step 2)

[0117]

[0118] The functional groups on the label vary depending on the type of label. For example, when using peptides or proteins in the label, amino, carboxyl, and thiol (SH) groups can be used. When using biotin in the label, the carboxyl groups of the biotin side chains can be used. The linker is preferably a chain-like compound (e.g., a polymer compound) with functional groups at both ends. When biotin is used as a label attachment, commercially available biotin-labeling reagents can also be used. These reagents contain biotin with spacer arms (e.g., PEG chains) of various lengths terminally bound to functional groups.

[0119] Next, the crosslinking reaction of representative functional groups will be explained. As a functional group, a compound having a carboxyl group can be combined with a compound having an amino group as a reactive group via three steps as shown in the diagram below. First, as shown in step 1 of the diagram below, a compound having a carboxyl group is reacted with a compound having a carbodiimide group (-N=C=N-) (in the diagram below, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC)). Next, as shown in step 2 of the diagram below, an unstable NHS ester is formed by reacting the product of step 1 with NHS. Furthermore, as shown in step 3 of the diagram below, the two can be crosslinked by reacting the product of step 2 with a compound having an amino group. For example, this crosslinking can be performed when combining a sterol or linker having a carboxyl group with a tag having an amino group. Additionally, when crosslinking a sterol or linker having a carboxyl group with a tag having a carboxyl group, a crosslinking agent having amino groups at both ends can also be used.

[0120]

Transformation 8

[0121] (Step 1)

[0122]

[0123] (Step 2)

[0124]

[0125] X = H or SO3Na

[0126] (Step 3)

[0127]

[0128] As a functional group, compounds having an amino group, as shown in the figure below, can be crosslinked with compounds having N-hydroxysuccinimide (NHS) ester or isothiocyanate as reactive groups. For example, when crosslinking a connector having an amino group and a label having an amino group, a crosslinking agent having NHS ester at both ends can also be used.

[0129]

Chemistry 9

[0130] [Reaction of NHS esters and amino groups]

[0131]

[0132] X = H or SO3Na

[0133] [Reaction between isothiocyanate and amino groups]

[0134]

[0135] As a functional group, compounds with thiol groups, as shown in the figure below, can be crosslinked with compounds having maleimide or bromo(or iodo)acetamide groups as reactive groups. For example, when crosslinking a connector with thiol groups and a label with thiol groups, a crosslinking agent having maleimide at both ends can also be used.

[0136]

Chemistry 10

[0137] The reaction between maleimide and thiol groups.

[0138]

[0139] The reaction between bromo(iodo)acetamide and thiol groups

[0140]

[0141] When a compound having a carboxyl group as a functional group and a compound having an amino group as a reactive group are covalently bonded, an amidation reaction using a known condensing agent can also be utilized. Examples of such condensing agents include, for instance, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphonate (HATU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine. Chloride, 2-chloro-1,3-dimethylimidazoline 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate, diphenylphosphonyl azide, chlorotripyrrolidinylphosphonium hexafluorophosphate, N,N'-diisopropylcarbodiimide, etc.

[0142] The crosslinking and amidation reactions described above can be carried out at room temperature and pressure. The solvent used in the reaction is not particularly limited as long as it is inactive for the above reactions and can dissolve or disperse the compounds attached to the reactants. 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 alone or in mixtures.

[0143] The first capture body, which specifically binds to the tag and has a labeled substance, is a capture body labeled with the labeled substance and specifically binding to the tag. The capture body specifically binding to the tag can be suitably determined according to the type of tag. For example, the combination of the tag described above and the substance specifically binding to that tag can be selected from antibodies, aptamers, ligand receptors, oligonucleotides, biotinylate derivatives, avidin derivatives, histidine tags, Ni-NTA, GST, glutathione, etc. Among these, avidin derivatives or antibodies are preferred. As an avidin derivative, avidin or streptavidin is preferred.

[0144] 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), biantibodies, trisomic antibodies, etc. Antibodies can also be either monoclonal or polyclonal antibodies. The source of the antibody is not particularly limited and can be an antibody derived from any mammal, such as a mouse, rat, hamster, rabbit, goat, horse, or camel. The isotype of the antibody can be any of IgG, IgM, IgE, or IgA, preferably IgG. The antibody that specifically binds to the tag can be a commercially available antibody or an antibody prepared by methods known in the art.

[0145] The labeling substance is not particularly limited, and examples include substances that generate a signal themselves (hereinafter also referred to as "signal generating substances"), substances that catalyze the reaction of other substances to generate a signal, etc. Examples of signal generating substances include fluorescent substances and radioactive isotopes. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Enzymes generate signals such as light and color through the reaction of enzymes with suitable substrates. Examples of enzymes include alkaline phosphatase (ALP), peroxidase (POD), β-galactosidase, luciferase, etc. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioactive isotopes include... 125 I, 14 C 32 P, etc. As labeling substances, enzymes are preferred, especially ALP and POD.

[0146] Labeling of a tagged body specifically binding to a tag can be achieved by directly or indirectly binding the tagged body to the tag. A tagged body directly bound to the tag is, for example, a covalently bound tagged body. This can be obtained by covalently binding the tag and the tagged body using, for example, a commercially available labeling kit or cross-linking agent. When both the tagged body and the tag are proteins, a tagged body directly bound to the tag can be a fusion protein of the tagged body and the tag. This can be produced using recombinant gene techniques known in the art.

[0147] When indirectly binding a labeled substance to a capture body, a substance covalently labeled and specifically binding to the capture body can be used. For example, when the capture body specifically binding to the tag is an avidin, a biotinylate covalently labeled substance (labeled biotinylate) can be used. Avidins typically form tetramers, binding to 4 molecules of biotinylate. By mixing avidins and labeled biotinylate at a ratio of 1 or 2 molecules of labeled biotinylate bound to the tetramer, avidins bound to labeled biotinylate are obtained. When the capture body specifically binding to the tag is an antibody, an antibody covalently labeled and specifically binding to said antibody (labeled second antibody) can be used.

[0148] In a preferred embodiment, the first capture body is a labeled antibody, labeled avidin, or labeled streptavidin that specifically binds to a tag. As the labeled antibody, antibodies covalently bonded to a labeled substance or antibodies fused with a labeled substance are preferred. As the labeled avidin and labeled streptavidin, avidin and streptavidin covalently bonded to a labeled substance, avidin and streptavidin fused with a labeled substance, and avidin and streptavidin bound with one or two molecules of labeled biotin are preferred.

[0149] Contact between the lipoprotein, the tagged sterol, and the first trap in the sample can be achieved by mixing the sample with the tagged sterol and the first trap. It is believed that through this mixing, the tagged sterol binds to the lipoprotein, and the first trap binds to the tag exposed on the outer surface of the lipoprotein. As a result, a complex is formed containing the lipoprotein with the tagged sterol and the first trap. The order of mixing is not particularly limited; the sample and the tagged sterol and the first trap can be mixed substantially simultaneously or sequentially.

[0150] The amount of tagged sterol added is not particularly limited. For example, tagged sterols 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 trapping agent added is not particularly limited and can be appropriately set according to the type of trapping agent and the labeled substance.

[0151] In a preferred embodiment, firstly, the lipoprotein in the sample is brought into contact with the tagged sterol. Thereby, the tagged sterol binds to the lipoprotein. Subsequently, the lipoprotein containing the tagged sterol is brought into contact with a first trap. Thereby, the first trap binds to the tag exposed on the outer surface of the lipoprotein, forming a complex containing the tagged sterol and the first trap. 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 can 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 can be left to stand, stirred, or shaken. The temperature and time conditions for mixing the lipoprotein containing the tagged sterol and the first trap are also not particularly limited and can be suitably determined from the above ranges.

[0152] In a preferred embodiment, a second trapping agent that specifically binds to the lipoprotein is used in the contact between the lipoprotein, the tagged sterol, and the first trapping agent. Specifically, the sample, the tagged sterol, the first trapping agent, and the second trapping agent are mixed. The order of mixing is not particularly limited. Furthermore, the amount of the second trapping agent added is not particularly limited and can be suitably set according to the type of trapping agent, etc.

[0153] The second trapping agent is not particularly limited to any substance that can specifically bind to a portion of the surface of a lipoprotein. Examples of such substances include antibodies and aptamers. Antibodies that specifically bind to lipoproteins are preferred as the second trapping agent, and antibodies that specifically bind to apolipoproteins, which are components of lipoproteins, are more preferred. Examples of such antibodies include anti-ApoA antibodies (anti-ApoAI and anti-ApoAII antibodies), anti-ApoB antibodies, and anti-ApoE antibodies (anti-ApoE2, anti-ApoE3, and anti-ApoE4 antibodies). Among these, anti-ApoAI antibodies are particularly preferred. Commercially available anti-lipoprotein antibodies or anti-ApoAI antibodies can also be used.

[0154] In the contact between the lipoprotein, the tagged sterol, and the first trapping body, a complex is formed by further contacting the second trapping body with the first trapping body, the lipoprotein bound to the tagged sterol, and the second trapping body. In this complex, the first trapping body binds to the tag exposed on the outer surface of the lipoprotein, and the second trapping body binds to the surface of the lipoprotein. That is, the lipoprotein containing the tagged sterol is in a sandwiched state between the first trapping body and the second trapping body. In this embodiment, the complex of the first trapping body, the lipoprotein containing the tagged sterol, and the second trapping body is hereinafter referred to as a "sandwich complex".

[0155] The preferred contact sequence in the formation of the sandwich complex is as follows: First, the lipoprotein in the sample is contacted with the tagged sterol. The tagged sterol then binds to the lipoprotein. Next, the lipoprotein containing the tagged sterol is contacted with a second trap. The second trap binds to the surface of the lipoprotein containing the tagged sterol, forming a complex of the lipoprotein containing the tagged sterol and the second trap. Subsequently, the complex of the lipoprotein containing the tagged sterol and the second trap is contacted with a first trap. The first trap binds to the tag exposed on the outer surface of the lipoprotein, forming a sandwich complex.

[0156] The temperature and time conditions for mixing the lipoprotein containing the tagged sterol and the second trap are not particularly limited. For example, the mixture of the lipoprotein containing the tagged sterol and the second trap can 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 can be left to stand, stirred, or shaken.

[0157] Complexes of lipoproteins containing tagged sterols and second traps can also be formed on a solid phase. For example, the aforementioned complexes can be formed on a solid phase by contacting the lipoproteins, tagged sterols, second traps, and solid phases in the sample. The order of contact is not particularly limited; for example, the lipoproteins and tagged sterols in the sample can be contacted first, followed by contacting the lipoproteins containing tagged sterols, the second traps, and the solid phase. Alternatively, the lipoproteins containing tagged sterols and the second traps can be contacted first, followed by contacting the complexes of the lipoproteins containing tagged sterols and the second traps with the solid phase.

[0158] The second trap can also be pre-immobilized on a solid phase. For example, after contacting the lipoproteins in the sample with the tagged sterols, the lipoproteins containing the tagged sterols can be contacted with the solid phase immobilized with the second trap. Alternatively, the lipoproteins containing the tagged sterols can be contacted with the solid phase immobilized with the second trap. The temperature and time conditions when using the solid phase are not particularly limited. For example, the same conditions as those for mixing the lipoproteins containing the tagged sterols and the second trap can be used.

[0159] The solid phase can be any insoluble carrier capable of immobilizing the second trap. For example, the trap can be immobilized on the solid phase by direct or indirect binding between the solid phase and the second trap. Examples of direct bonds between the solid phase and the second trap include adsorption to the solid phase surface via hydrophobic interactions or covalent bonding. For example, when the second trap is an antibody and the solid phase is a microplate for ELISA, the antibody can be immobilized in the wells of the plate by adsorption. Furthermore, when the second trap is an antibody and the solid phase has functional groups on its surface, the antibody can be immobilized on the solid phase surface using covalent bonds of these functional groups. For example, when the solid phase is a particle with carboxyl groups, a cross-linking reaction of a compound having the aforementioned carboxyl groups can be used. Specifically, the carboxyl groups on the particle surface are activated with WSC, and then reacted with NHS to form an NHS ester. Furthermore, when the particle with the NHS ester is brought into contact with the antibody, the NHS ester reacts with the amino groups of the antibody, and the antibody is immobilized on the particle surface by covalent bonds.

[0160] As an indirect binding between the solid phase and the second trap, examples include binding via molecules that specifically bind to the second trap. The second trap can be immobilized on the solid phase surface by pre-immobilizing such molecules. For example, when the second trap is an antibody, examples of molecules that specifically bind to the second trap include protein A, protein G, and antibodies that specifically recognize antibodies (second antibodies). Alternatively, a combination of substances intermediate between the second trap and the solid phase can be used to bind them. Examples of such combinations include biotinylate and avidin, haptens and anti-hapten antibodies, etc. For example, when the second trap is pre-modified with DNP, the second trap can be immobilized on the solid phase containing an immobilized anti-DNP antibody.

[0161] The raw materials for the solid phase can be selected from organic polymers, inorganic compounds, and biopolymers. Examples of organic polymers include latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylic acid copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymers, acrylic acid polymers, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, and polyvinyl acetate acrylate. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, cobalt, and ferrite), silicon oxide, alumina, and glass. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these can also be used in combination.

[0162] 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 shape of particles, a suspension of particles can be used as the solid phase in the formation of the aforementioned composite. When the solid phase is in the shape of a container such as a microplate, the formation of the aforementioned composite can be carried out within the container that serves as the solid phase. When using a second trapping body immobilized with magnetic particles, the assay can also be performed using commercially available fully automated immunoassay apparatuses such as the HISCL (registered trademark) series (Sysmex Co., Ltd.).

[0163] Bound / Free (BF) separation, which removes unreacted free components, can also be performed between the contact between a lipoprotein containing tagged sterols and a second trap, and between the contact between the formed complex and the first trap. Unreacted free components are those that do not constitute the complex of the lipoprotein containing tagged sterols and the second trap. Examples include free tagged sterols that do not bind to lipoproteins, free second traps that do not bind to lipoproteins, and impurities in the sample. The method of BF separation is not particularly limited; for example, the complex and unreacted free components can be separated by recovering only the complex using methods such as ultracentrifugation. When a complex forms on a solid phase, if the solid phase is particles, the complex and unreacted free components can be separated by recovering the particles by centrifugation or magnetic separation and removing the supernatant. When the solid phase is a container such as a microplate or microtube, the complex and unreacted free components can be separated by removing the liquid containing the unreacted free components.

[0164] After removing unreacted free components, the recovered complex or the solid phase of the immobilized complex can be washed with a suitable aqueous medium. Examples of such aqueous media include water, physiological saline, PBS, Tris-HCl, and Good buffer. Surfactants may also be added to the aqueous medium as needed. Surfactants are not particularly limited; for example, surfactants used in washing buffers from the fields of biochemistry or molecular biology can be appropriately selected.

[0165] In the measurement method of this embodiment, a signal generated by a labeled substance is detected. The labeled substance is a label present in a first trap that binds to a tag exposed on the outer surface of the lipoprotein. Thus, the signal generated by this labeled substance reflects the amount of tagged sterols in the lipoprotein. That is, the detection result of this signal becomes an indicator of the sterol harvesting energy of the lipoprotein.

[0166] In this specification, "detection signal" includes the presence or absence of a qualitative detection signal, the quantification of signal intensity, and the semi-quantitative detection of signal intensity. "Semi-quantitative detection of signal intensity" refers to detecting signal intensity in multiple stages, such as "no signal generation," "weak," and "strong." Preferably, the measured value is obtained by quantifying the signal intensity generated by the labeled substance contained in the aforementioned complex. Alternatively, as needed, a value obtained by subtracting the background value from the measured signal intensity value can be obtained. Examples of background values ​​include, for instance, the measured signal intensity value obtained by measuring any one of the following: without using a sample, with a labeled sterol, a first trapping agent, and a second trapping agent.

[0167] The method for detecting the signal is known in the prior art. In this embodiment, a suitable measurement method can be selected according to the type of signal originating from the labeled substance. For example, when the labeled substance is an enzyme, the signal of light, color, etc., generated by the reaction of the enzyme with a substrate against the enzyme can be measured using a known device. Examples of such measuring devices include spectrophotometers and luminometers.

[0168] The substrate for an enzyme can be appropriately selected from known substrates, corresponding to the type of enzyme. For example, when using peroxidase as an enzyme, chemiluminescent substrates such as LUMINOR and its derivatives, and chromogenic substrates such as 2,2'-azonobis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylene diamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB) can be cited as substrates. Furthermore, when using alkaline phosphatase as an enzyme, CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxane-3,2'-(5'-chloro)tricyclo[3.3.1.1]) can be cited as a substrate. 3,7 Chemiluminescent substrates such as sodium decane-4-yl)phenylphosphate, CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane-4-yl)phenylphosphate), and chromogenic substrates such as 5-bromo-4-chloro-3-indole phosphate (BCIP), sodium 5-bromo-6-chloro-indole phosphate, and p-nitrophenyl phosphate. In a preferred embodiment, the signal is a chemiluminescent signal generated by contacting the enzyme and the substrate.

[0169] When the labeling substance is a radioactive isotope, the radiation as a signal can be measured using a known device such as a scintillation counter. Furthermore, when the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescence microplate reader. Moreover, the excitation wavelength and fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.

[0170] B / F separation to remove unreacted free components can also be performed before signal detection. Examples of unreacted free components include, for instance, free first traps that do not bind to the tag. The specific methods and cleaning solutions for B / F separation are the same as the cleaning process described above.

[0171] The above-described steps are performed in vitro. Furthermore, the above-described steps are performed in a substantially cell-free system. A substantially cell-free system means that cells are not actively added for the purpose of determining sterols in lipoproteins. For example, in conventional methods for determining cholesterol excretion function, cells that accumulate cholesterol, such as macrophages, are used. However, in the determination method of this embodiment, since the tagged sterols are directly bound to the lipoproteins in the sample, there is no need to use such cells. Even when the sample contains cells derived from organisms, it is considered that the cells themselves have almost no impact on the binding of tagged sterols to lipoproteins, and the determination method is considered a cell-free system.

[0172] A further embodiment of the present invention relates to a reagent for the determination of sterols in lipoproteins (hereinafter also referred to as "the reagent of this embodiment"). The reagent of this embodiment contains labeled sterols and is used in the determination method of this embodiment described above. The details of the labeled sterols are the same as those described for the determination method of this embodiment.

[0173] The reagent of this embodiment can also be provided to the user in a container containing labeled sterols. An example of the reagent of this embodiment is shown below. Figure 1 . Reference Figure 1 10 indicates the reagent contained in the container of this embodiment. The labeled sterol in the reagent may be a solid (e.g., powder, crystal, freeze-dried product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). When the labeled sterol is contained in the reagent in liquid form, an aqueous medium, as described above, can be used as a solvent. Stabilizers such as casein and BSA may also be added to the aqueous medium as needed.

[0174] Further embodiments of the present invention relate to the use of tagged sterols in reagents for the determination of sterols in the manufacture of lipoproteins, wherein the tagged sterol is attached at the C3 position of the sterol backbone. Details of the tagged sterols are the same as those described for the determination method of this embodiment.

[0175] A further embodiment of the present invention relates to a kit for determining sterols in lipoproteins containing a reagent with a labeled sterol (hereinafter also referred to as "the kit of this embodiment"). The kit of this embodiment is used in the determination method of this embodiment described above. The details of the labeled sterol are the same as those described for the determination method of this embodiment. For example, a container holding a reagent with a labeled sterol may be bundled into a box and provided to the user as the kit of this embodiment. An accompanying document may also be included in the box. The accompanying document may describe the composition of the reagent, the structure of the labeled sterol, the method of using the reagent, the method of storing the reagent, etc. An example of the kit of this embodiment is shown below. Figure 2A . Reference Figure 2A 11 indicates the reagent kit of this embodiment, 12 indicates the container containing the reagent containing the labeled sterol, 13 indicates the packaging box, and 14 indicates the accompanying documents.

[0176] The kit of this embodiment may also include a first reagent containing a tagged sterol and a second reagent containing a first trapping agent that specifically binds to the tag and has a labeled substance. Additionally, the kit of this embodiment may also include a third reagent containing a second trapping agent that specifically binds to lipoproteins. Details of the first and second trapping agents are the same as those described in the assay method of this embodiment.

[0177] The kit of this embodiment can also be packaged in containers holding each reagent into a box and provided to the user. An accompanying document may also be included in the box. This document may describe the composition of each reagent, the structure of the labeled sterols, the method of use for each reagent, and the method of storage for each reagent. An example of the kit of this embodiment is shown below. Figure 2B . Reference Figure 2B 21 indicates the kit of this embodiment, 22 indicates the first container containing the first reagent containing the tagged sterol, 23 indicates the second container containing the second reagent containing the first capture body, 24 indicates the package box, and 25 indicates the accompanying documents.

[0178] In addition to the first and second reagents described above, the kit of this embodiment may also include a third reagent containing a second trapping body. (Refer to...) Figure 2C 31 indicates the kit of this embodiment, 32 indicates the first container containing the first reagent containing the tagged sterol, 33 indicates the second container containing the second reagent containing the first trap, 34 indicates the third container containing the third reagent containing the second trap, 35 indicates the package box, and 36 indicates the accompanying documents.

[0179] The labeled sterol, the first trap, and the second trap in the reagent can each be a solid (e.g., powder, crystal, freeze-dried product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). When the labeled sterol is contained in the reagent in liquid form, an aqueous medium, as described above, can be used as a solvent. Stabilizers such as casein and BSA can also be added to the aqueous medium as needed.

[0180] In the third reagent, the second trapping body may also be pre-immobilized in the solid phase. In this case, the third reagent contains the second trapping body immobilized in the solid phase. The details of the solid phase are the same as those described in the determination method of this embodiment. When the solid phase is a particle, in... Figure 2C In the diagram, 34 represents a container holding a reagent containing a second trapping body immobilized on particles. An example of the kit of this embodiment, where the solid phase is a microplate and the third reagent is a microplate immobilizing the second trapping body, is shown below. Figure 2D . Reference Figure 2D 41 represents the kit of this embodiment, 42 represents the first container containing the first reagent containing a tagged sterol, 43 represents the second container containing the second reagent containing the first trap, 44 represents the microplate immobilizing the second trap, 45 represents the packaging box, and 46 represents the accompanying documentation. Figure 2D In this context, the microplate is a 96-well plate.

[0181] The kit of this embodiment may also include calibrators. The calibrators include, for example, a buffer solution free of lipoproteins (negative control) and a buffer solution containing lipoproteins of known concentration. The kit of this embodiment may also include a washing solution. The details of the washing solution and the aqueous medium and surfactants contained therein are the same as those described for the assay method of this embodiment.

[0182] Further embodiments of the present invention relate to the use of a tagged sterol, a first trap having a labeling substance specifically bound to the aforementioned label, and a second trap having a labeling substance specifically bound to the aforementioned lipoprotein for the manufacture of a kit for the determination of sterols in lipoproteins, wherein the aforementioned tagged sterol has the label attached at the C3 position of the sterol backbone. The details of the tagged sterol, the first trap, and the second trap are the same as those described for the determination method of this embodiment.

[0183] The present invention will now be described in detail with reference to embodiments, but the present invention is not limited to these embodiments.

[0184]

Example

[0185] [Example 1: Modification of Tag-Added Sterols]

[0186] Modulated biotin-PEG, as a tagged sterol. n-Cholesterol, Biotin-(CH2) n -Cholesterol and biotin-PEG7(ether)-cholesterol. The biotin groups attached to these biotins in cholesterol are all D-biotin biotin groups. Their respective structural formulas are shown below.

[0187] [Chemical 11] Biotin-PEG n -cholesterol

[0188]

[0189] (In the formula, n is 1, 2, 3, 7, 11, or 23.)

[0190]

Chem. 12

[0191]

[0192] (In the formula, n is 2 or 5.)

[0193] [Chemical 13] Biotin-PEG7 (ether)-cholesterol

[0194]

[0195] (1) Biotin-PEG n - Modulation of cholesterol

[0196] Next, we will show biotin-PEG. n - Synthetic scheme for cholesterol.

[0197]

Chemistry 14

[0198] (Step 1)

[0199]

[0200] (Step 2)

[0201]

[0202] (Step 3)

[0203]

[0204] Following step 1, cholesterol (3 mmol) (Tokyo Chemical Industries, Ltd. (hereinafter also referred to as TCI)), ethyl bromoacetate (6 mmol), and sodium hydride (9 mmol) were dissolved in 15 mL of DMF. After stirring at room temperature under an argon atmosphere for 18 hours, the mixture was purified by silica gel column chromatography. Following 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. Next, crushed potassium hydroxide (0.22 mmol) was added, and the mixture was stirred at room temperature under an argon atmosphere for 1 hour. The solutions 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 a developing solvent (hexane:ethyl acetate = 10:1). The Rf value of the starting material was 0, the Rf value of the product in step 1 was 0.3, and the Rf value of the product in step 2 was 0.

[0205] Following step 3, dissolve the compound (0.03 mmol) obtained in step 2 and biotin-PEG in DMF (1 mL). 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 stirred at room temperature under an argon atmosphere for 18 hours. The reaction solutions obtained in step 2, step 3, and their mixtures were spotted onto silica gel plates and developed with a developing solvent (dichloromethane:methanol = 10:1). The product of step 3 (Biotin-PEG) n The Rf value of (-cholesterol) is 0.1–0.2. Biotin-PEG was recovered as labeled cholesterol by HPLC separation. n -Cholesterol. The following refers to the specified biotin-PEG. n In terms of cholesterol, the PEG chain is referred to as "PEG1", "PEG2", "PEG3", "PEG7", "PEG11" or "PEG23" depending on its length (the number of n).

[0206] (2) Biotin-(CH2) n - Modulation of cholesterol

[0207] In step 3 of (1) above, in addition to replacing 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), biotin-(CH2) is modulated in the same manner as described in (1) above. n-Cholesterol. The following refers to the specified biotin-(CH2). n - In the case of cholesterol, the carbon atom (n) between the nitrogen atoms of the two amide groups is called "C2-amide" or "C5-amide".

[0208] (3) Modulation of biotin-PEG7 (ether)-cholesterol

[0209] The following is a synthesis scheme for biotin-PEG7 (ether)-cholesterol.

[0210]

Chemistry 15

[0211] (Step 1)

[0212]

[0213] (Step 2)

[0214]

[0215] Cholesterol (1.94 mmol) (TCI) was dissolved in pyridine (6 mL), and the resulting solution was cooled to 0 °C. Toluenesulfonyl chloride (3.87 mmol) was dissolved in pyridine (1.2 mL). Following step 1, toluenesulfonyl chloride solution was added to the cholesterol solution, and the mixture was stirred overnight at room temperature. The reaction solution obtained in step 1 was concentrated by rotary evaporator, and the solid was recovered by dissolving it in 1.2 mL of chloroform. Methanol (15 mL) was added to precipitate cholester-5-ene-3β-toluenesulfonate. The precipitate was obtained by filtration, washed with methanol (15 mL) and acetonitrile (6 mL), and dried under vacuum. Following step 2, cholester-5-ene-3β-toluenesulfonate (0.02 mmol) and biotin-PEG7-ol (BroadPharm) obtained in step 1 were dissolved in 0.2 mL of 1,4-dioxane, and the mixture was heated under reflux at 110 °C for 18 hours. Biotin-PEG7(ether)-cholesterol (hereinafter also referred to as "PEG7(ether)") was recovered by silica gel column chromatography as a tagged cholesterol. As shown in the above synthetic scheme, due to the PEG7(ether), the toluenesulfonyl group is removed by a nucleophilic reaction of biotin-PEG7-ol, and the oxygen atom at the 3-position of the sterol skeleton is tagged, which is considered to be a stereoinversion of the oxygen atom at the 3-position.

[0216]

Example 2: Determination of sterols in lipoproteins (1)

[0217] In Example 1, C5-amide, PEG3, PEG7, PEG11, and PEG23 were used in the preparation of biotin-added cholesterol. Each biotin-added cholesterol was mixed with samples containing lipoproteins at various concentrations, and the biotin-added cholesterol in the lipoproteins was measured to investigate quantification. Furthermore, two lipoproteins with different activities were similarly measured to investigate whether results reflecting the qualitative activity of the lipoproteins were obtained. The measurements were performed using a research-grade fully automated high-sensitivity immunoassay device HI-1000 (Sysmex Corporation).

[0218] (1) Sample modulation

[0219] (1.1) Modulation of samples with different lipoprotein concentrations

[0220] An equal volume of 22% polyethylene glycol 4000 (NACALAI TESQUE Co., Ltd.) was mixed with the pooled serum of healthy individuals and allowed to stand at room temperature for 20 minutes. The mixture was then centrifuged at 860 × g at room temperature for 15 minutes. The resulting supernatant was recovered as the HDL fraction. The obtained HDL fraction was then used to prepare five samples with different HDL concentrations by stepwise dilution with PBS. These samples are hereby designated as "C1", "C2", "C3", "C4", and "C5" from lowest to highest HDL concentration. PBS was used as the calibrator "C0" which contained no HDL fraction.

[0221] (1.2) Preparation of samples containing cholesterol-containing HDLs with different collection energies

[0222] Two combined sera with known differences in cholesterol recovery energies of HDL were each treated in the same manner as described in (1.1) to recover HDL fractions. Each HDL fraction was diluted with PBS at the same ratio. Hereinafter, the HDL dilution fraction derived from the serum containing HDL with high recovery energy will be referred to as "Sample H," and the HDL dilution fraction derived from the serum containing HDL with low recovery energy will be referred to as "Sample L." Furthermore, the cholesterol recovery energy of HDL in the combined sera was determined by the method described in U.S. Patent Application Publication No. 2017 / 0315112.

[0223] (2) Preparation of reagents

[0224] (2.1) R1 reagent (a reagent containing labeled sterols)

[0225] As reagent R1, buffer solutions containing biotin-added cholesterol and 0.3% casein sodium were prepared. The concentration of biotin-added cholesterol in each R1 reagent was 0.5 μM.

[0226] (2.2) R2 reagent (a reagent containing the solid phase for immobilizing the second trap)

[0227] As reagent R2, magnetic particles immobilized with anti-ApoAI antibody were modulated. Specifically, the modulation was performed as follows: WSC (Dojin Chemical Research Institute Co., Ltd.) and NHS (Kishida Chemical Co., Ltd.) were added to the magnetic particles, followed by the addition of anti-ApoA1 antibody, causing the carboxyl groups on the surface of the magnetic particles to bind with the anti-ApoA1 antibody. Thus, as reagent R2, a suspension containing magnetic particles immobilized with anti-ApoAI antibody was obtained.

[0228] (2.3) R3 reagent (including reagent for the first captured body)

[0229] As the R3 reagent, a buffer containing alkaline phosphatase (ALP)-labeled streptavidin solution (Promega) was used.

[0230] (2.4) R4 reagent (buffer solution for assay) and R5 reagent (matrix solution)

[0231] As reagents R4 and R5, the HISCL (registered trademark) luminescent substrate group (Sysmex Co., Ltd.) is used, which contains a chemiluminescent substrate solution of assay buffer and ALP.

[0232] (2.5) Cleaning solution

[0233] As a cleaning solution, a buffer containing 0.1% (w / v) Kolliphor P188 was prepared by dissolving the surfactant Kolliphor P188 (a registered trademark) in a buffer solution.

[0234] (3) Measurement

[0235] (3.1) Contact between HDL and labeled sterols

[0236] Reagents R1 through R5 were prepared in an HI-1000 (Sysmex Corporation). As samples, calibrators C0 through C5, sample H, and sample L were used. Each sample (30 μL) was added to 90 μL of reagent R1 and mixed, then incubated at 42°C for 3 minutes. This allows HDL and biotin-added cholesterol to come into contact, resulting in the binding of HDL and biotin-added cholesterol.

[0237] (3.2) Formation of HDL and anti-ApoA1 antibody complex on magnetic particles

[0238] Add 30 μL of reagent R2 to a cuvette containing 120 μL of the mixture of reagent R1 and the sample, and incubate at 42 °C for 2 minutes. This allows HDL and anti-ApoA1 antibody to react. Subsequently, magnetic particles in the mixture are magnetized to remove the supernatant, and the magnetic particles are washed with washing buffer. After magnetizing the magnetic particles and removing the supernatant, add 100 μL of reagent R3 to the magnetic particles immobilized with the complex of biotin-added cholesterol bound to HDL and anti-ApoA1 antibody, and incubate at 42 °C for 3 minutes. This allows biotin-added cholesterol bound to HDL and ALP-labeled streptavidin to come into contact. Magnetic particles in the mixture are magnetized to remove the supernatant, and the magnetic particles are washed with washing buffer.

[0239] (3.3) Determination of labeled sterols in HDL

[0240] The supernatant was removed by magnetizing the cleaned magnetic particles. Reagent R4 (50 μL) and reagent R5 (100 μL) were added to the cuvette containing the magnetic particles, and the reaction was carried out at 37°C for 5 minutes. After the reaction, the chemiluminescence intensity (Count) was measured.

[0241] (4) Results

[0242] The test results of the calibrator are shown in Figures 3A-3E The determination results for samples H and L are shown in... Figures 4A-4E . Reference Figures 3A-3E Regardless of the type of sterol used, the measured values ​​of C1–C5 were consistently higher than those of CO without HDL. This indicates that signals from sterols labeled as binding to HDL can be detected. Therefore, it is demonstrated that sterols labeled in HDL can be measured regardless of the type of sterol used. Furthermore, the higher the HDL concentration in the calibrator, the higher the measured values. Since C1–C5 were modulated from the same pooled serum, it is believed that calibrators with high HDL concentrations contain multiple sterols in HDL. This suggests that quantitative determination of sterols labeled in HDL may be possible.

[0243] In the Figures 3A-3E When comparing the results, it was found that the method using sterols with attached connector length tags detected a higher signal intensity. Additionally, Figure 3A This indicates that even if the spacer portion in the connector is not PEG but a hydrocarbon chain, tagged sterols in HDL can still be determined.

[0244] Reference Figures 4A-4ERegardless of the type of sterol used, the measured value of sample H was significantly higher than that of sample L. Since samples L and H contain approximately the same concentration of HDL fraction, the difference in measured values ​​between samples L and H is considered to correspond to a difference in cholesterol absorption energy of HDL. This suggests that the determination of sterols in HDL provides a result reflecting the qualitative activity of lipoproteins.

[0245]

Example 3: Determination of sterols in lipoproteins (2)

[0246] In the biotin-added 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. Using each biotin-added cholesterol and samples L and H prepared in Example 2, it was investigated whether results reflecting lipoprotein activity were obtained. The determinations were performed in the same manner as in Example 2.

[0247] (1) Reagents, samples and determination

[0248] As reagent R1, a buffer solution containing biotin-added cholesterol and 0.3% casein sodium was prepared. The concentrations of biotin-added cholesterol in each reagent R1 were: C2-amide 6.2 μM, PEG3 0.5 μM, PEG7 0.2 μM, PEG11 0.1 μM, and PEG23 0.05 μM. Reagents R2 through R5 were the same as those used in Example 2. As samples, sample L and sample H prepared in Example 2 were used. Except for using the R1 reagents described above, the determination was performed in the same manner as in Example 2.

[0249] (2) Results

[0250] The measurement results are shown in Figures 5A-5E . Figures 5A-5E This indicates that, even when using C2-amides, as with other biotin-added cholesterol, the measured value of sample H is higher than that of sample L. This suggests that even when the spacer portion in the linker is a short-chain hydrocarbon, it is possible to determine the tagged sterols in HDL, yielding results reflecting the qualitative activity of lipoproteins. (Reference) Figures 5C to 5E By reducing the concentrations of PEG7, PEG11, and PEG23 in reagent R1, the chemiluminescence intensity (count) decreased, with the same result as in Example 2.

[0251]

Example 4: Determination of sterols in lipoproteins (3)

[0252] In the biotin-added cholesterol prepared in Example 1, PEG1 and PEG2 were used. Biotin-added cholesterol in lipoproteins was determined using the various biotin-added cholesterols and the calibrators, sample L, and sample H prepared in Example 2. The determination was performed in the same manner as in Example 2.

[0253] (1) Reagents, samples and determination

[0254] As reagent R1, a buffer solution containing biotin-added cholesterol and 0.3% casein sodium was prepared. The concentration of biotin-added cholesterol in each R1 reagent was 0.5 μM. Reagents R2 to R5 were the same as those in Example 2. As samples, calibrators C0 to C5, sample L, and sample H prepared in Example 2 were used. Except for using the R1 reagents described above, the determination was performed in the same manner as in Example 2.

[0255] (2) Results

[0256] The test results of the calibrator are shown in Figure 6A and Figure 6B The determination results for samples H and L are shown in... Figure 7A and Figure 7B . Reference Figure 6A and Figure 6B Even when using either PEG1 or PEG2, the measured values ​​for C1–C5 were higher than those for CO without HDL. Furthermore, it was shown that the higher the HDL concentration in the calibrator, the higher the measured value. (Reference) Figure 7A and Figure 7B Even when using either PEG1 or PEG2, the measured value of sample H was higher than that of sample L. This suggests that even when using PEG chains with short spacer portions as linkers, it is possible to quantify PEG-tagged sterols in HDL, yielding results reflecting the qualitative activity of lipoproteins.

[0257]

Example 5: Determination of sterols in lipoproteins (4)

[0258] In the biotin-added cholesterol prepared in Example 1, PEG7 (ether) was used. Biotin-added cholesterol in lipoproteins was determined using PEG7 (ether) and the calibrator prepared in Example 2, sample L, and sample H. The determination was performed in the same manner as in Example 2.

[0259] (1) Reagents, samples and determination

[0260] As reagent R1, a buffer solution containing 1 μM PEG7 (ether) and 0.3% casein sodium was prepared. Reagents R2 through R5 were the same as those in Example 2. As samples, calibrators C0 through C5, sample L, and sample H prepared in Example 2 were used. Except for reagent R1, the determination was performed in the same manner as in Example 2.

[0261] (2) Results

[0262] The test results of the calibrator are shown in Figure 8A The determination results for samples H and L are shown in... Figure 8B . Reference Figure 8A The measured values ​​for C1–C5 were higher than those for CO without HDL. This also indicates that the higher the HDL concentration in the calibrator, the higher the measured value. (Reference) Figure 8B The measured value of sample H was higher than that of sample L. As mentioned above, PEG7 (ether) differs from other tagged sterols in that the stereoconfiguration of the oxygen atom at the C3 position of the tagged sterol is reversed, which does not affect the determination. This suggests that even when using tagged sterols with ether bonds at the C3 position of the sterol backbone and the linker, it is possible to quantitatively determine tagged sterols in HDL, obtaining results reflecting the qualitative activity of lipoproteins.

[0263]

Example 6: Investigation of the effect of LCAT on sterol determination (1)

[0264] Cholesterol is collected at the center of the lipoprotein by esterification of its C3 hydroxyl group via LCAT. However, the tagged sterols of this embodiment do not have the aforementioned hydroxyl group. The determination of tagged sterols in the lipoprotein was investigated using recombinant LCAT to determine whether LCAT affects the results.

[0265] (1) Sample modulation

[0266] An equal volume of 22% polyethylene glycol 4000 (NACALAI TESQUE Co., Ltd.) was mixed with the pooled serum of healthy individuals and allowed to stand at room temperature for 20 minutes. The mixture was then centrifuged at 860 × g at room temperature for 15 minutes. The resulting supernatant was recovered as the HDL fraction. A portion of the HDL fraction was taken and the ApoAI concentration was determined using the ApoAI Assay Kit N-Assay TIA ApoAI-H (NITTOBO MEDICAL Co., Ltd.). The specific procedures for concentration determination were performed according to the manual accompanying the kit. For ApoA1 contained in the HDL fraction, recombinant LCAT (Sino Biological Co., Ltd.) was mixed with the HDL fraction at a weight ratio of 1:10 to prepare the LCAT-added sample. For comparison, the HDL fraction without LCAT was used as the LCAT-free sample in the assay.

[0267] (2) Reagents and assays

[0268] As reagent R1, buffer solutions containing C2-amide, PEG3, PEG7, PEG11, and PEG23, each with biotin-added cholesterol, and 0.3% casein sodium were prepared. The concentrations of biotin-added cholesterol in each R1 reagent were: C2-amide 6.2 μM, PEG3 0.5 μM, PEG7 0.2 μM, PEG11 0.1 μM, and PEG23 0.05 μM. Reagents R2 through R5 were the same as those in Example 2. Except for using the samples and R1 reagents described above, the measurements were performed in the same manner as in Example 2. Furthermore, for background determination, PBS was measured in the same way as for each sample.

[0269] (3) Results

[0270] The measurement results are shown in Figures 9A to 9E In the figure, "Net count" refers to the value obtained by subtracting the PBS measurement value from the measurement value of each sample; "rLCAT+" indicates that LCAT was added to the sample; and "rLCAT-" indicates that LCAT was not added to the sample. (Refer to...) Figures 9A to 9E Regardless of the type of tagged sterol used, the measured values ​​of rLCAT+ were significantly higher than those of rLCAT-. This indicates an increase in the signal of sterols tagged with HDL by the addition of recombinant LCAT. The presence of recombinant LCAT promotes the esterification of endogenous cholesterol present near the HDL surface and its migration into the HDL interior, suggesting an increase in the binding of tagged sterols at the HDL surface. Therefore, the determination of tagged sterols in HDL is indirectly influenced by LCAT.

[0271]

Example 7: Investigation of the effect of LCAT on sterol determination (2)

[0272] The determination of tagged sterols in lipoproteins was investigated using LCAT inhibitors to determine whether LCAT affects the assay.

[0273] (1) Sample modulation

[0274] N-ethylmaleimide (NEM) was added to the pooled serum of healthy individuals at a final concentration of 10 mM as an LCAT inhibitor, and incubated at 37°C for 45 minutes. Subsequently, the pooled serum was diluted 1600-fold with PBS to prepare the NEM-added sample. For comparison, diluted pooled serum without NEM was used as the NEM-unadded sample in the assay.

[0275] (2) Reagents and assays

[0276] As reagent R1, a buffer solution containing 0.5 μM PEG3 and 0.3% sodium caseinate was prepared. Reagents R2 through R5 were the same as those used in Example 2. Except for the sample and reagent R1 described above, the determinations were performed in the same manner as in Example 2. Each determination was performed independently twice.

[0277] (3) Results

[0278] The measurement results are shown in Figure 10 In the figure, "% of control" represents the proportion of NEM measurements with added samples when the measurements without NEM samples are set to 100%. (Refer to...) Figure 10 The measured values ​​of the NEM-added samples were lower than those of the NEM-unadded samples. This indicates that the addition of NEM results in a decrease in the signal of sterols with tags attached to HDL. It is believed that by inhibiting the activity of endogenous LCAT by NEM, the esterification of endogenous cholesterol in HDL and its migration into HDL are inhibited, thus reducing the amount of tagged sterols bound to HDL. Considering the results of Example 6, it is suggested that the method of this embodiment can evaluate the qualitative activity of lipoproteins that reflect LCAT activity.

[0279] [Example 8: Evaluation of the qualitative activity of lipoproteins determined by sterols]

[0280] The activity of lipoproteins can be evaluated by using tagged sterol assays on samples containing lipoproteins whose function has been reduced by oxidation.

[0281] (1) Sample modulation

[0282] Healthy serum was diluted 100-fold with PBS. Diethylenetriaminepentaacetic acid (final concentration 100 μM: Tokyo Chemical Industry Co., Ltd.), hydrogen peroxide (final concentration 20, 40, or 60 μM: Fujifilm and Wako Pure Chemical Industries Co., Ltd.), recombinant myeloperoxidase (final concentration 10 nM: R&D Systems Co., Ltd.), and sodium nitrite (200 μM: Fujifilm and Wako Pure Chemical Industries Co., 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 oxidation-treated samples. For comparison, samples prepared in the same manner as above were measured, except that no hydrogen peroxide was added.

[0283] (2) Reagents and assays

[0284] As reagent R1, a buffer solution containing 0.5 μM PEG3 and 0.3% sodium caseinate was prepared. Reagents R2 through R5 were the same as those used in Example 2. Except for the sample and reagent R1 described above, the determinations were performed in the same manner as in Example 2.

[0285] (3) Results

[0286] The measurement results are shown in Figure 11 In the figure, "% at 0 μM" represents the proportion of the measured values ​​of each oxidized sample when the measured value of the sample without added hydrogen peroxide is set to 100%. (Refer to...) Figure 11 The measured values ​​of the oxidized sample were lower than those of the sample without added hydrogen peroxide. Furthermore, it was shown that the higher the concentration of added hydrogen peroxide, the lower the measured values. By reducing the activity of HDL through oxidation treatment, the collection of endogenous cholesterol in HDL was inhibited, suggesting a decrease in the binding amount of tagged sterols in HDL. This indicates that the method of this embodiment can evaluate the qualitative activity of lipoproteins.

[0287] [Explanation of Symbols]

[0288] 10: Reagents for the determination of sterols in lipoproteins

[0289] 11, 21, 31, 41: Kits for the determination of sterols in lipoproteins

[0290] 12: Container

[0291] 22, 32, 42: Container 1

[0292] 23, 33, 43: Container 2

[0293] 34: Container 3

[0294] 44: Microplates for immobilizing the second capture body

[0295] 13, 24, 35, 45: Bundled boxes

[0296] 14, 25, 36, 46: Accompanying documents

Claims

1. A method for determining sterols in lipoproteins, comprising: The process of forming a complex containing the lipoprotein containing the labeled sterol and the first trap by contacting a lipoprotein, a tagged sterol, and a first trap that specifically binds to the tagged substance in a sample, and... A process for detecting the signal generated by the labeled substance contained in the complex. In the tagged sterol, the tag is attached at the C3 position of the sterol skeleton. The lipoproteins mentioned therein are high-density lipoproteins. The labeled sterol mentioned above is labeled cholesterol, and The tag and the first capture body are selected from the following combinations: Antigen and antibody that recognizes that antigen. Hapten and anti-hapten antibody Peptides or proteins and the aptamers that recognize them. ligands and their receptors, Oligonucleotides and oligonucleotides with their complementary chains, Biotin and avidin, Histidine tag and Ni-NTA, in which The histidine tag is a peptide containing 6 to 10 histidine residues. The Ni-NTA is a chelate that forms a chelate with nickel ions, and Glutathione S-transferase and glutathione.

2. The method of claim 1, wherein the tagged sterol is represented by the following formula (I): [Formula (I)] （I） In the formula, the solid and dashed double lines each independently represent a single bond or a double bond. R 1 It can be an alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms. Whether X and Y are the same or different is indicated by the following: -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 -, in, R 2 Each of the following is independently a linking bond: an alkylene group having 1 to 10 carbon atoms with a substituent; an aryl or heteroaryl group having 6 to 12 carbon atoms with a substituent; or a cycloalkyl or heterocycloalkyl group having 3 to 8 carbon atoms with a substituent. L is derived from -(CH2) d -[R 3 -(CH2) e ] f -、or-[(CH2)] e -R 3 ] f -(CH2) d -express, Among them, R 3 It is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH-, or a connecting bond. Z is a label. Whether a and c are the same or different, they are integers greater than 0 and less than 6. b is 0 or 1. d and e may be the same or different, and are integers greater than 0 and less than 12. f is an integer greater than 0 and less than 24.

3. The method of claim 2, wherein in formula (I), a is 0 or 1. b and c are 1. X is derived from -NH-(C=O)-R 2 -express, Y by-R 2 -(C=O)-NH- indicates that, R 2 Alkylene groups, each independently, without substituents and having 1 to 6 carbon atoms. L is derived from -[(CH2)2-O] f -(CH2) d -express, d is an integer greater than 1 and less than 6. f is an integer greater than 0 and less than 24.

4. The method of claim 1, wherein the label is biotinylated.

5. The method of claim 1, wherein the labeled sterol is represented by formula (II), formula (III) or formula (IV) below: 【Formula (II)】 (II) In the formula, n is an integer greater than 1 and less than 23; 【Formula (III)】 (III) In the formula, n is an integer greater than 1 and less than 7; or Formula (IV) (IV) In the formula, n is an integer between 2 and 5.

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

7. The method of claim 1, wherein in the formation step, the second trapping body that specifically binds to the lipoprotein is used in contact with the lipoprotein, the tagged sterol, and the first trapping body.

8. The method of claim 7, wherein In the formation process, the lipoprotein containing the tagged sterol is brought into contact with the second trap, thereafter The complex containing the lipoprotein with the added sterol and the second trapping body is brought into contact with the first trapping body.

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

10. The method of claim 7, wherein The second trapping body is immobilized in the solid phase. A complex containing the tagged sterol lipoprotein and the second trapping body is formed on the solid phase.

11. The method of claim 7, wherein the second trapping body is the antibody that specifically binds to the lipoprotein.

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

13. The method of claim 1, wherein a B / F separation to remove unreacted free components is performed between the forming step and the detecting step.

14. The method of claim 1, wherein The labeling substance is an enzyme. The signal is a chemiluminescent signal generated by contacting the enzyme and the substrate.

15. The method of claim 14, wherein the enzyme is an alkaline phosphatase or a peroxidase.

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

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