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WO2019177093A1 - Method for quantifying cholesterol in high-density lipoprotein - Google Patents

Method for quantifying cholesterol in high-density lipoprotein Download PDF

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Publication number
WO2019177093A1
WO2019177093A1 PCT/JP2019/010525 JP2019010525W WO2019177093A1 WO 2019177093 A1 WO2019177093 A1 WO 2019177093A1 JP 2019010525 W JP2019010525 W JP 2019010525W WO 2019177093 A1 WO2019177093 A1 WO 2019177093A1
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cholesterol
fraction
hdl
reagent
phospholipase
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PCT/JP2019/010525
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French (fr)
Japanese (ja)
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成則 内田
裕子 平尾
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デンカ生研株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a method for quantifying cholesterol in high-density lipoprotein (hereinafter sometimes referred to as “HDL cholesterol”) and a reagent kit therefor.
  • HDL cholesterol high-density lipoprotein
  • High density lipoprotein receives cholesterol from each tissue including the arteriosclerotic wall, and is therefore related to the removal action of cholesterol accumulated in the cells. Therefore, it is also called reverse cholesterol transfer system. It is known that there is a negative correlation with arteriosclerotic diseases such as coronary arteriosclerosis, and the low value of HDL is set as a low value limit as one of dyslipidemia and is useful as an index of arteriosclerosis It is known that
  • HDL is composed of apoprotein, phospholipid, cholesterol, and neutral fat.
  • Methods for measuring cholesterol in HDL include, for example, a method in which HDL is separated from other lipoproteins by ultracentrifugation and then subjected to cholesterol measurement, or the color intensity is measured by staining lipids after separation by electrophoresis.
  • the method of doing is known.
  • these methods have problems such as complicated operations and inability to process a large number of samples, and are rarely used on a daily basis.
  • a method for measuring cholesterol in HDL As a method for measuring cholesterol in HDL, a method generally used in the field of clinical examination at present is that a precipitant is added to a sample to aggregate lipoproteins other than HDL, and this is removed by centrifugation, and only the separated HDL is separated. Is a method for measuring cholesterol in a supernatant containing. Although this method is simpler than ultracentrifugation or electrophoresis, it involves an operation of adding a precipitant and separating it, so it is not satisfactory in terms of simplicity, and a relatively large amount of sample is required. I need.
  • an enzyme reaction is carried out in the presence of a bile salt or a nonionic surfactant (Patent Document 2). More recently, cholesterol esterase and cholesterol oxidase are chemically modified to include cyclodextrin and the like.
  • a method of specifically capturing cholesterol in HDL in the presence of a contact compound (Patent Document 3) or a method of forming an aggregate or complex with lipoproteins other than HDL, and then capturing cholesterol in HDL by an enzymatic reaction Patent Document 4 and Patent Document 5 are known, but both are problematic in terms of specificity, such as a deviation from the precipitation method being observed in some of the clinical specimens.
  • JP-A-6-242110 Japanese Patent Laid-Open No. 62-126498 JP-A-7-301636 JP-A-8-131197 JP-A-8-201393 Patent No. 5706418 publication
  • An object of the present invention is to selectively, easily and accurately quantify HDL cholesterol in a test sample containing HDL and other lipoproteins such as LDL, without requiring a complicated fraction separation operation. It is to provide a method for quantifying cholesterol in HDL.
  • the inventors of the present application select cholesterol in lipoproteins other than HDL lipoproteins by causing phospholipase and / or sphingomyelinase and a nonionic surfactant to act on the test sample. And the present invention has been reached.
  • the present invention provides the following.
  • a lipoprotein other than the high-density lipoprotein in the test sample comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant acting on the test sample To remove cholesterol from the reaction system.
  • the phospholipase is phospholipase C and / or phospholipase D.
  • the nonionic surfactant is a polyoxyethylene polyoxypropylene block copolymer.
  • the final concentration of the phospholipase is 0.1 to 200 U / mL, and the final concentration of the sphingomyelinase is 0.01 to 50 U / mL, according to any one of (1) to (3) the method of.
  • the final concentration of the phospholipase is 0.1 to 3 U / mL, the final concentration of the sphingomyelinase is 0.02 to 2 U / mL, and the final concentration of the nonionic surfactant is 0.01 to 6.
  • the method according to any one of (1) to (4) which is 1% by weight.
  • the elimination of cholesterol in lipoproteins other than high-density lipoproteins from the reaction system is carried out by eliminating cholesterol with an elimination system containing cholesterol esterase and cholesterol oxidase.
  • the method according to any one of the above.
  • a first step of erasing cholesterol in lipoproteins other than the high-density lipoprotein in the test sample out of the reaction system, and a second step of quantifying cholesterol in the high-density lipoprotein remaining in the reaction system A method for quantifying cholesterol in high-density lipoprotein in a test sample, wherein the first step is performed by the method according to any one of (1) to (6).
  • a kit for quantifying cholesterol in high-density lipoprotein comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant.
  • HDL cholesterol in a test sample can be quantified more selectively and accurately.
  • phospholipase and / or sphingomyelinase (hereinafter sometimes abbreviated as “phospholipase etc.”) and (ii) a nonionic surfactant are included in the test sample.
  • phospholipase etc. phospholipase and / or sphingomyelinase
  • a nonionic surfactant is included in the test sample.
  • a first step in a method for quantifying HDL cholesterol in a test sample comprising a first step of eliminating cholesterol other than HDL in the test sample and a second step of quantifying HDL cholesterol remaining in the reaction system
  • HDL cholesterol in the test sample can be quantified by applying the method of the present invention.
  • this two-step method will be described.
  • the first step of the two-step method is the method of the present invention.
  • the following 2nd processes are not essential, It is possible to quantify HDL cholesterol in a test sample also by applying another well-known method.
  • the method for quantifying HDL cholesterol including the first step and the second step is also the method of the present invention.
  • test sample to be subjected to the method of the present invention is not particularly limited as long as HDL cholesterol in the sample is to be quantified, but is preferably serum or plasma or a dilution thereof, particularly serum. Or a dilution thereof is preferred.
  • HDL to be measured in the present invention is composed of apoprotein, phospholipid, cholesterol, and neutral fat.
  • phospholipase or the like and a nonionic surfactant are allowed to act on the test sample. Further, a cholesterol-reactive enzyme such as cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase, or lipoprotein lipase as required is reacted. Each of these enzymes may be added alone or in combination of two or more.
  • the phospholipase used in the first step only needs to act on at least glycerophospholipid, and it is preferable if it has at least activity on phosphatidylcholine, but lysophosphaethanolamine other than phosphatidylcholine, or glycerophospholipid It may have activity against other sphingomyelin and ceramide. Phospholipase etc.
  • Phospholipase A2 Phospholipase A2
  • PLC Phospholipase C
  • PPD Phospholipase D
  • LYPL lysophospholipase
  • sphingomyelinase Sigma Aldrich
  • the phospholipase concentration at that time is 0. It is preferably 1 to 200 U / mL, more preferably 0.05 to 100 U / mL, and further preferably 0.1 to 3 U / mL.
  • the final concentration of sphingomyelinase is preferably about 0.01 to 50 U / mL, and more preferably 0.02 to 2 U / mL.
  • Sphingomyelinase is not particularly limited as long as it acts on at least sphingomyelin, and may have activity against phosphatidylinositol, which is a component constituting phospholipids other than sphingomyelin.
  • the nonionic surfactant used in the present invention is preferably added at a final concentration of 0.001 to 5% by weight (a total final concentration when a plurality of types of nonionic surfactants are used in combination). It is more preferably about 002 to 3.0% by weight, and further preferably about 0.01 to 1% by weight.
  • Nonionic surfactants include nonionic interfaces such as polyoxyethylene-polyoxypropylene copolymers, amide nonions, polyoxyethylene nonyl phenyl ethers, and polyoxyethylene polycyclic phenyl ethers having an HLB value of 14-17. Although an activator can be mentioned, it is not limited to these.
  • a polyoxyethylene polyoxypropylene block copolymer particularly a block copolymer in which a polyoxypropylene portion is sandwiched between polyoxyethylene portions is preferable.
  • Particularly preferred are those having a weight average molecular weight of 10,000 to 17,000 and an ethylene oxide content of 75 to 95 mol% in the molecule.
  • Pluronic P123 (Adeka), Pluronic F68 (Adeka), Pluronic F88 (Adeka), Pluronic P85 (Adeka), Pluronic 17R-3 (Adeka), Pluronic 17R-4 (Adeka), Pluronic TR-704 (Adeka), Pluronic PE6100 (Adeka), Pluronic PE6400 (Adeka), Pluronic PE6800 (Adeka), Adeka Carpole GH5 (Adeka), Adeka Carpole GH10 (Adeka), Adeka Carpole GH200 (Adeka), Adeka Carpole MH150 (Adeka), Adeka Carpol MH10000 (Adeka), Rebenol WX (Kao), Nonion HS220 (Nippon) Kashiwa, Nymid MT-215 (Nippon), Pronon 208 (Nippon), Newcol-723 (Nippon Emulsifier), Newcol -2614 (Japan emulsifier), Newcol
  • nonionic surfactants such as alkyl ether type nonionic surfactants such as Adecatol LB-83 (Adeka) and Adekatol LB-103 (Adeka), polyoxyethylene alkyl ether type nonionic surfactants Emulgen 109P (Kao), Emulgen TW0106 (Kao) and Emulgen 1108 (Kao), Neugen EA-207D (Daiichi Kogyo Seiyaku), polyoxyethylene alkylphenyl ether, Nonion HS- 240 (Nippon Oil), Newcol-CMP-1 (Nippon Emulsifier) and Newcol-CMP-11 (Nippon Emulsifier) which are polyoxyethylene p-cumylphenyl ether, Polystar OMP (Nippon Oil) which is sodium polycarboxylate, Fatty acid ester type nonionic surfactants, Rheodor TW-L120 (Kao), Rheodor 460 (Kao),
  • lipoprotein cholesterol other than HDL is eliminated by the action of phospholipase, cholesterol esterase and the like.
  • erasing means degrading lipoprotein cholesterol in a test sample so that it does not affect the reaction of cholesterol measurement in the subsequent steps.
  • Examples of a method for eliminating lipoprotein cholesterol include a method in which hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase is decomposed into water and oxygen using catalase.
  • the hydrogen donor and the generated hydrogen peroxide may be reacted with peroxidase to convert to colorless quinone, but is not limited thereto.
  • the method of cholesterol elimination itself is well known in the art.
  • the first step is to add phospholipase etc. and nonionic surfactant together with phospholipase etc. and nonionic surfactant by adding enzyme and surfactant to move out of the reaction system.
  • Decomposition of lipoproteins other than HDL by the agent and elimination of cholesterol contained therein can be simultaneously performed as a single step.
  • the cholesterol esterase concentration (in this specification, the concentration means the final concentration unless otherwise specified) is preferably about 0.1 to 10.0 U / mL. 0.2 to 2.0 U / mL is more preferable.
  • the concentration of cholesterol oxidase is preferably about 0.05 to 10.0 U / mL, more preferably about 0.1 to 1.0 U / mL.
  • the cholesterol esterase is not particularly limited as long as it acts on ester-type cholesterol.
  • cholesterol esterase (CEBP, CEN) manufactured by Asahi Kasei Co., Ltd.
  • cholesterol esterase COE-311, manufactured by Toyobo Co., Ltd.
  • COE-3113 Commercially available products such as COE-313) and cholesterol esterase (CHE-XE) manufactured by Kikkoman
  • the cholesterol oxidase is not particularly limited as long as it acts on free cholesterol.
  • cholesterol oxidase CONII
  • cholesterol oxidase COO-311, COO- 321 and COO-331
  • cholesterol oxidase CHO-CE, CHO-PEWL, CHO-BS
  • Kikkoman Corporation can be used.
  • cholesterol dehydrogenase When cholesterol dehydrogenase is used, it is preferably 0.01 to 200 U / mL, more preferably 0.1 to 100 U / mL. Cholesterol dehydrogenase is not particularly limited as long as it has the ability to oxidize cholesterol and reduce oxidized coenzyme.
  • cholesterol dehydrogenase (CHDH-5) manufactured by Amano Enzyme Co., Ltd. Commercial products can be used.
  • lipoprotein lipase can be further added, and one or two or more kinds may be used in combination, and the final concentration (the final concentration when two or more types are combined) is 0.01 to 0. About 5 U / mL is preferable, and 0.02 to 0.5 U / mL is more preferable.
  • Commercially available products can be used for lipoprotein lipase and phospholipase, such as lipoprotein lipase (LPL-311 and LPL-314) manufactured by Toyobo Co., Ltd., lipoprotein lipase (LPL-3 manufactured by Amano Enzyme, etc.), etc. ), Lipoprotein lipase (LPBP, LP, etc.) manufactured by Asahi Kasei Corporation can be used.
  • reaction solution used in the first step various buffer solutions used in normal biochemical reactions can be used, and the pH is preferably between 5 and 8.
  • a buffer solution of Good, Tris, phosphate, and glycine is preferable.
  • Good buffer solutions such as bis (2-hydroxyethyl) iminotris (hydroxyethyl) methane (Bis-Tris), piperazine-1, 4-bis ( 2-ethanesulfonic acid (PIPES), piperazine-1,4-bis (2-ethanesulfonic acid), 1.5 sodium salt, monohydrate (PIPES1.5Na), 2-hydroxy-3-morpholinopropanesulfone Acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and Piperazine-1,4-bis (2-hydroxy-3-propanesulf
  • a monovalent or divalent cation or a salt thereof can be added for the purpose of easily distinguishing lipoproteins other than HDL.
  • sodium chloride, potassium chloride, manganese chloride, calcium chloride, ammonium chloride, magnesium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and the like can be used.
  • the concentration is preferably 1 to 50.0 g / L, more preferably 5 to 30 g / L.
  • the reaction temperature in the first step is preferably about 25 to 40 ° C, more preferably 35 to 38 ° C, and most preferably 37 ° C.
  • the reaction time is not particularly limited, and is usually about 2 to 10 minutes.
  • HDL cholesterol in the HDL remaining in the reaction system is quantified.
  • the cholesterol quantification method itself is well known, and any known method can be adopted, and is specifically described in the following examples.
  • ester cholesterol in lipoproteins is hydrolyzed using cholesterol esterase to produce free cholesterol and fatty acids, and the resulting free cholesterol and free cholesterol originally present in lipoproteins using cholesterol oxidase Cholesteinone and hydrogen peroxide are generated and quantified by forming a quinone dye in the presence of peroxidase.
  • Examples of compounds that generate quinone dyes include HDAOS (N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline), DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl)) -3,5-dimethoxyaniline) or TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline) and 4-aminoantipyrine, which can generate quinone dyes. It is not limited to these as long as it is a possible combination.
  • cholesterol esterase and cholesterol oxidase are used in the first step
  • the cholesterol esterase and cholesterol oxidase used in the first step can be used as they are in the second step, and it is not necessary to add them newly.
  • NAD (P) H is generated from NAD (P) by the enzymatic reaction.
  • the generated NAD (P) H can quantitate cholesterol in HDL by measuring the absorbance at a wavelength of 330 to 400 nm.
  • the concentration of the compound that generates a quinone dye is preferably about 0.5 to 2.0 mmol / L for HDAOS, and preferably 0.1 to 2.0 mmol / L for 4-aminoantipyrine,
  • the concentration of peroxidase is preferably 0.4 to 5.0 U / mL.
  • sodium azide that is an inhibitor of catalase is used and added to the reaction solution in the second step.
  • the concentration of sodium azide is usually about 0.1 g / L to 1.0 g / L.
  • the concentration of peroxidase is preferably about 2.0 to 5.0 U / mL, more preferably about 3.0 to 4.0 U / mL.
  • the concentration is preferably about 0.4 to 0.8 mmol / L.
  • the presence of the surfactant is not essential and may or may not be added.
  • the surfactant that can be used in the first step is added within the same concentration or lower range in the first step.
  • reaction conditions reaction temperature, time, buffer solution, pH, etc.
  • reaction temperature, time, buffer solution, pH, etc. reaction temperature, time, buffer solution, pH, etc.
  • Example 1 Collect the CM, VLDL, IDL (CVI) fraction and LDL fraction using the ultracentrifugation method, and react by combining the following preparation reagent A or B as the first step and preparation reagent X as the second step.
  • PLDP phospholipase D
  • the ultracentrifugation method is a method of preparing each fraction of CM, VLDL, IDL, LDL, HDL, etc. by using a difference in lipoprotein density of a test sample such as serum by using a sodium bromide solution or the like. .
  • the CVI fraction has a density d ⁇ 1.019 g / mL
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent A BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer (trade name Pluronic F68, the same shall apply hereinafter) 0.25g / L Phospholipase D (PLDP) 0.5 U / mL
  • reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with Preparation Reagent C.
  • Phospholipase D and a nonionic surfactant By using Phospholipase D and a nonionic surfactant, CVI fraction was obtained in the first step. As a result, the erased amount of the image and LDL fraction was increased.
  • Example 2 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagent D or E as the first step and preparation reagent X as the second step. By using phospholipase C in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction was measured in the first step. As a result, the erased amount of the image and LDL fraction was increased.
  • Example 3 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents F to J as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 3. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent F BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L Sphingomyelinase 0.05U / mL
  • Preparation reagent G BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L Sphingomyelinase 0.10 U / mL
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
  • Example 4 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents K to O as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
  • Example 5 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents P to T as the first step and preparation reagent X as the second step. By using PLC in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 5.
  • the prepared reagent C was used as the HDL reagent.
  • the reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 6 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using an ultracentrifugation method, and the following preparation reagents U to Y were combined as a first step and prepared reagent X was combined as a second step. It was confirmed that the use of PLDP in one step increased the amount of CVI and LDL erased in the first step, and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 6. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent X BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDP 2.5U / mL
  • Preparation reagent Y BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDP 5.0U / mL
  • the reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 7 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents Z to AD as the first step and preparation reagent X as the second step. It was confirmed that the use of PLDPV in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
  • the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 7. In this example, the prepared reagent C was used as the HDL reagent.
  • Preparation reagent Z BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDPV 0.1U / mL
  • Preparation reagent AB BES buffer 100 mmol / L pH 6.6 TOOS 1.5mmol / L Catalase 600U / mL Cholesterol esterase 1.4 U / mL Cholesterol oxidase 0.8U / mL Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L PLDPV 1.0U / mL
  • the reaction rate of each preparation reagent with respect to the CVI fraction and the LDL fraction is lower than the reaction rate measured with the preparation reagent C.
  • the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
  • Example 8 Reagent (i) -1, reagent (i) -2 and reagent X having the following reagent composition were prepared, and various surfactants were added to reagent (i) -1 and reagent (i) -2 at 0.025% (W / V) Reagents with added concentrations were prepared.
  • all the various surfactants described in Table 8 below are the above-described commercially available nonionic surfactants.
  • reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with Preparation Reagent C.
  • SPC and a nonionic surfactant CVI fractionation and LDL are performed in the first step. As a result, the amount of elimination of the fraction was increased.

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Abstract

Disclosed are: a method for quantifying cholesterol in a HDL, whereby it becomes possible to selectively, simply and correctly quantify HDL cholesterol in a sample of interest containing an HDL as well as another lipoprotein such as a LDL without requiring any complicated fractionation/separation procedure; and a method for selectively eliminating cholesterol, which can be employed in the aforementioned method. A method for eliminating cholesterol in a lipoprotein other than a high-density lipoprotein in a sample of interest out of a reaction system includes allowing (i) phospholipase and/or sphingomyelinase and (ii) a nonionic surfactant to act on the sample.

Description

高密度リポ蛋白質中のコレステロールの定量方法Method for the determination of cholesterol in high density lipoproteins
　本発明は、高密度リポタンパク質中のコレステロール（以下、「ＨＤＬコレステロール」と呼ぶことがある）の定量方法およびそのための試薬キットに関する。 The present invention relates to a method for quantifying cholesterol in high-density lipoprotein (hereinafter sometimes referred to as “HDL cholesterol”) and a reagent kit therefor.
　高密度リポ蛋白（ＨＤＬ）は動脈硬化壁を含めた各組織からコレステロールを受け取るため細胞内に蓄積したコレステロールの除去作用に関係し、そのためコレステロール逆転送系ともいわれている。冠動脈硬化などの動脈硬化疾患と負の相関があることが知られており、ＨＤＬが低値であることは脂質異常症の一つとして低値限界が設けられており、動脈硬化の指標として有用であることが知られている。 High density lipoprotein (HDL) receives cholesterol from each tissue including the arteriosclerotic wall, and is therefore related to the removal action of cholesterol accumulated in the cells. Therefore, it is also called reverse cholesterol transfer system. It is known that there is a negative correlation with arteriosclerotic diseases such as coronary arteriosclerosis, and the low value of HDL is set as a low value limit as one of dyslipidemia and is useful as an index of arteriosclerosis It is known that
　ＨＤＬはアポタンパク質とリン脂質、コレステロール、中性脂肪から構成されている。ＨＤＬは密度がｄ＝１．０６３～１．２１０ｇ／ｍＬであり、さらにｄ＝１．０６３～１．１２５ｇ／ｍＬであるＨＤＬ２とｄ＝１．１２５～１．２１０ｇ／ｍＬであるＨＤＬ３の２つの分画に分類される。リポ蛋白の分布曲線によりｄ＝１．１２５ｇ／ｍＬ部分にノッチが認められ、ここから密度の重い部分をＨＤＬ３とされる。また、ＨＤＬ中のアポタンパク質のうちアポリポ蛋白Ｅ含有量差から、アポＥの含有量の多いＨＤＬをアポＥ－ｒｉｃｈ ＨＤＬとする亜分画の分け方も存在する。 HDL is composed of apoprotein, phospholipid, cholesterol, and neutral fat. HDL has a density of d = 1.063-1.210 g / mL, HDL2 with d = 1.063-1.125 g / mL, and HDL3 with d = 1.125-1.210 g / mL. It is classified into two fractions. According to the distribution curve of lipoprotein, a notch is observed in the portion of d = 1.125 g / mL, and a portion having a high density is designated as HDL3. In addition, there is a method of dividing a subfraction in which HDL having a high content of apo E is apo E-rich HDL due to the difference in the content of apolipoprotein E among apoproteins in HDL.
　ＨＤＬ中のコレステロールの測定方法としては、例えば超遠心分離によってＨＤＬを他のリポ蛋白と分離した後、コレステロール測定に供する方法や、電気泳動によって分離した後に脂質の染色を行なってその発色強度を測定する方法等が知られている。しかしながら、これらの方法は、いずれも操作が煩雑であったり、多数の検体を処理できない等の問題があり、日常的にはほとんど用いられていない。 Methods for measuring cholesterol in HDL include, for example, a method in which HDL is separated from other lipoproteins by ultracentrifugation and then subjected to cholesterol measurement, or the color intensity is measured by staining lipids after separation by electrophoresis. The method of doing is known. However, these methods have problems such as complicated operations and inability to process a large number of samples, and are rarely used on a daily basis.
　ＨＤＬ中のコレステロールの測定方法として現在臨床検査の領域で一般に用いられている方法は、検体に沈殿剤を加えてＨＤＬ以外のリポ蛋白質を凝集させ、これを遠心分離によって取り除き、分離されたＨＤＬのみを含む上清中のコレステロールを測定する方法である。この方法は、超遠心法や電気泳動法に比較して簡便であるものの、沈殿剤を加えて分離する操作を含むため、簡便性で満足できるものでなく、また、比較的多量の検体量を必要とする。 As a method for measuring cholesterol in HDL, a method generally used in the field of clinical examination at present is that a precipitant is added to a sample to aggregate lipoproteins other than HDL, and this is removed by centrifugation, and only the separated HDL is separated. Is a method for measuring cholesterol in a supernatant containing. Although this method is simpler than ultracentrifugation or electrophoresis, it involves an operation of adding a precipitant and separating it, so it is not satisfactory in terms of simplicity, and a relatively large amount of sample is required. I need.
　一方、酵素的にＨＤＬ中のコレステロールを分別定量する方法も既に検討されている。例えば、ＨＤＬ以外のリポ蛋白を抗体とポリアニオンで予め凝集させておき、ＨＤＬ中のコレステロールのみを酵素的に反応させた後に、酵素を失活させると同時に凝集体を再溶解して吸光度を測定するという方法（特許文献１）がある。しかしながら、この方法は少なくとも３回の試薬を添加する操作が必要なため、限定された分析装置にしか適用できず、汎用性の点で問題があった。 On the other hand, a method for enzymatically quantifying cholesterol in HDL has already been studied. For example, lipoproteins other than HDL are aggregated in advance with an antibody and a polyanion, and after reacting only cholesterol in HDL enzymatically, the enzyme is deactivated and at the same time the aggregate is re-dissolved and the absorbance is measured. (Patent Document 1). However, since this method requires an operation of adding at least three times of reagents, it can be applied only to a limited analyzer, and there is a problem in versatility.
　また、他の方法としては胆汁酸塩又は非イオン界面活性剤の存在下に、酵素反応を行なう方法（特許文献２）、さらに近年ではコレステロールエステラーゼやコレステロールオキシダーゼを化学修飾し、シクロデキストリン等の包接化合物存在下においてＨＤＬ中のコレステロールを特異的に捕える方法（特許文献３）やＨＤＬ以外のリポ蛋白と凝集体や複合体を形成させ、その後にＨＤＬ中のコレステロールを酵素的反応で捕える方法（特許文献４及び特許文献５）が知られているが、いずれも臨床検体の一部のもので沈殿法との乖離が認められる等、特異性の点で問題となっている。 As another method, an enzyme reaction is carried out in the presence of a bile salt or a nonionic surfactant (Patent Document 2). More recently, cholesterol esterase and cholesterol oxidase are chemically modified to include cyclodextrin and the like. A method of specifically capturing cholesterol in HDL in the presence of a contact compound (Patent Document 3) or a method of forming an aggregate or complex with lipoproteins other than HDL, and then capturing cholesterol in HDL by an enzymatic reaction ( Patent Document 4 and Patent Document 5) are known, but both are problematic in terms of specificity, such as a deviation from the precipitation method being observed in some of the clinical specimens.
　一方、本願出願人は、ホスフォリパーゼ及びスフィンゴミエリナーゼがＨＤＬ３以外のリポタンパク質に作用するが、ＨＤＬ３にはほとんど作用しないことを見出し、この現象を利用してＨＤＬ３中のコレステロールを定量する方法を発明し、特許を取得している（特許文献６）。 On the other hand, the applicant of the present application has found that phospholipase and sphingomyelinase act on lipoproteins other than HDL3, but hardly act on HDL3, and a method for quantifying cholesterol in HDL3 using this phenomenon. Invented and patented (Patent Document 6).
特開平６－２４２１１０号公報JP-A-6-242110 特開昭６２－１２６４９８号公報Japanese Patent Laid-Open No. 62-126498 特開平７－３０１６３６号公報JP-A-7-301636 特開平８－１３１１９７号公報JP-A-8-131197 特開平８－２０１３９３号公報JP-A-8-201393 特許第５７０６４１８号掲載公報Patent No. 5706418 publication
　本発明の目的は、煩雑な分画分離操作を必要とせず、ＨＤＬのほか、ＬＤＬ等の他のリポ蛋白を含む被検試料中のＨＤＬコレステロールを選択的に、簡便かつ正確に定量することができる、ＨＤＬ中のコレステロールの定量方法を提供することである。 An object of the present invention is to selectively, easily and accurately quantify HDL cholesterol in a test sample containing HDL and other lipoproteins such as LDL, without requiring a complicated fraction separation operation. It is to provide a method for quantifying cholesterol in HDL.
　本願発明者らは、鋭意研究の結果、被検試料に、ホスフォリパーゼ及び／又はスフィンゴミエリナーゼと、非イオン界面活性剤を作用させることにより、ＨＤＬリポ蛋白質以外のリポ蛋白質中のコレステロールが選択的に露出されることを見出し、本発明に到達した。 As a result of diligent research, the inventors of the present application select cholesterol in lipoproteins other than HDL lipoproteins by causing phospholipase and / or sphingomyelinase and a nonionic surfactant to act on the test sample. And the present invention has been reached.
　すなわち、本発明は以下のものを提供する。
(1)　被検試料に(i)ホスフォリパーゼ及び／又はスフィンゴミエリナーゼと、(ii)非イオン界面活性剤を作用させることを含む、被検試料中の高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去する方法。
(2)　前記ホスフォリパーゼがホスフォリパーゼＣ及び/又はホスフォリパーゼＤである(1)記載の方法。
(3)　前記非イオン界面活性剤が、ポリオキシエチレンポリオキシプロピレンブロック共重合体である(1)又は(2)記載の方法。
(4)　 前記ホスフォリパーゼの終濃度が０．１～２００Ｕ／ｍＬ、前記スフィンゴミエリナーゼの終濃度が０．０１～５０Ｕ／ｍＬである(1)～(3)のいずれか１項に記載の方法。
(5)　 前記ホスフォリパーゼの終濃度が０．１～３Ｕ／ｍＬ、前記スフィンゴミエリナーゼの終濃度が０．０２～２Ｕ／ｍＬであり、非イオン界面活性剤の終濃度が０．０１～１重量％である(1)～(4)のいずれか１項に記載の方法。
(6)　高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去することは、コレステロールエステラーゼ及びコレステロールオキシダーゼを含む消去系によりコレステロールを消去することにより行われる(1)～(5)のいずれか１項に記載の方法。
(7)　被検試料中の、高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去する第一工程と、反応系内に残存する高密度リポ蛋白質中のコレステロールを定量する第二工程を含み、前記第一工程を、(1)～(6)のいずれか１項に記載の方法により行う、被検試料中の高密度リポ蛋白質中のコレステロールの定量方法。
(8)　(i)ホスフォリパーゼ及び／又はスフィンゴミエリナーゼと、(ii)非イオン界面活性剤とを含む、高密度リポ蛋白質中のコレステロール定量用キット。 That is, the present invention provides the following.
(1) In a lipoprotein other than the high-density lipoprotein in the test sample, comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant acting on the test sample To remove cholesterol from the reaction system.
(2) The method according to (1), wherein the phospholipase is phospholipase C and / or phospholipase D.
(3) The method according to (1) or (2), wherein the nonionic surfactant is a polyoxyethylene polyoxypropylene block copolymer.
(4) The final concentration of the phospholipase is 0.1 to 200 U / mL, and the final concentration of the sphingomyelinase is 0.01 to 50 U / mL, according to any one of (1) to (3) the method of.
(5) The final concentration of the phospholipase is 0.1 to 3 U / mL, the final concentration of the sphingomyelinase is 0.02 to 2 U / mL, and the final concentration of the nonionic surfactant is 0.01 to 6. The method according to any one of (1) to (4), which is 1% by weight.
(6) The elimination of cholesterol in lipoproteins other than high-density lipoproteins from the reaction system is carried out by eliminating cholesterol with an elimination system containing cholesterol esterase and cholesterol oxidase. The method according to any one of the above.
(7) A first step of erasing cholesterol in lipoproteins other than the high-density lipoprotein in the test sample out of the reaction system, and a second step of quantifying cholesterol in the high-density lipoprotein remaining in the reaction system A method for quantifying cholesterol in high-density lipoprotein in a test sample, wherein the first step is performed by the method according to any one of (1) to (6).
(8) A kit for quantifying cholesterol in high-density lipoprotein, comprising (i) phospholipase and / or sphingomyelinase and (ii) a nonionic surfactant.
　本発明により、被検試料中のＨＤＬコレステロールを、より選択的に正確に定量することが可能となった。 According to the present invention, HDL cholesterol in a test sample can be quantified more selectively and accurately.
　上記のとおり、本発明の方法では、被検試料に(i)ホスフォリパーゼ及び／又はスフィンゴミエリナーゼ（以下、「ホスフォリパーゼ等」略すことがある）と、(ii)非イオン界面活性剤を作用させ、被検試料中の高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去する。生体から分離した被検試料から予めホスフォリパーゼ等と非イオン界面活性剤を作用させることにより、被検試料中のＨＤＬ以外のリポ蛋白を選択的に消去することができる。すなわち、被検試料中のＨＤＬ以外のコレステロールを消去する第一工程と、反応系内に残存するＨＤＬコレステロールを定量する第二工程を含む、被検試料中のＨＤＬコレステロールの定量方法における第一工程として、上記本発明の方法を適用することにより、被検試料中のＨＤＬコレステロールを定量することができる。以下、この２工程法について説明する。なお、以下の説明では、２工程法を説明するが、この２工程法の第一工程が本発明の方法である。また、本発明の方法を行うために、以下の第二工程は必須的ではなく、他の公知の方法を適用することによっても被検試料中のＨＤＬコレステロールを定量することが可能である。もっとも、第一工程と第二工程を含む、ＨＤＬコレステロールの定量方法も本発明の方法である。 As described above, in the method of the present invention, (i) phospholipase and / or sphingomyelinase (hereinafter sometimes abbreviated as “phospholipase etc.”) and (ii) a nonionic surfactant are included in the test sample. To eliminate cholesterol in lipoproteins other than the high density lipoprotein in the test sample from the reaction system. By allowing phospholipase or the like and a nonionic surfactant to act in advance from a test sample separated from a living body, lipoproteins other than HDL in the test sample can be selectively erased. That is, a first step in a method for quantifying HDL cholesterol in a test sample, comprising a first step of eliminating cholesterol other than HDL in the test sample and a second step of quantifying HDL cholesterol remaining in the reaction system As described above, HDL cholesterol in the test sample can be quantified by applying the method of the present invention. Hereinafter, this two-step method will be described. In the following description, a two-step method will be described. The first step of the two-step method is the method of the present invention. Moreover, in order to perform the method of this invention, the following 2nd processes are not essential, It is possible to quantify HDL cholesterol in a test sample also by applying another well-known method. However, the method for quantifying HDL cholesterol including the first step and the second step is also the method of the present invention.
　本発明の方法に供される被検試料としては、その試料中のＨＤＬコレステロールを定量しようとするものであれば特に限定されないが、好ましくは、血清若しくは血漿又はこれらの希釈物であり、特に血清又はその希釈物が好ましい。 The test sample to be subjected to the method of the present invention is not particularly limited as long as HDL cholesterol in the sample is to be quantified, but is preferably serum or plasma or a dilution thereof, particularly serum. Or a dilution thereof is preferred.
　本発明で測定しようとするＨＤＬは、アポタンパク質とリン脂質、コレステロール、中性脂肪から構成されている。ＨＤＬは密度がｄ＝１．０６３～１．２１０ｇ／ｍＬであり、さらにｄ＝１．０６３～１．１２５ｇ／ｍＬであるＨＤＬ２とｄ＝１．１２５～１．２１０ｇ／ｍＬであるＨＤＬ３の２つの分画に分類される。リポ蛋白の分布曲線によりｄ＝１．１２５ｇ／ｍＬ部分にノッチが認められ、ここから密度の重い部分をＨＤＬ３とされる。また、ＨＤＬ中のアポタンパク質のうちアポリポ蛋白Ｅ含有量差から、アポＥの含有量の多いＨＤＬをアポＥ－ｒｉｃｈ ＨＤＬとする亜分画の分け方も存在する。しかし、これらの定義はＨＤＬの連続した範囲の中での区切りであり、必ずしも上記の数値で明確に臨床的意義が限定されるものではない。一般的な報告でも密度の区切りが異なるものも存在するため、およそ上記の範囲とする。 HDL to be measured in the present invention is composed of apoprotein, phospholipid, cholesterol, and neutral fat. HDL has a density of d = 1.063-1.210 g / mL, HDL2 with d = 1.063-1.125 g / mL, and HDL3 with d = 1.125-1.210 g / mL. It is classified into two fractions. According to the distribution curve of lipoprotein, a notch is observed in the portion of d = 1.125 g / mL, and a portion having a high density is designated as HDL3. In addition, there is a method of dividing a subfraction in which HDL having a high content of apo E is apo E-rich HDL due to the difference in the content of apolipoprotein E among apoproteins in HDL. However, these definitions are delimiters within the continuous range of HDL, and the clinical significance is not necessarily clearly limited by the above numerical values. Since there are some general reports with different density delimiters, the above range is used.
　本発明の第一工程では、被検試料にホスフォリパーゼ等と非イオン界面活性剤とを作用させる。さらに、コレステロールエステラーゼ、コレステロールオキシダーゼまたはコレステロールデヒドロゲナーゼ等のコレステロール反応酵素や、所望によりリポプロテインリパーゼを添加し反応させる。これらの各酵素は１種でも２種以上組み合わせて添加してもよい。 In the first step of the present invention, phospholipase or the like and a nonionic surfactant are allowed to act on the test sample. Further, a cholesterol-reactive enzyme such as cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase, or lipoprotein lipase as required is reacted. Each of these enzymes may be added alone or in combination of two or more.
　第一工程で用いるホスフォリパーゼ等は少なくともグリセロリン脂質に対して作用すればよく、さらに少なくともホスファチジルコリンに活性を有するものであれば好ましいが、ホスファチジルコリン以外のリゾホスファエタノールアミン等や、または、グリセロリン脂質以外のスフィンゴミエリンやセラミドなどに対して活性を有していても構わない。ホスフォリパーゼ等は市販品を使用することが可能であり、具体的には、旭化成社製のホスフォリパーゼＡ２（ＰＬＡ２Ｌ）、ホスフォリパーゼＣ（ＰＬＣ）、ホスフォリパーゼＤ（ＰＬＤまたはＰＬＤＰまたはＰＬＤＰＶ）、リゾホスフォリパーゼ（ＬＹＰＬ）、スフィンゴミエリナーゼ（シグマアルドリッチ）等を用いることが可能である。 The phospholipase used in the first step only needs to act on at least glycerophospholipid, and it is preferable if it has at least activity on phosphatidylcholine, but lysophosphaethanolamine other than phosphatidylcholine, or glycerophospholipid It may have activity against other sphingomyelin and ceramide. Phospholipase etc. can use a commercial item, specifically, Asahi Kasei's Phospholipase A2 (PLA2L), Phospholipase C (PLC), Phospholipase D (PLD or PLDP or PLDPV), lysophospholipase (LYPL), sphingomyelinase (Sigma Aldrich) and the like can be used.
　第一工程においてホスフォリパーゼ等を用いる場合は、１種または２種以上を組み合わせて作用させてもよく、その際のホスフォリパーゼ濃度（２種以上組み合わせる場合はその合計終濃度）は０．１～２００Ｕ／ｍＬが好ましく、さらに０．０５～１００Ｕ／ｍＬがより好ましく、さらに０．１～３Ｕ／ｍＬがより好ましい。スフィンゴミエリナーゼを用いる場合は、スフィンゴミエリナーゼの終濃度は０．０１～５０Ｕ／ｍＬ程度が好ましく、０．０２～２Ｕ／ｍＬがより好ましい。スフィンゴミエリナーゼは少なくともスフィンゴミエリンに作用するものであれば特に制限されるものではなく、スフィンゴミエリン以外のリン脂質を構成する成分であるホスファチジルイノシトールなどに対する活性を有してもよい。 When phospholipase or the like is used in the first step, one kind or a combination of two or more kinds may be allowed to act, and the phospholipase concentration at that time (when two or more kinds are combined, the total final concentration) is 0. It is preferably 1 to 200 U / mL, more preferably 0.05 to 100 U / mL, and further preferably 0.1 to 3 U / mL. When sphingomyelinase is used, the final concentration of sphingomyelinase is preferably about 0.01 to 50 U / mL, and more preferably 0.02 to 2 U / mL. Sphingomyelinase is not particularly limited as long as it acts on at least sphingomyelin, and may have activity against phosphatidylinositol, which is a component constituting phospholipids other than sphingomyelin.
　本発明で用いる非イオン界面活性剤は、０．００１～５重量％終濃度（複数種類の非イオン界面活性剤を組み合わせて用いる場合にはその合計終濃度）で添加するのが好ましく、０．００２～３．０重量％程度がより好ましく、さらには０．０１～１重量％程度が好ましい。非イオン界面活性剤としては、ポリオキシエチレン－ポリオキシプロピレン共重合体、アミドノニオン、ポリオキシエチレンノニルフェニルエーテル、ＨＬＢ値が１４～１７のポリオキシエチレン多環フェニルエーテルのような非イオン系界面活性剤を挙げることができるがこれらに限定されるものではない。これらのうち、ポリオキシエチレンポリオキシプロピレンブロック共重合体、特にポリオキシプロピレン部分がポリオキシエチレン部分に挟まれた形態のブロック共重合体が好ましい。特に、重量平均分子量１０，０００～１７，０００であり、かつ分子中のエチレンオキシド含量が７５～９５モル％のものが好ましい。より具体的には、プルロニックＰ１２３（アデカ）、プルロニックＦ６８（アデカ）、プルロニックＦ８８（アデカ）、プルロニックＰ８５（アデカ）、プルロニック17R-3（アデカ）、プルロニック17R-4（アデカ）、プルロニックTR-704（アデカ）、プルロニックPE6100（アデカ）、プルロニックPE6400（アデカ）、プルロニックPE6800（アデカ）、アデカカーポールGH5（アデカ）、アデカカーポールGH10（アデカ）、アデカカーポールGH200（アデカ）、アデカカーポールMH150（アデカ）、アデカカーポールMH10000（アデカ）、レベノールＷＸ（花王)、ノニオンＨＳ２２０(日油) 、ナイミッドＭＴ－２１５（日油）、プロノン208（日油）、Ｎｅｗｃｏｌ-７２３（日本乳化剤）、Ｎｅｗｃｏｌ－２６１４（日本乳化剤）、Ｎｅｗｃｏｌ－７１４（日本乳化剤）、エパン485（第一工業製薬）、エパン785（第一工業製薬）、を挙げることができる。また、これら以外の非イオン界面活性剤として、アルキルエーテル型の非イオン界面活性剤であるアデカトールLB-83（アデカ）及びアデカトールLB-103（アデカ）、ポリオキシエチレンアルキルエーテル型の非イオン界面活性剤であるエマルゲン109P（花王）、エマルゲンTW0106（花王）及びエマルゲン1108（花王）、ポリオキシエチレンフェニルエーテルであるノイゲンEA-207D(第一工業製薬）、ポリオキシエチレンアルキルフェニルエーテルであるノニオンHS-240（日油）、ポリオキシエチレンp-クミルフェニルエーテルであるNewcol-CMP-1（日本乳化剤）及びNewcol-CMP-11（日本乳化剤）、ポリカルボン酸ナトリウムであるポリスターOMP（日油）、脂肪酸エステル型非イオン界面活性剤であるレオドールTW-L120（花王）、レオドール460（花王）、レオドールTW-105（花王）、ポリオキシエチレン(10)オクチルフェニルエーテル等も用いることができる。これらの非イオン界面活性剤は、単独でも２種以上を組み合わせても用いることができる。 The nonionic surfactant used in the present invention is preferably added at a final concentration of 0.001 to 5% by weight (a total final concentration when a plurality of types of nonionic surfactants are used in combination). It is more preferably about 002 to 3.0% by weight, and further preferably about 0.01 to 1% by weight. Nonionic surfactants include nonionic interfaces such as polyoxyethylene-polyoxypropylene copolymers, amide nonions, polyoxyethylene nonyl phenyl ethers, and polyoxyethylene polycyclic phenyl ethers having an HLB value of 14-17. Although an activator can be mentioned, it is not limited to these. Among these, a polyoxyethylene polyoxypropylene block copolymer, particularly a block copolymer in which a polyoxypropylene portion is sandwiched between polyoxyethylene portions is preferable. Particularly preferred are those having a weight average molecular weight of 10,000 to 17,000 and an ethylene oxide content of 75 to 95 mol% in the molecule. More specifically, Pluronic P123 (Adeka), Pluronic F68 (Adeka), Pluronic F88 (Adeka), Pluronic P85 (Adeka), Pluronic 17R-3 (Adeka), Pluronic 17R-4 (Adeka), Pluronic TR-704 (Adeka), Pluronic PE6100 (Adeka), Pluronic PE6400 (Adeka), Pluronic PE6800 (Adeka), Adeka Carpole GH5 (Adeka), Adeka Carpole GH10 (Adeka), Adeka Carpole GH200 (Adeka), Adeka Carpole MH150 (Adeka), Adeka Carpol MH10000 (Adeka), Rebenol WX (Kao), Nonion HS220 (Nippon) Kashiwa, Nymid MT-215 (Nippon), Pronon 208 (Nippon), Newcol-723 (Nippon Emulsifier), Newcol -2614 (Japan emulsifier), Newcol-714 (Japan emulsifier) ), Epan 485 (Daiichi Kogyo Seiyaku), and Epan 785 (Daiichi Kogyo Seiyaku). In addition, other nonionic surfactants such as alkyl ether type nonionic surfactants such as Adecatol LB-83 (Adeka) and Adekatol LB-103 (Adeka), polyoxyethylene alkyl ether type nonionic surfactants Emulgen 109P (Kao), Emulgen TW0106 (Kao) and Emulgen 1108 (Kao), Neugen EA-207D (Daiichi Kogyo Seiyaku), polyoxyethylene alkylphenyl ether, Nonion HS- 240 (Nippon Oil), Newcol-CMP-1 (Nippon Emulsifier) and Newcol-CMP-11 (Nippon Emulsifier) which are polyoxyethylene p-cumylphenyl ether, Polystar OMP (Nippon Oil) which is sodium polycarboxylate, Fatty acid ester type nonionic surfactants, Rheodor TW-L120 (Kao), Rheodor 460 (Kao), Rheodor TW- 105 (Kao), polyoxyethylene (10) octylphenyl ether, and the like can also be used. These nonionic surfactants can be used alone or in combination of two or more.
　本発明の方法の第一工程では、ＨＤＬ以外のリポ蛋白質コレステロールをホスフォリパーゼ、コレステロールエステラーゼ等の作用により消去する。ここで、「消去」とは、被検試料中のリポ蛋白のコレステロールを分解し、その後の工程において、コレステロール測定の反応に作用させないようにすることである。リポ蛋白コレステロールを消去するための方法としては、コレステロールエステラーゼ及びコレステロールオキシダーゼを作用させ発生した過酸化水素を、カタラーゼを用いて水と酸素に分解する方法が挙げられる。また、ペルオキシダーゼを用いて水素供与体と発生した過酸化水素を反応させ無色キノンに転化してもよいが、これらに限定されるものではない。コレステロールの消去の方法自体はこの分野において周知である。 In the first step of the method of the present invention, lipoprotein cholesterol other than HDL is eliminated by the action of phospholipase, cholesterol esterase and the like. Here, “erasing” means degrading lipoprotein cholesterol in a test sample so that it does not affect the reaction of cholesterol measurement in the subsequent steps. Examples of a method for eliminating lipoprotein cholesterol include a method in which hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase is decomposed into water and oxygen using catalase. In addition, the hydrogen donor and the generated hydrogen peroxide may be reacted with peroxidase to convert to colorless quinone, but is not limited thereto. The method of cholesterol elimination itself is well known in the art.
　第一工程は、ホスフォリパーゼ等と非イオン界面活性剤に加え、反応系外に移行させるための酵素や界面活性剤を併せて添加しておくことにより、ホスフォリパーゼ等と非イオン界面活性剤によるＨＤＬ以外のリポ蛋白質の分解と、これらの中に含まれるコレステロールの消去とを単一の工程として同時に行うこともできる。 The first step is to add phospholipase etc. and nonionic surfactant together with phospholipase etc. and nonionic surfactant by adding enzyme and surfactant to move out of the reaction system. Decomposition of lipoproteins other than HDL by the agent and elimination of cholesterol contained therein can be simultaneously performed as a single step.
　第一工程において、コレステロールエステラーゼ、コレステロールオキシダーゼを用いる場合、コレステロールエステラーゼの濃度（本明細書において、濃度は特に断りがない限り終濃度を意味する）は０．１～１０．０Ｕ／ｍＬ程度が好ましく、０．２～２．０Ｕ／ｍＬ程度がより好ましい。コレステロールオキシダーゼの濃度は０．０５～１０．０Ｕ／ｍＬ程度が好ましく、０．１～１．０Ｕ／ｍＬ程度がより好ましい。なお、コレステロールエステラーゼは、エステル型コレステロールに作用するものであれば、特に制限されるものではなく、例えば、旭化成社製のコレステロールエステラーゼ（ＣＥＢＰ、ＣＥＮ）や東洋紡社製のコレステロールエステラーゼ（ＣＯＥ－３１１、ＣＯＥ－３１３）やキッコーマン社製のコレステロールエステラーゼ（ＣＨＥ－ＸＥ）などの市販品を用いることができる。また、コレステロールオキシダーゼは、遊離型コレステロールに作用するものであれば、特に制限されるものではなく、例えば、旭化成社製のコレステロールオキシダーゼ（ＣＯＮII）や東洋紡社製のコレステロールオキシダーゼ（ＣＯＯ－３１１、ＣＯＯ－３２１、ＣＯＯ－３３１）やキッコーマン社製のコレステロールオキシダーゼ（ＣＨＯ－ＣＥ、ＣＨＯ－ＰＥＷＬ、ＣＨＯ－ＢＳ）などの市販品を用いることができる。 When cholesterol esterase or cholesterol oxidase is used in the first step, the cholesterol esterase concentration (in this specification, the concentration means the final concentration unless otherwise specified) is preferably about 0.1 to 10.0 U / mL. 0.2 to 2.0 U / mL is more preferable. The concentration of cholesterol oxidase is preferably about 0.05 to 10.0 U / mL, more preferably about 0.1 to 1.0 U / mL. The cholesterol esterase is not particularly limited as long as it acts on ester-type cholesterol. For example, cholesterol esterase (CEBP, CEN) manufactured by Asahi Kasei Co., Ltd. or cholesterol esterase (COE-311, manufactured by Toyobo Co., Ltd.) Commercially available products such as COE-313) and cholesterol esterase (CHE-XE) manufactured by Kikkoman can be used. The cholesterol oxidase is not particularly limited as long as it acts on free cholesterol. For example, cholesterol oxidase (CONII) manufactured by Asahi Kasei Co., Ltd. or cholesterol oxidase (COO-311, COO- 321 and COO-331) and cholesterol oxidase (CHO-CE, CHO-PEWL, CHO-BS) manufactured by Kikkoman Corporation can be used.
　コレステロールデヒドロゲナーゼを用いる場合は、０．０１～２００Ｕ/ｍＬが好ましく、さらに好ましくは０．１～１００Ｕ／ｍＬが好ましい。なお、コレステロールデヒドロゲナーゼとしては、コレステロールを酸化して酸化型補酵素を還元する能力を有する酵素であれば特に制限されるものではなく、例えば、天野エンザイム社製のコレステロールデヒドロゲナーゼ（ＣＨＤＨ－５）などの市販品を用いることが可能である。 When cholesterol dehydrogenase is used, it is preferably 0.01 to 200 U / mL, more preferably 0.1 to 100 U / mL. Cholesterol dehydrogenase is not particularly limited as long as it has the ability to oxidize cholesterol and reduce oxidized coenzyme. For example, cholesterol dehydrogenase (CHDH-5) manufactured by Amano Enzyme Co., Ltd. Commercial products can be used.
　本発明はさらにリポプロテインリパーゼを添加することが可能であり、１種または２種以上を組み合わせて作用させてもよく、終濃度（２種以上組み合わせる場合はその終濃度）は０．０１～０．５Ｕ／ｍＬ程度が好ましく、０．０２～０．５Ｕ／ｍＬがさらに好ましい。リポプロテインリパーゼ、ホスフォリパーゼは市販品を用いることが可能であり、例えば、東洋紡社製のリポプロテインリパーゼ（ＬＰＬ－３１１やＬＰＬ－３１４）、天野エンザイム社製のリポプロテインリパーゼ（ＬＰＬ－３など）、旭化成社製のリポプロテインリパーゼ（ＬＰＢＰやＬＰなど）を用いることができる。 In the present invention, lipoprotein lipase can be further added, and one or two or more kinds may be used in combination, and the final concentration (the final concentration when two or more types are combined) is 0.01 to 0. About 5 U / mL is preferable, and 0.02 to 0.5 U / mL is more preferable. Commercially available products can be used for lipoprotein lipase and phospholipase, such as lipoprotein lipase (LPL-311 and LPL-314) manufactured by Toyobo Co., Ltd., lipoprotein lipase (LPL-3 manufactured by Amano Enzyme, etc.), etc. ), Lipoprotein lipase (LPBP, LP, etc.) manufactured by Asahi Kasei Corporation can be used.
　第一工程で用いる反応液には通常の生化学反応に用いられる各種の緩衝液を使用することができ、ｐＨが５～８の間であるのが好ましい。溶液としては、グッド、トリス、リン酸、グリシンの緩衝溶液が好ましく、グッド緩衝液であるビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシエチル）メタン（Ｂｉｓ－Ｔｒｉｓ）、ピペラジン－１、４－ビス（２－エタンスルフォン酸）（ＰＩＰＥＳ）、ピペラジン－１、４－ビス（２－エタンスルフォン酸）、１．５ナトリウム塩、一水和物（ＰＩＰＥＳ１．５Ｎａ）、２－ヒドロキシ－3－モルホリノプロパンスルホン酸（ＭＯＰＳＯ）、Ｎ、Ｎ－ビス（２－ヒドロキシエチル）―２―アミノエタンスルフォン酸（ＢＥＳ）、２－［４－（２－ヒドロキシエチル）－１－ピペラジニル］エタンスルフォン酸（ＨＥＰＥＳ）およびピペラジン－１、４－ビス（２－ヒドロキシ－３－プロパンスルフォン酸）（ＰＯＰＳＯ）が好ましい。 As the reaction solution used in the first step, various buffer solutions used in normal biochemical reactions can be used, and the pH is preferably between 5 and 8. As the solution, a buffer solution of Good, Tris, phosphate, and glycine is preferable. Good buffer solutions such as bis (2-hydroxyethyl) iminotris (hydroxyethyl) methane (Bis-Tris), piperazine-1, 4-bis ( 2-ethanesulfonic acid (PIPES), piperazine-1,4-bis (2-ethanesulfonic acid), 1.5 sodium salt, monohydrate (PIPES1.5Na), 2-hydroxy-3-morpholinopropanesulfone Acid (MOPSO), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO) is preferred That's right.
　第一工程においては、ＨＤＬ以外のリポ蛋白質を差別しやすくする目的で、一価またはニ価の陽イオンまたはそれらの塩を添加することができる。具体的には、塩化ナトリウム、塩化カリウム、塩化マンガン、塩化カルシウム、塩化アンモニウム、硫酸マグネシウム、硫酸カリウム、硫酸リチウム、硫酸アンモニウム、酢酸マグネシウム等を用いることが可能である。濃度は１～５０．０ｇ／Ｌが好ましく、さらに５～３０ｇ／Ｌが好ましい。 In the first step, a monovalent or divalent cation or a salt thereof can be added for the purpose of easily distinguishing lipoproteins other than HDL. Specifically, sodium chloride, potassium chloride, manganese chloride, calcium chloride, ammonium chloride, magnesium sulfate, potassium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and the like can be used. The concentration is preferably 1 to 50.0 g / L, more preferably 5 to 30 g / L.
　第一工程の反応温度は２５～４０℃程度が好ましく、さらに３５～３８℃が好ましく、３７℃が最も好ましい。反応時間は特に限定されず、通常、２～１０分程度である。 The reaction temperature in the first step is preferably about 25 to 40 ° C, more preferably 35 to 38 ° C, and most preferably 37 ° C. The reaction time is not particularly limited, and is usually about 2 to 10 minutes.
　続く第二工程では、反応系内に残存するＨＤＬのうちＨＤＬコレステロールを定量する。　コレステロールの定量方法自体は周知であり、周知のいずれの方法をも採用することができ、下記実施例にも具体的に記載されている。例えば、リポ蛋白中のエステル型コレステロールにコレステロールエステラーゼを用いて加水分解し、遊離型コレステロールと脂肪酸が生じ、生じた遊離型コレステロールと元来リポ蛋白中に存在する遊離コレステロールとをコレステロールオキシダーゼを用いてコレステノンと過酸化水素を発生させ、これをペルオキシダーゼの存在下でキノン色素を形成させ定量する。キノン色素を発生させる化合物として、例えばＨＤＡＯＳ（Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメトキシアニリン）、ＤＡＯＳ（Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメトキシアニリン）又はＴＯＯＳ（Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３－メチルアニリン）と４－アミノアンチピリンが挙げられるが、キノン色素を発生させることができる組み合わせであればこれらに限定されるものではない。第一工程でコレステロールエステラーゼ及びコレステロールオキシダーゼを用いる場合には、第二工程では、第一工程で用いたコレステロールエステラーゼ及びコレステロールオキシダーゼをそのまま用いることができ、新たに添加する必要はない。 In the subsequent second step, HDL cholesterol in the HDL remaining in the reaction system is quantified. The cholesterol quantification method itself is well known, and any known method can be adopted, and is specifically described in the following examples. For example, ester cholesterol in lipoproteins is hydrolyzed using cholesterol esterase to produce free cholesterol and fatty acids, and the resulting free cholesterol and free cholesterol originally present in lipoproteins using cholesterol oxidase Cholesteinone and hydrogen peroxide are generated and quantified by forming a quinone dye in the presence of peroxidase. Examples of compounds that generate quinone dyes include HDAOS (N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline), DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl)) -3,5-dimethoxyaniline) or TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline) and 4-aminoantipyrine, which can generate quinone dyes. It is not limited to these as long as it is a possible combination. When cholesterol esterase and cholesterol oxidase are used in the first step, the cholesterol esterase and cholesterol oxidase used in the first step can be used as they are in the second step, and it is not necessary to add them newly.
　コレステロールに反応するコレステロール測定用酵素としてコレステロールエステラーゼおよびコレステロールオキシダーゼを用いる場合、酵素反応により過酸化水素が発生する。発生した過酸化水素からペルオキシダーゼの存在下で水素供与体と水素受容体とのカップリング反応により形成した色素は波長４００～７００ｎｍでの吸光度を測定することによりＨＤＬ中のコレステロールを定量することができる。 When cholesterol esterase and cholesterol oxidase are used as cholesterol measuring enzymes that react with cholesterol, hydrogen peroxide is generated by the enzymatic reaction. Dye formed by the coupling reaction between a hydrogen donor and a hydrogen acceptor from the generated hydrogen peroxide in the presence of peroxidase can measure cholesterol in HDL by measuring absorbance at a wavelength of 400 to 700 nm. .
　コレステロールに反応するコレステロール測定用酵素としてコレステロールエステラーゼおよびコレステロールデヒドロゲナーゼを用いる場合、酵素反応によりＮＡＤ（Ｐ）からＮＡＤ（Ｐ）Ｈが発生する。発生したＮＡＤ（Ｐ）Ｈは波長３３０～４００ｎｍでの吸光度を測定することによりＨＤＬ中のコレステロールを定量することができる。 When cholesterol esterase and cholesterol dehydrogenase are used as cholesterol measuring enzymes that react with cholesterol, NAD (P) H is generated from NAD (P) by the enzymatic reaction. The generated NAD (P) H can quantitate cholesterol in HDL by measuring the absorbance at a wavelength of 330 to 400 nm.
　キノン色素を発生させる化合物の濃度は、例えば、ＨＤＡＯＳであれば濃度は０．５～２．０ｍｍｏｌ／Ｌ程度が好ましく、４－アミノアンチピリンであれば０．１～２．０ｍｍｏｌ／Ｌが好ましく、また、ペルオキシダーゼの濃度は０．４～５．０Ｕ／ｍＬが好ましい。また、第一工程で発生した過酸化水素をカタラーゼで分解する工程ではカタラーゼの阻害剤であるアジ化ナトリウムを使用し、第二工程の反応液中へ添加する。この場合のアジ化ナトリウムの濃度は、通常、０．１ｇ／Ｌ～１．０ｇ／Ｌ程度である。 The concentration of the compound that generates a quinone dye is preferably about 0.5 to 2.0 mmol / L for HDAOS, and preferably 0.1 to 2.0 mmol / L for 4-aminoantipyrine, The concentration of peroxidase is preferably 0.4 to 5.0 U / mL. Further, in the step of decomposing hydrogen peroxide generated in the first step with catalase, sodium azide that is an inhibitor of catalase is used and added to the reaction solution in the second step. In this case, the concentration of sodium azide is usually about 0.1 g / L to 1.0 g / L.
　第一工程において、ペルオキシダーゼを用いる場合、ペルオキシダーゼの濃度は２．０～５．０Ｕ/ｍＬ程度が好ましく、さらに３．０～４．０Ｕ/ｍＬ程度が好ましい。また、無色キノンに転化する化合物を用いる場合、その濃度は０．４～０．８ｍｍoｌ／Ｌ程度が好ましい。 In the first step, when peroxidase is used, the concentration of peroxidase is preferably about 2.0 to 5.0 U / mL, more preferably about 3.0 to 4.0 U / mL. When a compound that converts to colorless quinone is used, the concentration is preferably about 0.4 to 0.8 mmol / L.
　第二工程においては界面活性剤の存在は必須ではなく、添加していてもしていなくてもよい。添加する場合は、第一工程で用いる事のできる界面活性剤を第一工程の同濃度もしくはそれ以下の範囲内で添加する。 In the second step, the presence of the surfactant is not essential and may or may not be added. In the case of adding, the surfactant that can be used in the first step is added within the same concentration or lower range in the first step.
　第二工程の他の反応条件（反応温度、時間、緩衝液、ｐＨ等）は上記した第一工程の反応条件と同様でよい。 Other reaction conditions (reaction temperature, time, buffer solution, pH, etc.) in the second step may be the same as those in the first step.
　以下、本発明を実施例に基づき具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.
実施例１
　超遠心法を用いてＣＭ、ＶＬＤＬ、ＩＤＬ（ＣＶＩ）分画とＬＤＬ分画を回収し、下記調製試薬ＡまたはＢを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でホスフォリパーゼＤ（ＰＬＤＰ）を用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。すなわち、この方法は具体的には次のようにして行った。超遠心法は臭化ナトリウム溶液などを用いることにより、血清などの被検試料のリポ蛋白の密度の差を利用しＣＭ、ＶＬＤＬ、ＩＤＬ、ＬＤＬ、ＨＤＬなどの各分画を調製する方法である。本発明の実施例ではＣＶＩ分画は密度ｄ＜１．０１９ｇ/ｍＬ、ＬＤＬ分画は密度ｄ＝１．０１９～１．０６３ｇ／ｍＬ、ＨＤＬ分画は密度＝１．０６３～１．２１ｇ／ｍＬの密度範囲中のリポ蛋白を含む分画である。測定は超遠心分画２μＬに対し第一工程用調製試薬を１５０μＬ混合し３７℃５分反応の後、第二工程用調製試薬を５０μＬの割合で混合し３７℃５分反応させた。波長は主波長６００ｎｍ、副波長７００ｎｍを用いた。 Example 1
Collect the CM, VLDL, IDL (CVI) fraction and LDL fraction using the ultracentrifugation method, and react by combining the following preparation reagent A or B as the first step and preparation reagent X as the second step. By using phospholipase D (PLDP) in the process, it was confirmed whether the amount of CVI and LDL erased in the first process increased and the reaction of these fractions in the second process decreased. That is, this method was specifically performed as follows. The ultracentrifugation method is a method of preparing each fraction of CM, VLDL, IDL, LDL, HDL, etc. by using a difference in lipoprotein density of a test sample such as serum by using a sodium bromide solution or the like. . In the examples of the present invention, the CVI fraction has a density d <1.019 g / mL, the LDL fraction has a density d = 1.018 to 1.063 g / mL, and the HDL fraction has a density = 1.063-1.21 g / mL. Fraction containing lipoproteins in the density range of mL. For the measurement, 150 μL of the first step preparation reagent was mixed with 2 μL of the ultracentrifugation fraction and reacted at 37 ° C. for 5 minutes, and then the second step preparation reagent was mixed at a ratio of 50 μL and reacted at 37 ° C. for 5 minutes. As the wavelength, a main wavelength of 600 nm and a sub wavelength of 700 nm were used.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表１に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ａ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体（商品名プルロニックＦ６８、以下同じ）
０．２５ｇ／Ｌ
ホスフォリパーゼＤ（ＰＬＤＰ） ０．５Ｕ／ｍＬ Preparation reagent A
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer (trade name Pluronic F68, the same shall apply hereinafter)
0.25g / L
Phospholipase D (PLDP) 0.5 U / mL
調製試薬Ｂ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ホスフォリパーゼＤ（ＰＬＤＰ） ２．０Ｕ／ｍＬ Preparation reagent B
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Phospholipase D (PLDP) 2.0 U / mL
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ホスフォリパーゼＤと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with Preparation Reagent C. By using Phospholipase D and a nonionic surfactant, CVI fraction was obtained in the first step. As a result, the erased amount of the image and LDL fraction was increased.
実施例２
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬ＤまたはＥを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でホスフォリパーゼＣを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 2
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagent D or E as the first step and preparation reagent X as the second step. By using phospholipase C in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表１に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 1. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｄ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ホスフォリパーゼＣ（ＰＬＣ） ０.５Ｕ／ｍＬ Preparation reagent D
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Phospholipase C (PLC) 0.5 U / mL
調製試薬Ｅ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ホスフォリパーゼＣ（ＰＬＣ） ２．０Ｕ／ｍＬ Preparation reagent E
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Phospholipase C (PLC) 2.0 U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ホスフォリパーゼＣと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction was lower than the reaction rate measured with the prepared reagent C. By using phospholipase C and a nonionic surfactant, the CVI fraction was measured in the first step. As a result, the erased amount of the image and LDL fraction was increased.
実施例３
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬Ｆ～Ｊを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でスフィンゴミエリナーゼを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 3
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents F to J as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表３に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 3. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｆ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.０５Ｕ／ｍＬ Preparation reagent F
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.05U / mL
調製試薬Ｇ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.１０Ｕ／ｍＬ Preparation reagent G
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.10 U / mL
調製試薬Ｈ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.２５Ｕ／ｍＬ Preparation reagent H
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.25U / mL
調製試薬Ｉ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.５０Ｕ／ｍＬ Preparation reagent I
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.50U / mL
調製試薬Ｊ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／mＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ １．００Ｕ／ｍＬ Preparation reagent J
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 1.00U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、スフィンゴミエリナーゼと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C. By using sphingomyelinase and a nonionic surfactant, the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
実施例４
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬Ｋ～Ｏを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でスフィンゴミエリナーゼを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 4
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents K to O as the first step and preparation reagent X as the second step. It was confirmed that the use of sphingomyelinase in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表４示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. Table 4 shows the results. In this example, the prepared reagent C was used as the HDL reagent.
　調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｋ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.１Ｕ／ｍＬ Preparation reagent K
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.1U / mL
調製試薬Ｌ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ０.５Ｕ／ｍＬ Preparation reagent L
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 0.5U / mL
調製試薬Ｍ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ １．０Ｕ／ｍＬ Preparation reagent M
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 1.0 U / mL
調製試薬Ｎ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ２.５Ｕ／ｍＬ Preparation reagent N
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 2.5U / mL
調製試薬Ｏ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
スフィンゴミエリナーゼ ５．０Ｕ／ｍＬ Preparation reagent O
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
Sphingomyelinase 5.0U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、スフィンゴミエリナーゼと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C. By using sphingomyelinase and a nonionic surfactant, the CVI fraction is used in the first step. As a result, the amount of erasure of the LDL fraction was increased.
実施例５
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬Ｐ～Ｔを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でＰＬＣを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 5
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents P to T as the first step and preparation reagent X as the second step. By using PLC in one step, it was confirmed whether the amount of CVI and LDL erased in the first step increased and the reaction of these fractions in the second step decreased.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表５に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 5. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｐ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＣ ０.１Ｕ／ｍＬ Preparation reagent P
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLC 0.1U / mL
調製試薬Ｑ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＣ ０.５Ｕ／ｍＬ Preparation reagent Q
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLC 0.5U / mL
調製試薬Ｒ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＣ １．０Ｕ／ｍＬ Preparation reagent R
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLC 1.0U / mL
調製試薬Ｓ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＣ ２.５Ｕ／ｍＬ Preparation reagent S
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLC 2.5U / mL
調製試薬Ｔ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／mＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＣ ５．０Ｕ／ｍＬ Preparation reagent T
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLC 5.0U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ＰＬＣと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C. By using PLC and a nonionic surfactant, the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
実施例６
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬Ｕ～Ｙを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でＰＬＤＰを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 6
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using an ultracentrifugation method, and the following preparation reagents U to Y were combined as a first step and prepared reagent X was combined as a second step. It was confirmed that the use of PLDP in one step increased the amount of CVI and LDL erased in the first step, and reduced the reaction of these fractions in the second step.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表６に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 6. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｕ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰ ０.１Ｕ／ｍＬ Preparation reagent U
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDP 0.1U / mL
調製試薬Ｖ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰ ０.５Ｕ／ｍＬ Preparation reagent V
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDP 0.5U / mL
調製試薬Ｗ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰ １．０Ｕ／ｍＬ Preparation reagent W
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDP 1.0U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰ ２.５Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDP 2.5U / mL
調製試薬Ｙ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／mＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰ ５．０Ｕ／ｍＬ Preparation reagent Y
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDP 5.0U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ＰＬＤＰと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each prepared reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with the prepared reagent C. By using PLDP and a nonionic surfactant, the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
実施例７
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、下記調製試薬Ｚ～ＡＤを第一工程として調製試薬Ｘを第二工程として組み合わせて反応させ、第一工程でＰＬＤＰＶを用いることにより第一工程におけるＣＶＩ、ＬＤＬの消去量が増大し、第二工程でのこれら分画の反応が低減するか確認を行った。 Example 7
In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using ultracentrifugation, and reacted by combining the following preparation reagents Z to AD as the first step and preparation reagent X as the second step. It was confirmed that the use of PLDPV in one step increased the amount of CVI and LDL erased in the first step and reduced the reaction of these fractions in the second step.
　測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、ＨＤＬ試薬で測定した場合との比較を行った。結果を表７に示す。ＨＤＬ試薬は本実施例では調製試薬Ｃを使用した。 By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction is calculated, and compared with the case measured with the HDL reagent. It was. The results are shown in Table 7. In this example, the prepared reagent C was used as the HDL reagent.
調製試薬Ｃ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ Preparation reagent C
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
調製試薬Ｚ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰＶ ０.１Ｕ／ｍＬ Preparation reagent Z
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDPV 0.1U / mL
調製試薬ＡＡ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰＶ ０.５Ｕ／ｍＬ Preparation reagent AA
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDPV 0.5U / mL
調製試薬ＡＢ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰＶ １．０Ｕ／ｍＬ Preparation reagent AB
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDPV 1.0U / mL
調製試薬ＡＣ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰＶ ２.５Ｕ／ｍＬ Preparation reagent AC
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDPV 2.5U / mL
調製試薬ＡＤ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／mＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
ポリオキシエチレン・ポリオキシプロピレンブロック共重合体 ０．２５ｇ／Ｌ
ＰＬＤＰＶ ５．０Ｕ／ｍＬ Preparation reagent AD
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Polyoxyethylene / polyoxypropylene block copolymer 0.25 g / L
PLDPV 5.0U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ＰＬＤＰＶと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each preparation reagent with respect to the CVI fraction and the LDL fraction is lower than the reaction rate measured with the preparation reagent C. By using PLDPV and a nonionic surfactant, the CVI fraction and LDL are used in the first step. As a result, the amount of elimination of the fraction was increased.
実施例８
　以下の試薬組成の試薬(i)－１、試薬(i)－２と試薬Ｘを調製し、試薬(i)－１及び試薬(i)－２に各種界面活性剤を０．０２５％（Ｗ／Ｖ）濃度添加した試薬をそれぞれ調製した。なお、下記表８に記載される各種界面活性剤は、いずれも上記した市販の非イオン界面活性剤である。 Example 8
Reagent (i) -1, reagent (i) -2 and reagent X having the following reagent composition were prepared, and various surfactants were added to reagent (i) -1 and reagent (i) -2 at 0.025% (W / V) Reagents with added concentrations were prepared. In addition, all the various surfactants described in Table 8 below are the above-described commercially available nonionic surfactants.
　実施例１と同様にして、超遠心法を用いてＣＶＩ分画とＬＤＬ分画を回収し、各種界面活性剤入りの試薬(i)－１または試薬(i)－２を第一工程として、試薬Ｘを第二工程として組み合わせて反応させた。測定した各分画の測定値を分画の総コレステロール値で除し、１００を乗ずる事により、分画１００ｍｇ／ｄＬあたりに生じる反応率を算出し、各界面活性剤においてＳＰＣを添加した場合とＳＰＣ未添加の場合の比較を行った。結果を表８に示す。 In the same manner as in Example 1, the CVI fraction and the LDL fraction were collected using an ultracentrifugation method, and reagent (i) -1 or reagent (i) -2 containing various surfactants was used as the first step. Reagent X was combined and reacted as the second step. By dividing the measured value of each measured fraction by the total cholesterol value of the fraction and multiplying by 100, the reaction rate generated per 100 mg / dL of the fraction was calculated, and when SPC was added to each surfactant Comparison was made when no SPC was added. The results are shown in Table 8.
調製試薬(i)－１
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
界面活性剤 ０．２５ｇ／Ｌ Preparation reagent (i) -1
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Surfactant 0.25g / L
調製試薬(i)－２
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ６．６
ＴＯＯＳ １．５ｍｍｏｌ／Ｌ
カタラーゼ ６００Ｕ／ｍＬ
コレステロールエステラーゼ １．４Ｕ／ｍＬ
コレステロールオキシダーゼ ０．８Ｕ／ｍＬ
界面活性剤 ０．２５ｇ／Ｌ
ＳＰＣ ０.２５Ｕ／ｍＬ Preparation reagent (i) -2
BES buffer 100 mmol / L pH 6.6
TOOS 1.5mmol / L
Catalase 600U / mL
Cholesterol esterase 1.4 U / mL
Cholesterol oxidase 0.8U / mL
Surfactant 0.25g / L
SPC 0.25U / mL
調製試薬Ｘ
ＢＥＳ緩衝液 １００ｍｍｏｌ／Ｌ ｐＨ７．０
アジ化ナトリウム ０．１％
４－アミノアンチピリン ４．０ｍｍｏｌ／Ｌ
ペルオキシダーゼ ２．４Ｕ／ｍＬ Preparation reagent X
BES buffer 100 mmol / L pH 7.0
Sodium azide 0.1%
4-aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 U / mL
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　各調製試薬のＣＶＩ分画、ＬＤＬ分画に対する反応率は調製試薬Ｃで測定した反応率よりも低い値を示し、ＳＰＣと非イオン界面活性剤を用いることにより第一工程でＣＶＩ分画、ＬＤＬ分画の消去量が増大している結果が得られた。 The reaction rate of each preparation reagent with respect to the CVI fraction and LDL fraction is lower than the reaction rate measured with Preparation Reagent C. By using SPC and a nonionic surfactant, CVI fractionation and LDL are performed in the first step. As a result, the amount of elimination of the fraction was increased.

Claims (8)

  1. 　被検試料に(1)ホスフォリパーゼ及び／又はスフィンゴミエリナーゼと、(2)非イオン界面活性剤を作用させることを含む、被検試料中の高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去する方法。 (1) Phospholipase and / or sphingomyelinase on the test sample, and (2) cholesterol in lipoproteins other than the high-density lipoprotein in the test sample, including the action of a nonionic surfactant. Erasing out of the reaction system.
  2. 　前記ホスフォリパーゼがホスフォリパーゼＣ及び/又はホスフォリパーゼＤである請求項１記載の方法。 The method according to claim 1, wherein the phospholipase is phospholipase C and / or phospholipase D.
  3. 　前記非イオン界面活性剤が、ポリオキシエチレンポリオキシプロピレンブロック共重合体である請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the nonionic surfactant is a polyoxyethylene polyoxypropylene block copolymer.
  4. 　前記ホスフォリパーゼの終濃度が０．１～２００Ｕ／ｍＬ、前記スフィンゴミエリナーゼの終濃度が０．０１～５０Ｕ／ｍＬである請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the final concentration of the phospholipase is 0.1 to 200 U / mL, and the final concentration of the sphingomyelinase is 0.01 to 50 U / mL.
  5. 　前記ホスフォリパーゼの終濃度が０．１～３Ｕ／ｍＬ、前記スフィンゴミエリナーゼの終濃度が０．０２～２Ｕ／ｍＬであり、非イオン界面活性剤の終濃度が０．０１～１重量％である請求項１～４のいずれか１項に記載の方法。 The final concentration of the phospholipase is 0.1 to 3 U / mL, the final concentration of the sphingomyelinase is 0.02 to 2 U / mL, and the final concentration of the nonionic surfactant is 0.01 to 1% by weight. The method according to any one of claims 1 to 4, wherein
  6. 　高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去することは、コレステロールエステラーゼ及びコレステロールオキシダーゼを含む消去系によりコレステロールを消去することにより行われる請求項１～５のいずれか１項に記載の方法。 6. The method according to any one of claims 1 to 5, wherein the elimination of cholesterol in lipoproteins other than the high-density lipoprotein is performed by eliminating cholesterol with an elimination system containing cholesterol esterase and cholesterol oxidase. The method described.
  7. 　被検試料中の、高密度リポ蛋白質以外のリポ蛋白質中のコレステロールを反応系外へ消去する第一工程と、反応系内に残存する高密度リポ蛋白質中のコレステロールを定量する第二工程を含み、前記第一工程を、請求項１～６のいずれか１項に記載の方法により行う、被検試料中の高密度リポ蛋白質中のコレステロールの定量方法。 Includes a first step of eliminating cholesterol in a lipoprotein other than the high-density lipoprotein in the test sample from the reaction system and a second step of quantifying cholesterol in the high-density lipoprotein remaining in the reaction system. A method for quantifying cholesterol in high-density lipoprotein in a test sample, wherein the first step is performed by the method according to any one of claims 1 to 6.
  8. 　(1)ホスフォリパーゼ及び／又はスフィンゴミエリナーゼと、(2)非イオン界面活性剤とを含む、高密度リポ蛋白質中のコレステロール定量用キット。 (1) A kit for quantifying cholesterol in high-density lipoprotein, comprising phospholipase and / or sphingomyelinase and (2) a nonionic surfactant.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH09299A (en) * 1995-06-21 1997-01-07 Internatl Reagents Corp Determination of cholesterol in lipoprotein fraction having high specific gravity and determination reagent kit
WO1998026090A1 (en) * 1996-12-09 1998-06-18 Denka Seiken Co., Ltd. Method of determining cholesterol content of high-density lipoproteins
WO2012011554A1 (en) * 2010-07-23 2012-01-26 デンカ生研株式会社 Method for quantifying the amount of cholesterol in high-density lipoprotein 3

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Publication number Priority date Publication date Assignee Title
JPH09299A (en) * 1995-06-21 1997-01-07 Internatl Reagents Corp Determination of cholesterol in lipoprotein fraction having high specific gravity and determination reagent kit
WO1998026090A1 (en) * 1996-12-09 1998-06-18 Denka Seiken Co., Ltd. Method of determining cholesterol content of high-density lipoproteins
WO2012011554A1 (en) * 2010-07-23 2012-01-26 デンカ生研株式会社 Method for quantifying the amount of cholesterol in high-density lipoprotein 3

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