WO2006118199A1 - 高密度リポ蛋白中のコレステロールの測定方法 - Google Patents
高密度リポ蛋白中のコレステロールの測定方法 Download PDFInfo
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- WO2006118199A1 WO2006118199A1 PCT/JP2006/308850 JP2006308850W WO2006118199A1 WO 2006118199 A1 WO2006118199 A1 WO 2006118199A1 JP 2006308850 W JP2006308850 W JP 2006308850W WO 2006118199 A1 WO2006118199 A1 WO 2006118199A1
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- 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
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- 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
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- 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 measuring cholesterol in a high-density lipoprotein in a sample, a measuring reagent, and a measuring kit.
- HDL high density lipoprotein
- LDL low density lipoprotein
- VLDL very low density lipoprotein
- CM Chylomicron
- HDL is related to the removal of cholesterol accumulated in cells because each tissue force including arterial wall also receives cholesterol, and is a risk prevention factor for various arteriosclerosis such as coronary arteriosclerosis.
- the blood level is known to be a useful guide for predicting the onset of arteriosclerotic diseases.
- HDL cholesterol Conventional methods for measuring cholesterol in HDL (hereinafter abbreviated as HDL cholesterol) include fractionation operations such as ultracentrifugation, immunochemical methods, electrophoresis, and precipitation, and cholesterol determination procedures. There is also a stage power. However, the fractionation operation is complicated and requires a long time, and there is a problem in terms of safety. Therefore, these measurement methods involving separation operations are extremely inefficient and unsuitable for practical use.
- a method for measuring HDL cholesterol by reacting with the enzyme in a buffer containing a rusterase, cholesterol oxidase, a bile acid group surfactant, and a nonionic surfactant (see Patent Document 2) It has been known. In the measurement method described in Patent Document 2, first, the reaction between LDL cholesterol and the enzyme proceeds, and then the reaction between HDL cholesterol and the enzyme proceeds, whereby HDL cholesterol can be measured.
- these measuring methods require a long time for the measurement, and are not always effective in measuring methods specific to HD L cholesterol.
- Methods for measuring HDL cholesterol by aggregating lipoproteins other than HDL include, for example, reagents that aggregate non-HDL lipoproteins such as dextran sulfate, divalent metal salts, and chemically modified Measurement method using enzyme (see Patent Document 3), reagent that forms a complex with lipoproteins other than HDL such as polyanion, and lipoprotein such as polyoxyethylene polyoxypropylene condensate (See Patent Document 4), polyions such as dextran sulfate, divalent metal salts, specific nonionic surfactants, and albumin different from albumin derived from the sample.
- non-HDL lipoproteins such as dextran sulfate, divalent metal salts, and chemically modified Measurement method using enzyme
- reagent that forms a complex with lipoproteins other than HDL such as polyanion
- lipoprotein such as polyoxyethylene polyoxypropylene condensate
- polyions such as dextran sulfate, divalent metal
- Examples of methods for measuring HDL cholesterol without aggregating lipoproteins other than HDL include biological samples, spleen-derived cholesterol esterase and cholesterol
- a method for measuring HDL cholesterol in a biological sample by reacting oxidase with bile acid or a salt thereof and albumin and measuring a compound consumed or produced by the enzyme reaction see Patent Document 7
- HDL fraction In the presence of a nonionic surfactant with an HLB value of 16 or more that has selectivity for lipase, lipoprotein lipase and Z or cholesterol esterase and cholesterol acidase that preferentially act on the HDL fraction.
- a method of measuring HDL cholesterol in a specimen by reacting with an enzyme is known.
- cholesterol in lipoproteins other than HDL is preferentially converted to hydrogen peroxide using the acyl polyoxyethylene sorbitan ester, and the generated peroxyhydrogen is removed.
- a method of enzymatically measuring HDL cholesterol by addition is also known.
- a sample is analyzed with i) cholesterol ester hydrolase and cholesterol acid enzyme, or ii) cholesterol ester hydrolase, acid type. It is characterized by reacting coenzyme and cholesterol dehydrogenase in an aqueous medium containing nonionic surfactant, polyion and albumin, and measuring the generated hydrogen peroxide or reduced coenzyme.
- a method for measuring HDL cholesterol in a sample (see Patent Document 10), a sample, cholesterol ester hydrolase and cholesterol oxidase, or cholesterol ester hydrolase, acid ⁇ type coenzyme and cholesterol dehydration Enzyme produced in the reaction with an aqueous medium containing a bile acid derivative.
- Measurement method (patent documents HDL cholesterol, which comprises measuring the hydrogen or - reducing coenzyme
- Patent Document 1 Japanese Patent Laid-Open No. 62-69999
- Patent Document 2 Japanese Patent Laid-Open No. 63-126498
- Patent Document 3 JP-A-8-131197
- Patent Document 4 JP-A-8-201393
- Patent Document 5 Japanese Patent Laid-Open No. 9-285298
- Patent Document 6 JP-A-8-116996
- Patent Document 7 International Publication No. 97Z40376 Pamphlet
- Patent Document 8 International Publication No. 00Z52480 Pamphlet
- Patent Document 9 JP-A-9 299
- Patent Document 10 International Publication No. 2004Z035816 Pamphlet
- Patent Document 11 Pamphlet of International Publication No. 2004Z035817
- An object of the present invention is to provide a method for measuring HDL cholesterol, a measuring reagent, and a measuring kit, which can be measured easily and accurately.
- the inventor of the present invention diligently studied to solve the above-mentioned problems, and cholesterol ester hydrolysis was carried out in the presence of a specific nitrogen-containing surfactant having a structure of an amine or an ammonium salt and a polyone.
- the inventors have found that a degrading enzyme and cholesterol oxidase or cholesterol dehydrogenase specifically act on HDL, and have completed the present invention. That is, the present invention relates to the following (1) to (13).
- Specimen and i) cholesterol ester hydrolase and cholesterol acid fermentase, or ii) cholesterol ester hydrolase, oxidized coenzyme and cholesterol dehydrogenase are represented by the general formula (I)
- R 1 represents a linear or branched alkyl group or alkenyl group having 6 to 30 carbon atoms
- R 2 represents a linear or branched carbon group having 1 to 30 carbon atoms.
- X represents an anion
- R 5 represents a linear or branched alkyl group having 6 to 30 carbon atoms or a alkenyl group
- R 6 and R 7 are the same or different and each represents a hydrogen atom
- a linear or branched alkyl group having 1 to 30 carbon atoms or a alkenyl group having 2 to 30 carbon atoms and at least one substance selected from the group consisting of substance forces, and polyion
- a method for measuring cholesterol in a high-density lipoprotein in a specimen which comprises reacting in a water-containing aqueous medium and measuring the produced hydrogen peroxide or reduced coenzyme.
- aqueous medium further contains at least one substance selected from the group consisting of albumin, polyoxyethylene alkylamine, and polyoxyethylene alkenylamine.
- R 1 represents a linear or branched alkyl group or alkenyl group having 6 to 30 carbon atoms
- R 2 represents a linear or branched carbon group having 1 to 30 carbon atoms.
- X represents an anion
- R 5 represents a linear or branched alkyl group having 6 to 30 carbon atoms or an alkenyl group
- R 6 and R 7 are the same or different and each represents a hydrogen atom
- R 1 represents a linear or branched alkyl group or alkenyl group having 6 to 30 carbon atoms
- R 2 represents a linear or branched carbon group having 1 to 30 carbon atoms.
- R 5 represents a linear or branched alkyl group or alkyl group having 6 to 30 carbon atoms
- R 6 and R 7 are the same or different and each represents a hydrogen atom
- a kit for measuring cholesterol in high-density lipoprotein comprising a first reagent and a second reagent, having the general formula (I)
- R 1 represents a linear or branched alkyl group having 6 to 30 carbon atoms or a alkenyl group
- R 2 represents a linear or branched alkyl group having 1 to 30 carbon atoms.
- R 3 and R 4 are the same or different and are each linear or A branched alkyl group having 1 to 6 carbon atoms or a alkenyl group having 2 to 6 carbon atoms, and X represents an anion
- X represents an anion
- R 5 represents a linear or branched alkyl group or alkyl group having 6 to 30 carbon atoms
- R 6 and R 7 are the same or different and each represents a hydrogen atom
- a linear or branched alkyl group having 1 to 30 carbon atoms or a alkenyl group having 2 to 30 carbon atoms and at least one substance selected from the group consisting of material forces, and polyion Is contained in the first reagent
- cholesterol oxidase is contained in the second reagent
- the hydrogen peroxide measurement reagent is contained in either the first reagent, the second reagent, or both
- the cholesterol ester hydrolase is contained in the first reagent.
- a kit for measuring cholesterol in high-density lipoprotein comprising a first reagent and a second reagent, having the general formula (I)
- R 1 represents a linear or branched alkyl group having 6 to 30 carbon atoms or a alkenyl group
- R 2 represents a linear or branched alkyl group having 1 to 30 carbon atoms.
- an alkenyl group having 2 to 30 carbon atoms, and R 3 and R 4 are the same or different and each represents a linear or branched alkyl group having 1 to 6 carbon atoms or an alkenyl group having 2 to 6 carbon atoms.
- R 5 represents a linear or branched alkyl group having 6 to 30 carbon atoms or alkenyl group
- R 6 and R 7 are the same or different and each represents a hydrogen atom
- a linear or branched alkyl group having 1 to 30 carbon atoms or a alkenyl group having 2 to 30 carbon atoms and at least one substance selected from the group consisting of material forces, and polyion Is contained in the first reagent
- cholesterol dehydrogenase is contained in the second reagent
- oxidized coenzyme is contained in either the first reagent, the second reagent, or both
- the cholesterol esterase hydrolyzing enzyme is contained in the first reagent.
- At least one substance selected from the group consisting of albumin, polyoxyethylene alkylamine, and polyoxyethylene alkylamine is contained in either or both of the first reagent, the second reagent, and cholesterol ester hydrolysate.
- the present invention provides a simple and accurate method for measuring HDL cholesterol, a reagent for measurement, and a kit for measurement.
- the method for measuring HDL cholesterol according to the present invention is a method for measuring HDL cholesterol without eliminating cholesterol in lipoproteins other than HDL and without aggregating cholesterol in lipoproteins other than HDL. It is.
- force plasma and serum including whole blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, spleen and the like are preferable.
- the cholesterol esterase-degrading enzyme in the present invention is not particularly limited as long as it is an enzyme having the ability to hydrolyze cholesterol ester.
- cholesterol esterase and lipoprotein lipase produced by genetic engineering techniques can be used.
- cholesterol ester water-degrading enzyme unmodified cholesterol ester hydrolase or chemically modified cholesterol ester hydrolase can be used.
- cholesterol esterase hydrolyzing enzymes include cholesterol esterase “Amano” 2 (CHE2; manufactured by Amano Enzyme), cholesterol esterase “Amano” 3 (CHE3; manufactured by Amano Enzym), lipoprotein lipase.
- LPL311 manufactured by Toyobo Co., Ltd.
- lipoprotein lipase "Amano” 3 LPL3; manufactured by Amano Enzym
- 43kDa esterase manufactured by Amano Enzym
- 40kDa esterase manufactured by Amano Enzym
- ES T “Amano” 2 (Manufactured by Amano Enzyme)
- COE313 chemically modified cholesterol esterase
- Examples of the (chemically modifying group) include a group having polyethylene glycol as a main component, a group having polypropylene glycol as a main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group containing a water-soluble polysaccharide, Forces such as sulfopropyl group, sulfobutyl group, polyurethane group, group having chelate function, etc. Groups based on coal are preferred.
- Examples of the water-soluble polysaccharide include dextran, pullulan, and soluble starch.
- a functional group capable of reacting with the above-mentioned chemical modification group and the amino group, carboxyl group, sulfhydryl group, etc. of the enzyme examples thereof include compounds having both a group and a structure.
- Examples of functional groups or structures that can react with amino groups in the enzyme include carboxyl groups, active ester groups (N-hydroxysuccinimide groups, etc.), acid anhydrides, acid chlorides, aldehydes, epoxide groups, 1, 3 Examples include propane sultone and 1,4 butane sultone.
- the functional group or structure capable of reacting with a carboxyl group in the enzyme include an amino group.
- groups or structures reactive with sulfhydryl groups in enzymes include maleimide groups
- Chemical modifiers include Sunbright VFM—4101, Sunbright ME—050AS, and Sunbright DE—030 AS (both containing polyethylene glycol as the main component and N-hydroxysuccinimide group) Manufactured by Nippon Oil & Fats Co., Ltd., Sunbright AKM series (for example, Sunbright AKM-1510, etc.) having a group mainly composed of polyalkylene glycol and an acid anhydride structure, Sunbright ADM series, Sunbright ACM series (all Nippon Oil & Fats Co., Ltd.), EPOX-3400 with polyethylene glycol as the main component and epoxide group, M-EPOX-5000 (also from Sheawater Polymers), group with chelate function and acid anhydride structure Diethylenetriamine-N, N, ⁇ ', ⁇ ", ⁇ " pentahydrodiacetic anhydride (DTPA anhydride; manufactured by
- the chemical modification of cholesterol esterase is not limited to this method, for example, by the following method.
- a pH 8.0 or higher buffer solution for example, HEPES buffer solution
- 0 to 1 to 500 times the molar amount of a chemical modifier is added at 0 to 55 ° C. Stir for minutes to 5 hours.
- a chemically modified cholesterol ester hydrolase not only the reaction solution itself, but also unreacted chemical modifiers, etc. were removed by ultrafiltration membranes, etc. as necessary. Can be used as well.
- the concentration of the cholesterol ester hydrolase used in the method of this reaction is not particularly limited as long as it enables the measurement of HDL cholesterol of the present invention, but is 0.01 to 400 UZmL in the reaction solution. A concentration of 0.02 to 200 U / mL is more preferable.
- the cholesterol oxidase enzyme in the present invention is not particularly limited as long as it is an enzyme having the ability to oxidize cholesterol to produce hydrogen peroxide, for example, an animal, plant or microorganism-derived cholesterol oxidase.
- cholesterol oxidase produced by genetic engineering techniques can also be used, such as cholesterol oxidase “Amano” l (CHOD1; Amano Enzyme), cholesterol oxidase (CH OPE; Kikkoman).
- Commercially available products such as cholesterol oxidase (C00321; manufactured by Toyobo Co., Ltd.), cholesterol oxidase Kyowa (manufactured by Kyowa Hakko), etc.
- the cholesterol acid enzyme may be an unmodified enzyme or a chemically modified enzyme.
- the chemically modified cholesterol oxidase enzyme can be prepared by the above-described chemical modification method using, for example, the above-described chemical modifier.
- the concentration of cholesterol acid enzyme used in the reaction method is not particularly limited as long as it is a concentration that enables measurement of HDL cholesterol of the present invention, but is 0.01 to 400 UZmL in the reaction solution.
- the concentration is preferably 0.02 to 200 UZmL.
- the cholesterol dehydrogenase in the present invention is not particularly limited as long as it is an enzyme having the ability to oxidize cholesterol in the presence of an oxidized coenzyme to produce a reduced coenzyme.
- cholesterol dehydrogenase produced by genetic engineering techniques and the like can also be used.
- Commercial products such as cholesterol dehydrogenase “Amano” 5 (CHDH5; manufactured by Amano Enzyme Co., Ltd.) can also be used.
- two or more kinds of cholesterol dehydrogenase can be used in combination.
- Cholesterol dehydrogenase may be an unmodified enzyme or a chemically modified enzyme. scientific Cholesterol dehydrogenase modified in the above manner can be prepared by the above-described chemical modification method using, for example, the above-described chemical modifier.
- the concentration of cholesterol dehydrogenase used in the method of the present reaction is not particularly limited as long as it is a concentration that enables measurement of HDL cholesterol of the present invention.
- a concentration of 400 UZmL is preferred.
- a concentration of 0.02 to 200 UZmL is more preferred.
- oxidized coenzyme In the measurement method using cholesterol dehydrogenase of the present invention, oxidized coenzyme is used.
- the oxidized coenzyme include NAD, NADP, thio-NAD, thio-NADP and the like.
- R 1 represents a linear or branched alkyl group or alkenyl group having 6 to 30 carbon atoms
- R 2 represents a linear or branched carbon group having 1 to 30 carbon atoms.
- R 5 represents a linear or branched alkyl group having 6 to 30 carbon atoms or an alkenyl group.
- R 6 and R 7 are the same or different and each represents a hydrogen atom, a linear or branched alkyl group having 1 to 30 carbon atoms, or a alkenyl group having 2 to 30 carbon atoms.
- Substance to be used [hereinafter referred to as compound ( ⁇ )] is used.
- Examples of the linear or branched alkyl group having 6 to 30 carbon atoms in compound (I) and compound ( ⁇ ⁇ ⁇ ) include hexyl, heptyl, octyl, isooctyl, nonyl, decyl, undecyl, dodecyl (lauryl), Tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, henicosyl, docosyl (beher), tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl , Nonacosyl, triaconsil, and the like, and an alkyl group having 8 to 24 carbon atoms is preferred, and an alkyl group having 10
- Examples of the straight chain or branched alkenyl group having 6 to 30 carbon atoms in the compound (I) and the compound ( ⁇ ) include hexyl, heptul, otatur, nonel, dece- , Citronellyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadesel, heptadecyl, oleil, nonadecele, eicosole, hen eicosenore, dococennole, Examples include tricoseninoles, tetracoseninoles, pentacoceninoles, hexacosels, heptacoses, octacoses, nonacoses, triacontails, etc., preferably alkenyl groups having 8 to 24 carbon atoms. More preferably, it is an alkenyl group having 10 to 18 carbon atom
- Examples of the linear or branched alkyl group having 1 to 30 carbon atoms in the compound (I) and the compound ( ⁇ ) include, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl.
- the group include methyl, ethyl, propyl, butyl, pentyl, hexyl and the like, and it is more preferably a methyl group that is preferably a methyl, ethyl, or propyl group.
- Examples of the straight chain or branched alkenyl group having 2 to 30 carbon atoms in the compound (I) and the compound ( ⁇ ) include, for example, bur, probe, allyl, butenyl, pentyl, hexyl. , Heptul, otatur, nonel, deseal, citronellyl, undecyl, dodecyl, tridecenyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, oleil, nonadece -E.g., eicosel, henecosyl, docosel, tricocennenole, tetracoseninole, pentacosennenole, hexacoseninole, heptacoceinole, octacocele, nonacoseol, triaconte, etc.
- Examples of the straight-chain or branched alkenyl group having 2 to 6 carbon atoms in the compound (I) and the compound ( ⁇ ⁇ ⁇ ) include, for example, bur, probe, allyl, butyl, pentale, and the like.
- Xenyl and the like can be mentioned, and it is more preferable that it is a vinyl group, which is preferably either a bull or a probe group.
- Examples of the anion in compound (I) include a hydroxide ion, a halogen ion, an anion derived from an inorganic acid, an anion derived from an organic acid, and the like.
- Examples of the halogen ion include fluorine ion, chlorine ion, bromine ion and iodine ion.
- Examples of the anion derived from an inorganic acid include nitrate ion, sulfate ion, phosphate ion, carbonate ion and the like.
- Examples of the anions derived from organic acids include formate ions, acetate ions, lactate ions, citrate ions, carboxylate ions such as glutamate ions, and the like.
- R 1 represents a linear or branched alkyl group or alkyl group having 6 to 30 carbon atoms
- R 2 represents a linear or branched group.
- Preferred is a compound which represents a branched alkyl group having 1 to 30 carbon atoms or an alkenyl group having 2 to 30 carbon atoms
- R 3 and R 4 both represent methyl
- X represents an anion.
- Specific examples (products) of compound (I) include cation AB, cation BB, cation 2 ABT, cation 2DB-500E, cation 2-OLR (all manufactured by NOF Corporation) and the like.
- Specific examples (products) of compound ( ⁇ ) include Ammin BB, Ammin AB, Ammin 2 — OLR, Grade 3 Ammin BB, Grade 3 Amine FB (all manufactured by NOF Corporation).
- the concentration of the compound (I) or the compound ( ⁇ ⁇ ) is not particularly limited as long as the concentration of the HDL cholesterol of the present invention can be measured, but the concentration in the reaction solution is 0.0001 to 1%. It is preferable that 0.001 to 0.1% is more preferable.
- the polyion used in the present invention is not particularly limited as long as it is a polyion capable of measuring the HDL cholesterol of the present invention.
- dextran sulfate or a salt thereof henone or a salt thereof, phosphorus Tungstic acid or its salt, sulfated cyclodextrin or its salt, sulfated oligosaccharide or its salt, and the like.
- Dextrane sulfuric acid or its salt is preferable.
- Examples of dextran sulfate include dextran sulfate having a molecular weight of 40,000, 80,000, 200,000, 500,000, 1 million, 2 million, and the like.
- sulfated oligosaccharides include diagarose sulfate, sulfated trehalose, and chondroitin sulfate.
- the salt include sodium salt, potassium salt, lithium salt, ammonium salt, magnesium salt and the like.
- two or more polyones may be used.
- the polyaion concentration in the measurement of HDL cholesterol of the present invention is not particularly limited as long as it can measure the HDL cholesterol of the present invention, but the concentration in the reaction solution is 0.001 to 10. % Is preferred. 0.01-1% is more preferred.
- the albumin used in the present invention is not particularly limited as long as it is an albumin capable of measuring the HDL cholesterol of the present invention, such as ushi, horse, hidge, human-derived albumin and the like. Forced serum albumin (BSA) is preferred.
- BSA Forced serum albumin
- albumin produced by genetic engineering techniques can also be used.
- two or more types of albumin can be used in combination.
- the concentration of albumin in the measurement of HDL cholesterol of the present invention is not particularly limited as long as it enables the measurement of HDL cholesterol of the present invention, but the concentration in the reaction solution is 0.001 to 10%. Preferably, 0.01 to 1% is more preferable.
- polyoxyethylene alkylamine or polyoxyethylenealkylamine used in the present invention is, for example, represented by the general formula (III)
- R 8 represents a linear or branched alkyl group or a alkenyl group
- R 9 represents a hydrogen atom or (CH 2 CH 2 O) H
- m and n are the same or different, 1 to 100
- M + n is an integer of 2 to 200] [hereinafter referred to as compound (III)].
- alkyl group and alkenyl group in the compound ( ⁇ ) include, for example, the above-described linear or branched alkyl group having 6 to 30 carbon atoms, and the above-described linear or branched carbon group having 6 to 6 carbon atoms. 30 alkyl groups and the like, and preferably an alkyl group having 8 to 24 carbon atoms or an alkyl group, preferably an alkyl group having 10 to 18 carbon atoms or an alkenyl group. I like it!
- polyoxyethylene alkylamine or polyoxyethylene alkylamine include, for example, Niimine L201 (oxyethylene dodecylamine; manufactured by Nippon Oil & Fats Co., Ltd.), Naimine L207 (Polyoxyethylene dodecyl) Nymeen S204, Nymeen S210 (Polyoxyethylene Octadecylamine; produced by Nihon Yushi Co., Ltd.), New Coal OD420 (Polyoxyethylene Octadecylamine; Nippon Milky Co., Ltd.) ), Bionine D3104 (polyoxyethylene laurylamine; manufactured by Takemoto Yushi Co., Ltd.), Bionine D3110 (polyoxyethylene laurylamine; manufactured by Takemoto Yushi Co., Ltd.), Nyonin D3605 [Polyoxyethylene alkyl (soy) Amamine; Takemoto Yushi Co., Ltd.) Bionin D3615T [Polyoxyethylene alkyl (beef
- the degree of polymerization of the oxyethylene chain in the polyoxyethylene alkylamine and polyoxyethylene alkyleneamine is preferably 1 to 100, more preferably 1 to 60.
- two or more types of polyoxyethylene alkylamine and polyoxyethylene alkylamine may be used.
- the concentration of the oxyethylene alkyleneamine is not particularly limited as long as it enables the measurement of HDL cholesterol of the present invention, but the concentration in the reaction solution is preferably 0.0001 to 1%. Mashi 0. 001-0. 1% is more preferable.
- the aqueous medium used in the HDL cholesterol measurement method of the present invention is preferably a force buffer solution such as deionized water, distilled water, or buffer solution.
- a force buffer solution such as deionized water, distilled water, or buffer solution.
- the buffer used in the buffer include tris (hydroxymethyl) aminomethane buffer, phosphate buffer, borate buffer, Good's buffer, and the like.
- Good buffering agents include, for example, 2 morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminonic acid (ADA), piperazine 1 N, N, 1 bis (2-ethanesulfonic acid) (PIPE S), N— (2 acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino 1 2-hydroxypropanesulfone Acid (MOPSO), N, N bis (2-hydroxyethyl) 2 aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] 2 aminoethanesulfonic acid (TES), 2- [4- (2-Hydroxyethyl) 1-piperadyl] ethanesulfonic acid (HEPES), 3- [N, N bis
- TAPS N cyclohexyl 2 Aminoethanesulfonic acid
- CHES N-cyclohexyl 3 Amino 2 hydroxypropane sulfonic acid
- CAPSO propanesulfonic acid
- the concentration of the buffer solution is not particularly limited as long as it is suitable for the measurement, but is preferably 0.001 to 2. Omol /! L force, more preferably 0.005 to 1.0 mol /: L force ⁇ ! / ⁇ .
- Examples of the method for measuring HDL cholesterol of the present invention include the methods of the following aspects.
- HDL cholesterol in a sample can be measured by calculating the HDL cholesterol concentration in the sample from the value measured in (2) and a calibration curve prepared in advance.
- the reaction of (1) is performed, for example, at 10 to 50 ° C, preferably at 20 to 40 ° C, for 1 to 60 minutes, preferably 2 to 30 minutes.
- the amount of hydrogen peroxide generated can be measured, for example, with a reagent for measuring hydrogen peroxide, which can be directly measured with a hydrogen peroxide electrode.
- the reagent for measuring hydrogen peroxide is a reagent for converting the produced hydrogen peroxide to a detectable substance.
- the detectable substance include a dye and luminescence, and a dye is preferable.
- the reagent for measuring hydrogen peroxide contains an oxidative coloring type chromogen and a peroxidase active substance such as peroxidase.
- the oxidative coloring type chromogen include the following oxidative coloring type chromogen.
- the reagent for hydrogen peroxide measurement contains a chemiluminescent substance.
- the chemiluminescent substance include luminol, isoluminol, lucigenin, and atalydium ester.
- a reagent containing a peroxide active substance such as an oxidative coloring type chromogen and peroxidase
- hydrogen peroxide is oxidized in the presence of the peroxide active substance. Reacts with chromogenic chromogen to produce dye, and quantifies the dye produced
- peroxyhydrogen can be quantified.
- hydrogen peroxide measuring reagent containing a chemiluminescent substance hydrogen peroxide peroxygen reacts with the chemiluminescent substance to generate photon, and the generated photon is quantified. Acidic hydrogen can be quantified.
- Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.
- Leuco-type chromogens are substances that are converted to pigments alone in the presence of peroxide active substances such as hydrogen peroxide and peroxidase.
- peroxide active substances such as hydrogen peroxide and peroxidase.
- CCAP 10-N-carboxymethylcarbamoyl-3,7 bis (dimethylamino) -10H phenothiazine
- M CDP 10-N-methylcarbamoyl-3,7 bis (dimethylamino) -10H phenothiazine
- Oxidative coupling chromogenic chromogen is a substance that produces a dye by acid coupling of two compounds in the presence of a peroxidation active substance such as hydrogen peroxide and peroxidase.
- Examples of the combination of the two compounds include a combination of a coupler and an arylene, a combination of a coupler and a phenol.
- Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine.
- the concentration of the peracid active substance is not particularly limited as long as it is a concentration suitable for the measurement, but peroxidase is used as the peracid active substance. When used, 1 to: LOOkUZL is preferable.
- the concentration of the oxidative coloring type chromogen is not particularly limited as long as it is a concentration suitable for measurement, but is preferably 0.01 to 10 gZL.
- Examples of the method for measuring reduced coenzyme include a method of measuring the absorbance of the produced reduced coenzyme, a method using a reagent for measuring reduced coenzyme, and the like.
- the absorbance in the method for measuring the absorbance of the reduced coenzyme is preferably 300 to 500 nm, more preferably 330 to 400 nm, and particularly preferably around 340 nm.
- the reagent for measuring reduced coenzyme is a reagent for converting the produced reduced coenzyme into a detectable substance. Examples of the detectable substance include a dye.
- Examples of the reagent for reducing coenzyme when the detectable substance is a dye include a reagent containing diaphorase, an electron carrier, and a reduced color developing chromogen.
- Examples of the electron carrier include 1-methoxy 5-methylphenmethyl methyl sulfate and the like.
- Examples of reducing chromogens include 3- (4,5 dimethyl-2-thiazolyl) 2,5-diphenyl-1,2H-tetrazolium bromide (MTT) ⁇ 2- (4-iodophenol). ) 3— (4-Trophele) 5— (2, 4 Disulfophenol) 2H— Tetrazolium mononatri UM salt (WST— 1), 2— (4—Edophenol) —3— (2, 4-Dinitrophenol) — 5— (2, 4-Disulfophenol) — 2H—Tetrazolium monosodium Examples include salt (WST-3).
- the reagent for measuring HDL cholesterol of the present invention comprises at least one substance selected from the group consisting of compound (I) and compound (i), polyion, cholesterol ester hydrolase, cholesterol acid enzyme and peracid acid.
- the reagent for measurement includes at least one substance selected from the group consisting of compound (I) and compound ( ⁇ ), polyoxyethylene alkylamine and polyoxyethylene alkylamine-group.
- a reagent containing at least one substance, polyan-one, cholesterol esterase, cholesterol acid enzyme and peracid hydrogen measuring reagent, compound (I) and compound ( ⁇ ) A reagent containing at least one substance selected from the group consisting of polyanone, albumin, cholesterol ester-hydrolyzing enzyme, cholesterol acid enzyme and peracid hydrogen measuring reagent is preferred.
- the measurement reagent at least one substance selected from the group consisting of the compound (I) and the compound ( ⁇ ) force, polyoxyethylene alkylamine and a group force including the polyoxyethylene alkylamine force are also selected.
- the reagent for measuring HDL cholesterol of the present invention comprises at least one substance selected from the group consisting of compound (I) and compound (v), polyion, cholesterol ester hydrolase, cholesterol dehydrogenase, and oxidation. Containing a coenzyme, and, if necessary, containing at least one substance selected as a reduced coenzyme measuring reagent, albumin, polyoxyethylene alkylamine and polyoxyethylene alkylamine as well as group power .
- the measurement reagent is a compound selected from the group consisting of Compound (I) and Compound (I). At least one substance, polyoxyethylene alkylamine and polyoxyethylene alkylkeamine, which are also selected for their group strength, polyone, cholesterol ester hydrolase, cholesterol dehydrogenase, oxidized coenzyme And a reagent containing a reagent for measuring reduced coenzyme, at least one substance selected from the group consisting of compound (I) and compound (II), polyone, albumin, cholesterol ester carohydrolyzing enzyme, cholesterol dehydrogenation Reagents containing enzymes, oxidized coenzymes and reduced coenzyme measuring reagents are preferred.
- the reagent for measurement at least one substance selected from the group consisting of compound (I) and compound ( ⁇ ) force, polyoxyethylene alkylamine and group force consisting of polyoxyethylene alkylamine force are also selected. Particularly preferred are those containing at least one substance, polyone, albumin, cholesterol ester hydrolase, cholesterol dehydrogenase, acid coenzyme, and reduced coenzyme measuring reagent.
- Examples of the reagent for measuring HDL cholesterol of the present invention include the reagents of the following embodiments, but these do not limit the scope of the present invention.
- at least one substance selected from compound (I) and compound ( ⁇ ) group power is hereinafter referred to as “compound A”, and albumin, polyoxyethylene alkylamine, and polyoxyethylene alkylamine strength.
- the group power of at least one selected substance is hereinafter referred to as “Compound B”.
- one or more compounds A can be used, and one or more compounds B can be used.
- a reagent containing Compound A, polyone, cholesterol ester-hydrolyzing enzyme, cholesterol oxidase and a reagent for measuring hydrogen peroxide is provided.
- a reagent containing Compound A, Polyone, Compound B, cholesterol esterase, cholesterol acid enzyme and peracid hydrogen reagent is a reagent containing Compound A, Polyone, Compound B, cholesterol esterase, cholesterol acid enzyme and peracid hydrogen reagent.
- a reagent containing Compound A, Polyone, Compound B, cholesterol esterase, acid coenzyme and cholesterol dehydrogenase is provided.
- a reagent containing Compound A, polyion, cholesterol ester hydrolase, oxidized coenzyme, cholesterol dehydrogenase and a reagent for measuring reduced coenzyme is provided.
- a reagent containing Compound A, Polyone, Compound B, cholesterol esterase, acid coenzyme, cholesterol dehydrogenase enzyme, and reduced coenzyme measurement reagent is provided.
- the reagent for measuring HDL cholesterol of the present invention may be stored, distributed and used in the form of a kit.
- the form of the kit is not particularly limited, and may be a two-reagent system or a three-reagent system, but a two-reagent system is preferred.
- the cholesterol esterase-hydrolyzing enzyme and the cholesterol oxidase or cholesterol dehydrogenase include the first reagent and the second reagent. It may be contained separately in the reagent or together in the second reagent, but when it is contained separately in the first reagent and the second reagent, cholesterol esterase is added to the first reagent.
- An embodiment in which cholesterol acid zyme enzyme or cholesterol dehydrogenase is contained in the second reagent is preferred.
- the acid type coenzyme used in the measurement method using cholesterol dehydrogenase may be contained in either or both of the first reagent and the second reagent.
- the compound (I) and the compound (v) force group power At least one substance selected (compound A) may be contained in either or both of the first reagent and the second reagent. It is preferably contained in the reagent. Polyone may be contained in either the first reagent, the second reagent, or both, but is preferably contained in the first reagent.
- the group power of albumin, polyoxyethylene alkylamine and polyoxyethylene alkenyl amine is selected from the group consisting of a first reagent, a second reagent, and a second reagent. May be contained in either or both of the reagents.
- the hydrogen peroxide measurement reagent may be contained in one or both of the first reagent and the second reagent.
- the reagent contains an oxidatively coupled chromogen
- the reducing coenzyme measuring reagent may be contained in either or both of the first reagent and the second reagent, but is preferably contained in both the first reagent and the second reagent.
- kits for measuring HDL cholesterol of the present invention include the following kits, but these do not limit the scope of the present invention.
- Compound A polyone, hydrogen peroxide measuring reagent, cholesterol ester hydrolase
- Compound B reagent for measuring hydrogen peroxide, cholesterol acid enzyme
- Compound B reagent for measuring hydrogen peroxide, cholesterol esterase, cholesterol esterase
- Ki, t9 First reagent
- Compound A polyanion, oxidized coenzyme, reagent for measuring reduced coenzyme, cholesterol esterase
- Reductive coenzyme measurement reagent cholesterol esterase, cholesterol dehydrogenase
- Reductive coenzyme measurement reagent cholesterol esterase, cholesterol dehydrogenase
- Compound A polyanion, oxidized coenzyme, reagent for measuring reduced coenzyme, cholesterol ester hydrolase second reagent
- Compound B reagent for measuring reduced coenzyme, cholesterol dehydrogenase
- Compound B reagent for measuring reduced coenzyme, cholesterol esterase, cholesterol dehydrogenase
- the above-mentioned present invention is provided.
- Compound (I), Compound ( ⁇ ), Polyanion, Albumin, Polyoxyethylene alkylamine, Polyoxyethylene alkylamine, Cholesterol ester-hydrolyzing enzyme mentioned in the method for measuring HDL cholesterol Cholesterol oxidase, cholesterol dehydrogenase, oxidized coenzyme, hydrogen peroxide measuring reagent, and reduced coenzyme measuring reagent can be used.
- the reagent for measuring HDL cholesterol and the kit for measurement of the present invention may contain an aqueous medium, a stabilizer, a preservative, an influential substance erasing agent, a reaction accelerator and the like, if necessary.
- an aqueous medium the above-mentioned aqueous medium etc. are mentioned, for example.
- the stabilizer include ethylene diamine amine acetic acid (EDTA), sucrose, calcium chloride and the like.
- antiseptics include sodium azide and antibiotics.
- Examples of the influence substance elimination agent include ascorbate oxidase for eliminating the influence of ascorbic acid.
- the reaction accelerator include enzymes such as colipase and phospholipase, and salts such as sodium sulfate and sodium chloride.
- the HDL cholesterol measurement reagent and measurement kit of the present invention may be either lyophilized or dissolved in an aqueous medium.
- the reagent is used after being dissolved in an aqueous medium.
- the content of cholesterol esterase, cholesterol oxidase, and cholesterol dehydrogenase in the HDL cholesterol measurement reagent and measurement kit of the present invention is such that the concentration in a state dissolved in an aqueous medium is 0.01 to 1200 U /
- the content of mL is preferable.
- the content of 0.02 to 600 U / mL is more preferable.
- the content of the compound (I) or the compound ( ⁇ ⁇ ⁇ ⁇ ) in the HDL cholesterol measurement reagent and measurement kit of the present invention is such that the concentration in a state dissolved in an aqueous medium is 0.0001 to 3%. A content of 0.001-0.3% is more preferred.
- the polyaone content in the reagent for measuring HD L cholesterol and the measurement kit of the present invention is preferably such that the concentration in the state dissolved in an aqueous medium is 0.001 to 30%. A content of ⁇ 3% is more preferred.
- Albumin in the HDL cholesterol measurement reagent and measurement kit of the present invention The content is preferably such that the concentration in a state dissolved in an aqueous medium is 0.001 to 30%, more preferably 0.01 to 3%.
- the content of polyoxyalkylamine or polyoxyethylene alkylamine in the HDL cholesterol measurement reagent and measurement kit of the present invention is preferably such that the concentration in a state dissolved in an aqueous medium is 0.0001 to 3%. A content of 0.001 to 0.3% is more preferable.
- HEPES (manufactured by BDH Laboratory), EMSE (manufactured by Daito Chemix), sodium sulfate (manufactured by Kanto Yigaku Co., Ltd.), Amine BB (Dodecylamine; manufactured by Nippon Oil & Fats Co., Ltd.), Class 3 Ammine BB (Dodecyldimethylamine) ; Nippon Oil & Fats Co., Ltd.), Cationic BB (Dodecyltrimethylammonium chloride; Nippon Oil & Fats Co., Ltd.), Dextran Sodium Sulfate (Molecular Weight 500,000) (manufactured by Pharmacia), Usi Serum Albumin (BSA; Proliantone Earth) , 4-Aminoantipyrine (Saikyo Kasei Co., Ltd.), Peroxidase (Toyobo Co., Ltd.), 43 kDa esterase (Cholesterol ester hydrolase; Amano Enzyme),
- first reagent (reagent A) and second reagent (reagent a) powerful HDL cholesterol measurement kits were prepared.
- first reagent (reagent B) and second reagent (reagent a) powerful HDL cholesterol measurement kits were prepared.
- first reagent (reagent C) and second reagent (reagent a) powerful HDL cholesterol measurement kits were prepared.
- An HDL cholesterol measurement kit comprising the following first reagent (reagent D) and second reagent (reagent a) was prepared.
- An HDL cholesterol measurement kit comprising the following first reagent (reagent C) and second reagent (reagent b) was prepared.
- the following kit for measuring HDL cholesterol was also prepared with the ability of the following first reagent (reagent D) and second reagent (reagent b).
- kits for measuring HDL cholesterol were prepared, including the following first reagent (reagent E) and second reagent (reagent a).
- absorbance represents a value obtained by subtracting E1 from E2 based on two absorbances (E1 and E2) measured in the following reaction.
- the ⁇ absorbance '' in the sample was determined in the same manner as the (absorbance) calculation method in (1). Calculated.
- HDL cholesterol in 30 samples of human serum was measured by Hitachi 7170 automatic analyzer in the same manner as in the measurement method of Example 7, except that the kit of Comparative Example 1 was used instead of the kit of Example 1.
- the present invention provides a method for measuring HDL cholesterol, a reagent for measurement and a kit for measurement useful for diagnosis of diseases such as arteriosclerosis.
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Abstract
Description
Claims
Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
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BRPI0610378A BRPI0610378A2 (pt) | 2005-04-27 | 2006-04-27 | método, reagente e kit para a medição de colesterol em lipoproteína de densidade alta |
CA2605329A CA2605329C (en) | 2005-04-27 | 2006-04-27 | Method for determination of cholesterol in high-density lipoprotein |
CN2006800142148A CN101166831B (zh) | 2005-04-27 | 2006-04-27 | 高密度脂蛋白中的胆固醇的测定方法 |
ES06732406T ES2396143T3 (es) | 2005-04-27 | 2006-04-27 | Método para la medida de colesterol en lipoproteína de alta densidad |
US11/911,029 US7811780B2 (en) | 2005-04-27 | 2006-04-27 | Method for measurement of cholesterol in high-density lipoprotein |
AU2006241793A AU2006241793B2 (en) | 2005-04-27 | 2006-04-27 | Method for determination of cholesterol in high-density lipoprotein |
JP2007514809A JP5112861B2 (ja) | 2005-04-27 | 2006-04-27 | 高密度リポ蛋白中のコレステロールの測定方法 |
KR1020077024676A KR101153759B1 (ko) | 2005-04-27 | 2006-04-27 | 고밀도 리포 단백질 중의 콜레스테롤의 측정 방법 |
EP06732406A EP1876243B1 (en) | 2005-04-27 | 2006-04-27 | Method for determination of cholesterol in high-density lipoprotein |
HK08104126.1A HK1109916A1 (en) | 2005-04-27 | 2008-04-11 | Method for determination of cholesterol in high-density lipoprotein |
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JP2005-129319 | 2005-04-27 | ||
JP2005129319 | 2005-04-27 |
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US (1) | US7811780B2 (ja) |
EP (1) | EP1876243B1 (ja) |
JP (1) | JP5112861B2 (ja) |
KR (1) | KR101153759B1 (ja) |
CN (1) | CN101166831B (ja) |
AU (1) | AU2006241793B2 (ja) |
BR (1) | BRPI0610378A2 (ja) |
CA (1) | CA2605329C (ja) |
ES (1) | ES2396143T3 (ja) |
HK (1) | HK1109916A1 (ja) |
RU (1) | RU2007143970A (ja) |
TW (1) | TWI372783B (ja) |
WO (1) | WO2006118199A1 (ja) |
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WO2008143159A1 (ja) * | 2007-05-18 | 2008-11-27 | Kyowa Medex Co., Ltd. | 低密度リポ蛋白中のコレステロールの測定方法及び測定用キット |
WO2009048143A1 (ja) * | 2007-10-10 | 2009-04-16 | Denka Seiken Co., Ltd. | small,dense LDLコレステロールの定量方法およびキット |
EP2108961A1 (en) * | 2008-04-10 | 2009-10-14 | Fujifilm Corporation | Dry analytical element for measurement of high density lipoprotein cholesterol |
WO2012124340A1 (ja) | 2011-03-16 | 2012-09-20 | 協和メデックス株式会社 | Hdl亜分画中のコレステロールの測定方法、測定用試薬及び測定用キット |
JPWO2011136316A1 (ja) * | 2010-04-30 | 2013-07-22 | 協和メデックス株式会社 | 低密度リポ蛋白中のコレステロールの測定方法、測定用試薬及び測定用キット |
WO2014034823A1 (ja) | 2012-08-31 | 2014-03-06 | 協和メデックス株式会社 | 高密度リポ蛋白中のコレステロールの測定方法 |
WO2015030236A1 (ja) | 2013-08-30 | 2015-03-05 | 積水メディカル株式会社 | 高比重リポ蛋白中のコレステロール測定方法および該方法に用いる試薬 |
JP2016082880A (ja) * | 2014-10-23 | 2016-05-19 | 東洋紡株式会社 | 高密度リポ蛋白コレステロールの測定方法 |
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EP2604657A4 (en) * | 2010-08-11 | 2015-08-19 | Kyowa Medex Co Ltd | METHOD FOR PRESERVING AQUEOUS SOLUTION COMPRISING A LEUCO TYPE CHROMOGEN |
TWI456199B (zh) | 2011-12-29 | 2014-10-11 | Univ Nat Chiao Tung | 生物檢測裝置及檢測方法 |
WO2015077776A1 (en) * | 2013-11-25 | 2015-05-28 | University Of Washington Through Its Center For Commercialization | Methods for calibrated ion mobility analysis and uses thereof |
CN117491644B (zh) * | 2023-10-31 | 2024-10-22 | 深圳上泰生物工程有限公司 | 一种载脂蛋白e的检测试剂盒及其应用 |
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JPWO2014034823A1 (ja) * | 2012-08-31 | 2016-08-08 | 協和メデックス株式会社 | 高密度リポ蛋白中のコレステロールの測定方法 |
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WO2015030236A1 (ja) | 2013-08-30 | 2015-03-05 | 積水メディカル株式会社 | 高比重リポ蛋白中のコレステロール測定方法および該方法に用いる試薬 |
JP2016082880A (ja) * | 2014-10-23 | 2016-05-19 | 東洋紡株式会社 | 高密度リポ蛋白コレステロールの測定方法 |
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AU2006241793B2 (en) | 2011-03-24 |
CN101166831B (zh) | 2011-07-20 |
BRPI0610378A2 (pt) | 2016-11-08 |
CA2605329C (en) | 2014-08-05 |
KR20080012854A (ko) | 2008-02-12 |
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