Classifications

• • 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
• • 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 high-density lipoprotein in a sample, a reagent for measurement, and a kit for measurement.
• HDL high-density lipoprotein
• LDL low-density lipoprotein
• VLDL ultra-low-density lipoprotein
• CM chylomicrons
• HDL cholesterol Conventional methods for measuring cholesterol in HDL (hereinafter abbreviated as HDL cholesterol) include two procedures: ultracentrifugation, immunochemical methods, electrophoresis, precipitation, etc., and cholesterol quantification. Gradual power is also available.
• the fractionation operation is complicated, takes a long time, and has a problem in terms of safety. Therefore, the measurement methods involving these separation operations are extremely inefficient and are not suitable for practical use.
• Patent Document 2 A method for measuring HDL cholesterol by reacting the enzyme in a buffer containing lusterase, 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, then the reaction between HDL cholesterol and the enzyme proceeds, and HDL cholesterol can be measured. However, these assays took a long time to measure and were not necessarily specific to HD L cholesterol.
• Methods for measuring HDL cholesterol by aggregating lipoproteins other than HDL include, for example, reagents for aggregating lipoproteins other than HDL, such as dextran sulfate, divalent metal salts, and chemically modified lipoproteins.
• Assay using enzyme see Patent Document 3
• poly-ones such as dextran sulfate, divalent metal salts, certain nonionic surfactants, and albumin different from sample-derived albumin.
• Solution to be used a solution containing serum or plasma containing a lipoprotein fraction (a combination of polyanion such as dextran sulfate and divalent cation such as magnesium ion) Cholesterol esterase and cholesterol oxidase in the presence of a ionic surfactant (alkyl sulfonic acid or bile acid or a derivative thereof) without separating the resulting mixture into a solid and a liquid.
• a ionic surfactant alkyl sulfonic acid or bile acid or a derivative thereof
• Methods for measuring HDL cholesterol without aggregating lipoproteins other than HDL include, for example, a biological sample, 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
• an HDL fraction Lipoprotein lipase and Z or cholesterol esterase and cholesterol esterase which preferentially act on the HDL fraction, in the presence of a nonionic detergent with an HLB value of 16 or more
• a method of measuring HDL cholesterol in a sample by reacting with a dani enzyme see Patent Document 8) and the like are known.
• cholesterol in lipoproteins other than HDL is preferentially converted to hydrogen peroxide by acyl polyoxyethylene sorbitan ester, and the generated hydrogen peroxide is eliminated.
• a method of enzymatically measuring HDL cholesterol by addition is also known.
• Patent Document 1 JP-A-62-69999
• Patent Document 2 JP-A-63-126498
• Patent Document 3 JP-A-8-131197
• Patent Document 4 JP-A-8-201393
• Patent Document 5 JP-A-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-9299
• An object of the present invention is to provide a method for measuring HDL cholesterol, a reagent for measurement and a kit for measurement, which can be simply and accurately measured. Means for solving the problem
• the present invention relates to the following [1] to [19].
• the aqueous medium further comprises at least one substance selected from the group consisting of polyaone, bile acid derivatives, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine and polyoxyethylenealkenylamine.
• Bile acid derivative power Cholic acid or a salt thereof, taurocholic acid or a salt thereof, glycocholic acid or a salt thereof, lithocholic acid or a salt thereof, deoxycholic acid or a salt thereof, chenodeoxycholic acid or a salt thereof , Ursodeoxycholic acid or a salt thereof, 7-oxolithocholic acid or a salt thereof, 12-oxolithocholic acid or a salt thereof, 12-oxokenodeoxycholic acid or a salt thereof, 7-oxodoxychol Acid or a salt thereof, hoocholic acid or a salt thereof, hydroxycholic acid or a salt thereof, dehydrocholic acid or a salt thereof, general formula (I)
• R 1 is a 3- (3-cholamidopropyl) dimethylammo group
• R 2 is a hydrogen atom or a hydroxyl group
• a reagent for measuring cholesterol in high-density lipoprotein characterized by containing at least one substance selected from the group consisting of pyrene diamine derivatives, cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme.
• poly-ones, bile acid derivatives, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamines and polyoxyethylenealkenylamines comprising at least one substance selected from the group consisting of:
• the bile acid derivative is cholic acid or a salt thereof, taurocholic acid or a salt thereof, glycocholic acid or a salt thereof, lithocholic acid or a salt thereof, deoxycholic acid or a salt thereof, chenodeoxycholic acid or a salt thereof , Ursodeoxycholic acid or a salt thereof, 7-oxolithocholic acid or a salt thereof, 12-oxolithocholic acid or a salt thereof, 12-oxochenodeoxycholic acid or a salt thereof, 7-oxodoxychol Acid or a salt thereof, insectolic acid or a salt thereof, hydroxycholic acid or a salt thereof, dehydrocholic acid or a salt thereof, and a compound represented by the general formula (I)
• R 1 is a 3- (3-cholamidopropyl) dimethylammo group
• R 2 is a hydrogen atom or a hydroxyl group
• a cholesterol ester in a high-density lipoprotein characterized by containing at least one substance selected from the group and cholesterol ester hydrolase in one or both of the first reagent and the second reagent.
• kits comprising a first reagent and a second reagent, wherein the kit comprises an oxidized coenzyme in the first reagent, a cholesterol dehydrogenase in the second reagent, and an alkylamine polyoxygen.
• kits for measuring cholesterol in high-density lipoprotein comprising at least one substance and a cholesterol ester hydrolase in one or both of a first reagent and a second reagent.
• kit according to [14] further comprising a reduced coenzyme measurement reagent in one or both of the first reagent and the second reagent.
• At least one substance selected from the group consisting of polyaone, bile acid derivative, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine and polyoxyethylenealkenylamine is also used as a first reagent, a second reagent.
• the bile acid derivative is cholic acid or a salt thereof, taurocholic acid or a salt thereof, glycocholic acid or a salt thereof, lithocholic acid or a salt thereof, deoxycholic acid or a salt thereof, chenodeoxycholic acid or a salt thereof , Ursodeoxycholic acid or a salt thereof, 7-oxolithocholic acid or a salt thereof, 12-oxolithocholic acid or a salt thereof, 12-oxochenodeoxycholic acid or a salt thereof, 7-oxodoxychol Acid or a salt thereof, insectolic acid or a salt thereof, hydroxycholic acid or a salt thereof, dehydrocholic acid or a salt thereof, and a compound represented by the general formula (I) [0027] [Formula 5]
• R 1 is a 3- (3-cholamidopropyl) dimethylammo group, and R 2 is a hydrogen atom or a hydroxyl group
• kits [0030] (X represents a hydrogen atom or a hydroxyl group, and R 3 and R 4 are the same or different and represent a substituted or unsubstituted alkyl group or a substituted or unsubstituted alkanol group.)
• kit according to any one of [16] to [18], further comprising albumin in one or both of the first reagent and the second reagent.
• 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 of the present invention is a method for measuring HDL cholesterol without eliminating cholesterol in lipoproteins other than HDL.
• specimen used in the measurement method of the present invention are preferably plasma and serum, including whole blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, spleen, and the like.
• the cholesterol esterase hydrolyzing enzyme in the present invention is not particularly limited as long as it is an enzyme capable of hydrolyzing cholesterol ester, such as cholesterol esterase and lipoprotein lipase derived from animals, plants or microorganisms. , Heredity Cholesterol esterase, lipoprotein lipase, etc., produced by an elementary engineering technique can also be used.
• cholesterol esterase hydrolase an unmodified cholesterol esterase or a chemically modified cholesterol esterase can be used. Also, it is better to use a commercially available cholesterol ester hydrolase.
• cholesterol esterase hydrolyzing enzymes include cholesterol esterase "Amano” 2 (CHE2; manufactured by Amano Enzym), cholesterol esterase "Amano” 3 (CHE3; manufactured by Amano Enzym), lipoprotein lipase ( LPL311; manufactured by Toyobo Co., Ltd.), lipoprotein lipase "Amano” 6 (LPL6; manufactured by Amano Enzym), 43 kDa esterase (manufactured by Amano Enzym), cholesterol esterase [COE313 (chemically modified cholesterol esterase); Company-made].
• two or more cholesterol esterases can be used in combination.
• Examples of the (chemically modifying group) include a group mainly composed of polyethylene glycol, a group mainly composed of polypropylene glycol, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group containing a water-soluble polysaccharide, Examples of the group include a sulfopropyl group, a sulfobutyl group, a polyurethane group, and a group having a chelating function. Examples of the water-soluble polysaccharide include dextran, pullulan, and soluble starch.
• the reagent (chemical modifying agent) for modifying the cholesterol ester hydrolase in a diagonal manner includes a functional group capable of reacting with the above-mentioned chemical modifying group and an amino group, a carboxyl group, a sulfhydryl group or the like of the enzyme.
• a functional group capable of reacting with the above-mentioned chemical modifying group and an amino group, a carboxyl group, a sulfhydryl group or the like of the enzyme Compounds having both a group and a structure are exemplified.
• the functional group or structure capable of reacting with an amino group in the enzyme include a carboxyl group, an active ester group (such as an N-hydroxysuccinimide group), an acid anhydride, an acid chloride, an aldehyde, an epoxide group, And propane sultone and 1,4-butane sultone.
• Examples of the functional group or structure capable of reacting with the carboxyl group in the enzyme include an amino group.
• Examples of the group or structure reactive with the sulfhydryl group in the enzyme include a maleimide group, a disulfide, an a-haloester (such as ⁇ - ode ester), and the like.
• Chemical modifiers include Sunbright VFM-4101, Sunbright ME-050AS, and Sunbright DE-030AS (each having a group mainly composed of polyethylene glycol and an N-hydroxysuccinimide group).
• Sunbright AKM series having a group consisting mainly of polyalkylene glycol and an acid anhydride structure (eg, Sunbright AKM-1510, etc.), Sunbright ADM series, and Sunbright ACM series (all EPOX-3400 and M-EPOX-5000 (manufactured by Sheawater Polymers) having a group mainly composed of polyethylene glycol and an epoxide group, and a group having a chelating function and an acid anhydride structure.
• Diethylenetriamine-N, N, ⁇ ', ⁇ ", ⁇ " pentaacetic anhydride (DTPA anhydride; manufactured by Dojin Chemical Co., Ltd.).
• the chemical modification of the cholesterol esterase is not limited to the method described below, which can be performed, for example, by the following method.
• a buffer having a pH of 8.0 or more eg, HEPES buffer
• a chemical modifier of 0.01 to 500 times the molar amount is added at 0 to 55 ° C. Stir for minutes to 5 hours.
• the reaction solution itself not only the reaction solution itself but also the unreacted chemical modifier etc. were removed by ultrafiltration membrane as needed, as a chemically modified cholesterol esterase.
• the concentration of cholesterol ester hydrolase used in the reaction method is not particularly limited as long as the concentration of the HDL cholesterol of the present invention can be measured, but is 0.01 to 400 UZmL in the reaction solution.
• the concentration is more preferably 0.02 to 200 U / mL.
• the cholesterol oxidase according to the present invention is not particularly limited as long as it is an enzyme capable of oxidizing cholesterol to generate hydrogen peroxide, for example, cholesterol oxidase derived from animals, plants or microorganisms.
• cholesterol oxidase and the like produced by genetic engineering techniques can also be used.
• Cholesterol oxidase "Amano"(CHOD1; manufactured by Amano Enzym), cholesterol oxidase (CO — PE; commercially available products such as Kikkoman Co., Ltd., cholesterol oxidase (C00321; Toyobo Co., Ltd.), cholesterol oxidase Kyowa (Kyowa Hakko Co., Ltd.) and the like can also be used.
• the cholesterol oxidase may be an unmodified enzyme or a chemically modified enzyme.
• the chemically modified cholesterol oxidase can be produced, for example, by using the above-mentioned chemical modifier and the above-mentioned chemical modification method.
• the concentration of the cholesterol oxidase used in the reaction method is not particularly limited as long as the concentration enables measurement of the HDL cholesterol of the present invention, but it is 0.01 to 400 UZmL in the reaction solution.
• the concentration is preferably 0.02 to 200 UZmL, more preferably the concentration.
• the cholesterol dehydrogenase in the present invention is not particularly limited as long as it is an enzyme capable of oxidizing cholesterol in the presence of an oxidized coenzyme to generate a reduced coenzyme.
• cholesterol dehydrogenase produced by a genetic engineering technique and the like can be used.
• Commercial products such as cholesterol dehydrogenase "Amano" 5 (CHDH5; manufactured by Amano Enzym) can also be used.
• two or more cholesterol dehydrogenases can be used in combination.
• Cholesterol dehydrogenase may be an unmodified enzyme or a chemically modified enzyme.
• a chemically modified cholesterol dehydrogenase can be produced, for example, by using the above-mentioned chemical modifier and the above-mentioned chemical modification method.
• the concentration of cholesterol dehydrogenase used in the reaction method is not particularly limited as long as the concentration of HDL cholesterol of the present invention can be measured.
• the concentration is preferably 400 UZmL, more preferably 0.02 to 200 UZmL.
• an oxidized coenzyme is used.
• the oxidized coenzyme include NAD, NADP, thio-NAD, thio-NADP and the like.
• the amine oxide and alkylpropylene diamine derivatives are not particularly limited as long as the HDL cholesterol of the present invention can be measured.
• alkylamine oxide examples include alkyldimethylamine oxide, dihydroxyethylalkylamine oxide, polyoxyethylene dimethylamine oxide, and the like.
• alkyl propylene diamine derivative examples include alkyl propylene diamine, polyoxyethylene alkyl propylene diamine, and the like.
• alkyl in alkylamine polyoxyethylene polyoxypropylene condensate polyoxyethylene alkylamine sulfate, polyoxyethylene benzyl-alkyl quaternary ammonium salt, polyoxyethylene amide, alkylamine oxide and alkylpropylene diamine derivative Is a straight or branched chain having 6 to 30 carbon atoms such as hexyl, heptyl, octyl, isootatyl, nonyl, decyl, pendecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, Xadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, henecosyl, docosyl (behyl), tricosyl, tetracosyl, pentacosyl,
• the degree of polymerization of the oxyethylene chain in oxyethylenealkylpropylenediamine is preferably from 1 to 100, more preferably from 1 to 50.
• alkylamine polyoxyethylene polyoxypropylene condensate examples include BLAUNON SAP3004 and BLAUNON SAP3010 (tallow amine polyoxyethylene polyoxypropylene condensate; both manufactured by Aoki Yushi Co., Ltd.) C16EO30P
• polyoxyethylene alkylamine sulfate examples include, for example, Levelon A-625X (manufactured by Fukusha Yushi Co., Ltd.), Mignol PA-30 (manufactured by Fukusha Yushi Co., Ltd.), and PE—61 (manufactured by Sanyo Chemical Industries) and the like.
• polyoxyethylenebenzyl-alkyl quaternary ammonium salt examples include Bisnol SK (manufactured by Ogata Yushi Co., Ltd.).
• Specific examples (products) of polyoxyethylene acid amide include, for example, -kkol TAMDS15 (manufactured by Nikko Chemicals Co., Ltd.) and Nimit MT-215 (manufactured by NOF Corporation).
• Specific examples (products) of alkylamine oxides include, for example, u-safe A-LE (dihydroxyxyl lauryl amine oxide; manufactured by NOF Corporation) and u-safe A-LM (alkyl dimethylamine oxide; Japan).
• Y-Safe A-LY polyoxyethylene cache oil alkyldimethylamine oxide; manufactured by NOF Corporation) and the like.
• alkylpropylenediamine derivative examples include, for example, BLAUNON
• BLAUNON DTI 5 alkyl propylene diamine; both manufactured by Aoki Oil & Fat Co., Ltd.
• Asphazol # 10 polyoxyethylene tallow propylene diamine; manufactured by Nippon Oil & Fat Co., Ltd.
• alkylamine polyoxyethylene polyoxypropylene condensate polyoxyethylene alkylamine sulfate, polyoxyethylene benzylalkyl quaternary ammonium salt, polyoxyethylene acid amide, alkylamine oxide
• at least one substance selected from the group consisting of an alkyl propylene diamine derivative and an alkyl propylene diamine derivative can be used in any combination.
• alkylamine polyoxyethylene polyoxypropylene condensate polyoxyethylene alkylamine sulfate, polyoxyethylene Group ethylene benzyl-alkyl quaternary ammonium salt, polyoxyethylene amide, alkylamine oxide and alkyl propylene diamine derivative power
• concentration of at least one selected substance may be There is no particular limitation as long as the concentration enables measurement of HDL cholesterol, but the concentration in the reaction solution is preferably 0.0001 to 1%, more preferably 0.001 to 0.1%. ⁇ .
• the polyaone used in the present invention is not particularly limited as long as it is a polyadion capable of measuring HDL cholesterol of the present invention, for example, dextran sulfate or a salt thereof, henone or a derivative thereof.
• dextran sulfate or a salt thereof including salts, phosphotungstic acid or a salt thereof, sulfated cyclodextrin or a salt thereof, sulfated oligosaccharide or a salt thereof, and the like.
• Examples of the dextran sulfate include dextran sulfate having a molecular weight of 40,000, 80,000, 200,000, 500,000, 1,000,000, 2,000,000, or the like.
• sulfated oligosaccharide examples include sulfated garagarose, sulfated trehalose, and chondroitin sulfate.
• the salt examples include a sodium salt, a potassium salt, a lithium salt, an ammonium salt, a magnesium salt and the like.
• the concentration of poly-one in the measurement of HDL cholesterol of the present invention 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 preferably 0.001 to 10%. % Is preferred 0.01-1% is more preferred.
• the bile acid derivative of the present invention is not particularly limited as long as it is a bile acid derivative of the present invention that enables measurement of HDL cholesterol.
• a bile acid derivative having an anionic surfactant activity examples include bile acid derivatives having an amphoteric surfactant, bile acid derivatives having a nonionic surfactant, and the like.
• Bile acid derivatives having an anionic surfactant include, for example, cholic acid or a salt thereof, taurocholic acid or a salt thereof, glycocholic acid or a salt thereof, lithocholic acid or a salt thereof, dexocholic acid or a salt thereof, and kenode.
• Oxocholic acid or a salt thereof ursodeoxycholic acid or a salt thereof, 7-oxolithocholic acid or a salt thereof, 12 oxolithocholic acid or a salt thereof, 12-oxokenedoxycholic acid or a salt thereof, 7 —Oxodoxycholic acid or its salt, hyocholic acid or its salt, Oxycholic acid or a salt thereof, dehydrocholic acid or a salt thereof, and the like.
• the salt include an ammonium salt, a lithium salt, a sodium salt, a potassium salt, a magnesium salt, a calcium salt and the like.
• concentration of the bile acid derivative having an anionic surfactant activity 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 0.001 to 10%. It is preferably 0.01-1%.
• the bile acid derivative having an amphoteric surfactant has, for example, the general formula (I)
• R 1 is a 3- (3-cholamidopropyl) dimethylammo group, and R 2 is a hydrogen atom or a hydroxyl group] I)].
• the compound (I) in which R 2 is a hydrogen atom is referred to as CHAPS
• the compound (I) in which R 2 is a hydroxyl group is referred to as CHAPSO.
• the concentration of the bile acid derivative having an amphoteric surfactant effect 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 0.001 to 10%. It is preferably 0.01-1%.
• the bile acid derivative having a nonionic surfactant has, for example, the general formula (II)
• Alkyl and alkyl in alkanoyl include linear or branched carbon atoms having 1 to: LO such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and pentyl. , Neopentyl, hexyl, heptyl, octyl, noel, decyl and the like.
• substituent in the substituted alkyl and the substituted alkanoyl include a hydroxyl group and a halogen atom.
• Halogen atom means each atom of fluorine, chlorine, bromine and iodine.
• R 3 and R 4 are both
• substituent A Compounds (hereinafter, referred to as substituent A) are preferred.
• a compound in which X, R 3 and R 4 are each a hydrogen atom, a substituent A and a substituent A are referred to as deoxy-BIGCHAP
• a compound in which X, R 3 and R 4 are a hydroxyl group, a substituent A and a substituent A are referred to as BIGCHAP.
• the concentration of the bile acid derivative having a nonionic surfactant 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 0.001 to 10%. It is more preferable that 0.01-1% is more preferable.
• two or more bile acid derivatives may be used.
• Examples of the ethylenediaminetetrapolyoxyalkylene include ethylenediaminepolyoxypropylenepolyoxyethylene condensate, ethylenediaminetetrapolyoxyethylene, and ethylenediaminepolyoxypropylene.
• ethylenediaminetetrapolyoxyalkylene examples include, for example, Adecapul Kick TR704, Adecapul Kick TR701, and Adecapul Kick TR913R (Ethylenediamine polyoxyethylene polyoxypropylene condensate; Dulusha), Loop 32T Y-65BI (ethylenediaminetetrapolyoxyalkylene; manufactured by NOF Corporation), ethylenediamine PO52EO60 (total degree of polymerization of oxyethylene chain: 60; total degree of polymerization of oxypropylene chain: 52) ; Manufactured by NOF CORPORATION).
• Adecapul Kick TR704, Adecapul Kick TR701, and Adecapul Kick TR913R Ethylenediamine polyoxyethylene polyoxypropylene condensate; Dulusha
• Loop 32T Y-65BI ethylenediaminetetrapolyoxyalkylene; manufactured by NOF Corporation
• ethylenediamine PO52EO60 total degree of polymerization
• the polymerization number of ethylenediaminetetrapolyoxyalkylene is preferably 1 to 100, more preferably 1 to 60. In the present invention, two or more ethylenediaminetetrapolyoxyalkylenes may be used.
• the concentration of ethylenediaminetetrapolyoxyalkylene 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 0.001 to 10%. It is preferably 0.01-1%.
• Examples of the polyoxyethylene alkylamine and polyoxyethylenealkamine include compounds represented by the general formula (III) [0061]
• R represents a linear or branched alkyl group or an alkyl group
• X represents a hydrogen atom or (CH 2 CH 2 O) H
• m and n are the same or different, and 1 Integer of ⁇ 100
• alkyl is a straight-chain or branched carbon.
• alkyl is a straight-chain or branched carbon.
• polyoxyethylene alkylamine or polyoxyethylenealkenylamine include, for example, Nymin L201 (oxyethylene dodecylamine; manufactured by NOF Corporation) and Nymin L207 (polyoxyethylene dodecyl).
• the degree of polymerization of the oxyshethylene chain in polyoxyethylene alkylamine and polyoxyethylene alkyleneamine is preferably 1 to: LOO, more preferably 1 to 60.
• two or more kinds of polyoxyethylene alkylamine and polyoxyethylene alkyleneamine may be used.
• the concentration of polyoxyethylene alkylamine and polyoxyethylene alkyleneamine 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 % and it is good preferred, from 0.001 to 0.1 0/0 power preferable than S ⁇ .
• 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, and examples include albumin, poma, hidge, and albumin derived from human. Potassium serum albumin (BSA) is preferred. Albumin produced by a genetic engineering technique can also be used. In the present invention, two or more kinds of albumins 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%. 0.01 to 1% is more preferable.
• the aqueous medium used in the HDL cholesterol measurement method of the present invention is preferably a force buffer such as deionized water, distilled water, a buffer and the like.
• a force buffer such as deionized water, distilled water, a buffer and the like.
• the buffer used for the buffer include tris (hydroxymethyl) aminomethane buffer, phosphate buffer, borate buffer, Good's buffer and the like.
• Good's buffer examples include 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetoamide) iminoniacetic acid (ADA) , Piperazine-N, N, monobis (2-ethanesulfonic acid) (PIPES), N- (2-acetoamide) 2-aminoethanesulfonic acid (ACES), 3 morpholino- 1-hydroxypropanesulfonic acid (MOPSO), N, N bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] 1-2 aminoethanesulfonic acid (TES ), 2- [4- (2-hydroxyethyl) -1-piperazyl] ethanesulfonic acid (HEPES), 3- [N, N bis (2-hydroxye
• Examples of the method for measuring HDL cholesterol of the present invention include the following embodiments.
• a sample and cholesterol esterase and cholesterol oxidase, or cholesterol esterase, oxidise type coenzyme, and cholesterol dehydrogenase are reacted with alkylamine polyoxyethylene polyoxypropylene.
• the HDL cholesterol in the 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 (1) is carried out, for example, at 10 to 50 ° C., preferably 20 to 40 ° C., for 1 to 60 minutes, preferably 2 to 30 minutes.
• the amount of generated hydrogen peroxide can be measured, for example, using a reagent for measuring hydrogen peroxide.
• the reagent for measuring hydrogen peroxide is a reagent for converting the generated hydrogen peroxide into a detectable substance.
• a detectable substance a power dye such as a dye or luminescence is preferable.
• the detectable substance is a dye
• the reagent for measuring hydrogen peroxide contains a peroxidase active substance such as a peroxidase such as a peroxidase.
• the oxidative color-forming chromogen include the acid chromogenic chromogen described below.
• the reagent for measuring hydrogen peroxide contains a chemiluminescent substance.
• the chemiluminescent substance include luminol, isoluminol, lucigenin, atalidium-dumester and the like.
• a reagent containing an oxidative coloring type chromogen and a peroxide active substance such as peroxidase is used as a reagent for measuring hydrogen peroxide
• the hydrogen peroxide is oxidized in the presence of the active peroxide substance.
• the coloring type chromogen By reacting with the coloring type chromogen to generate a dye and quantifying the generated dye, hydrogen peroxide can be quantified.
• the hydrogen peroxide reacts with the chemiluminescent substance to generate photons, and the generated photons are quantified to determine the amount of the photons. It is possible to quantitatively determine the amount of hydrogen.
• Examples of the oxidative coloring type chromogen include a leuco type chromogen, an oxidative coupling coloring type chromogen, and the like.
• a leuco-type chromogen is a substance that is converted into a dye alone in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase.
• CCAP 10-N-carboxymethylcarbamoyl-3,7bis (dimethylamino) 10H phenothiazine
• MCDP 10-N-methylcarbamoyl-3,7bis (dimethylamino) -10H-phenothiazine
• DA-64 4,4,1-bis (dimethylamino) diphenamine
• BCMA bis [3- Bis (4-chlorophenol) methyl 4-dimethylaminophen Enyl] amine
• the oxidative coupling color-forming chromogen is a substance that produces a dye by oxidative coupling of two compounds in the presence of a peroxidation active substance such as hydrogen peroxide and peroxidase.
• a peroxidation active substance such as hydrogen peroxide and peroxidase.
• the combination of the two compounds include a combination of a coupler and an arrin, a combination of a coupler and a phenol, and the like.
• the coupler include 4-aminoantipyrine (4AA), 3-methyl-2-benzothiazolinone hydrazine and the like.
• the concentration of the peroxide is not particularly limited as long as the concentration is suitable for the measurement.
• peroxidase is used as the peroxide.
• 1 to: LOOkUZL is preferred.
• 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 the reduced coenzyme include a method for measuring the absorbance of the generated reduced coenzyme, a method using a reagent for measuring the reduced coenzyme, and the like.
• the absorbance in the method for measuring the absorbance of the reduced coenzyme is preferably around 300 to 500 nm, more preferably 330 to 400 nm, and more preferably around 340 nm.
• the reduced coenzyme measurement reagent is a reagent for converting the generated reduced coenzyme into a detectable substance.
• the detectable substance include a dye.
• examples of the reagent for measuring a reduced coenzyme include a reagent containing diaphorase, an electron carrier, and a reduced chromogen.
• the electron carrier include 1-methoxy-5-methylphenazine methyl sulfate and the like.
• the dye formed by converting the reduced chromogenic chromogen is quantified to determine The reduced coenzyme can be quantified.
• Examples of the reduced chromogenic type chromogen include 3- (4,5 dimethyl-2 thiazolyl) 2,5-diphenyl-1 2H-tetrazolium bromide (MTT) (2- (4-odophenol). 3) (4-Trophenyl) 5— (2,4 disulfophenol) 2H—tetrazolium mononatrium salt (WST—1), 2— (4 phenol) — 3— (2,4) Dinitrophenyl) -5- (2,4 disulfophenol) 2H-tetrazolium monosodium salt (WST-3).
• the reagent for measuring HDL cholesterol of the present invention includes alkylamine polyoxyethylene polyoxypropylene condensate, polyoxyethylene alkylamine sulfate, polyoxyethylene benzyl-alkyl quaternary ammonium salt, polyoxyethylene amide, It contains at least one substance selected from the group consisting of alkylamine oxide and alkyl propylene diamine derivatives, cholesterol esterase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
• -One a bile acid derivative, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine and at least one substance selected from the group consisting of polyoxyethylenealkenylamines.
• the reagent for measuring HDL cholesterol of the present invention includes alkylamine polyoxyethylene polyoxypropylene condensate, polyoxyethylene alkylamine sulfate, polyoxyethylene benzyl-alkyl quaternary ammonium salt, polyoxyethylene.
• Examples of the reagent for measuring HDL cholesterol of the present invention include the following reagents, but these do not limit the scope of the present invention at all.
• alkylamine polyoxyethylene polyoxypropylene condensate polyoxyethylene alkylamine sulfate, polyoxyethylene benzyl-alkyl quaternary ammonium salt, polyoxyethylene amide, alkylamine oxide, and alkylamine pyrylenediamine derivative
• I-Dragon A At least one substance to be selected is hereinafter referred to as "I-Dragon A”.
• At least one substance selected from the group consisting of polyaone, bile acid derivatives, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine and polyoxyethylenealkenylamine is referred to as "di-animate compound" below.
• B a substance selected from the group consisting of polyaone, bile acid derivatives, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine and polyoxyethylenealkenylamine.
• a reagent containing Compound A, cholesterol esterase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide is a reagent containing Compound A, cholesterol esterase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
• a reagent containing Compound A, Compound B, cholesterol esterase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide is a reagent containing Compound A, Compound B, cholesterol esterase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
• Reagent 3 A reagent containing Compound A, Compound B, albumin, cholesterol esterase hydrolase, cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
• Reagent containing compound A, cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme Reagent containing compound A, cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme.
• a reagent containing Compound A, Compound B, cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme A reagent containing Compound A, Compound B, cholesterol esterase, cholesterol dehydrogenase and oxidized coenzyme.
• a reagent containing compound A, compound B, albumin, cholesterol esterase hydrolase, cholesterol dehydrogenase, and oxidized coenzyme is a reagent containing compound A, compound B, albumin, cholesterol esterase hydrolase, cholesterol dehydrogenase, and oxidized coenzyme.
• a reagent containing compound A, cholesterol esterase, cholesterol dehydrogenase, oxidized coenzyme and reduced coenzyme is provided.
• a reagent containing Compound A, Compound B, cholesterol esterase, cholesterol dehydrogenase, oxidized coenzyme 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 any of a two-reagent system and a three-reagent system, but a two-reagent system is preferred.
• a two-reagent HDL cholesterol measurement kit comprising a first reagent and a second reagent
• cholesterol esterase hydrolase and cholesterol oxidase or cholesterol dehydrogenase are composed of the first reagent and the second reagent. It may be contained separately in the reagents or together in the second reagent, but when it is contained separately in the first and second reagents, cholesterol ester hydrolase is included in the first reagent. An embodiment in which cholesterol oxidase or cholesterol dehydrogenase is contained in the second reagent is preferred.
• the bile acid derivative, ethylenediaminetetrapolyoxyalkylene, polyoxyethylenealkylamine, polyoxyethylenealkenylamine and albumin may be contained in one or both of the first reagent and the second reagent.
• the oxidized coenzyme used in the assay using cholesterol dehydrogenase may be contained in either or both of the first reagent and the second reagent.
• the reagent for measuring hydrogen peroxide may be contained in either or both of the first reagent and the second reagent. However, when the reagent contains an oxidative coupling type chromogen, the reagent may be oxidized. Preferably, each compound of the coupling type chromogen is contained in a separate reagent.
• the reducing coenzyme measurement 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 of the kit for measuring HDL cholesterol of the present invention include the kits of the following embodiments, but these do not limit the scope of the present invention at all.
• Cholesterol acid enzyme reagent for measuring hydrogen peroxide, compound A, compound B
• Cholesterol acid enzyme reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Second reagent Cholesterol acid enzyme reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Cholesterol acid enzyme reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Cholesterol ester hydrolase reagent for measuring hydrogen peroxide, compound B, albumin
• Cholesterol ester hydrolase reagent for measuring hydrogen peroxide, compound A, compound Thing B, albumin
• Cholesterol ester hydrolase reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Cholesterol acid enzyme reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Cholesterol ester hydrolase reagent for measuring hydrogen peroxide, compound B, albumin, compound A
• Cholesterol ester hydrolase reagent for measuring hydrogen peroxide, compound A, compound B, albumin
• Kit 57 First reagent
• Cholesterol esterase cholesterol acid enzyme, reagent for measuring hydrogen peroxide, compound B, albumin
• Ki 1 First reagent
• Oxidized coenzyme compound A, compound B, albumin
• Oxidized coenzyme compound A, compound B, albumin
• Oxidized coenzyme compound A, compound B, albumin
• kit 101 First reagent
• Oxidized coenzyme compound A, compound B, albumin
• Oxidized coenzyme cholesterol esterase, compound A, compound B, albumin
• Oxidized coenzyme cholesterol esterase, compound A, compound B, albumin
• Oxidized coenzyme cholesterol esterase, compound A, compound B, albumin
• Oxidized coenzyme cholesterol esterase, compound A, compound B, albumin
• Reagents for measuring oxidized coenzyme, cholesterol esterase, and reduced coenzyme Compound B, albumin
• Oxidized coenzyme cholesterol dehydrogenase

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