WO2011136316A1 - Method for measuring low-density lipoprotein cholesterol, measurement reagent and measurement kit - Google Patents

Method for measuring low-density lipoprotein cholesterol, measurement reagent and measurement kit Download PDF

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WO2011136316A1
WO2011136316A1 PCT/JP2011/060352 JP2011060352W WO2011136316A1 WO 2011136316 A1 WO2011136316 A1 WO 2011136316A1 JP 2011060352 W JP2011060352 W JP 2011060352W WO 2011136316 A1 WO2011136316 A1 WO 2011136316A1
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reagent
polyoxyethylene
cholesterol
concentration
ldl
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PCT/JP2011/060352
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French (fr)
Japanese (ja)
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英之 桑田
知子 荒武
健太 金城
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協和メデックス株式会社
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Priority to JP2012512903A priority Critical patent/JP5832995B2/en
Publication of WO2011136316A1 publication Critical patent/WO2011136316A1/en

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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
    • 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
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the present invention relates to a method for measuring cholesterol (hereinafter referred to as LDL-C) in a low density lipoprotein (hereinafter referred to as LDL) contained in a specimen, a reagent for measurement, and a kit for measurement.
  • LDL-C cholesterol-lowering lipoprotein
  • LDL has a role of supplying cholesterol to peripheral cells, and is a direct factor of various arteriosclerosis including coronary arteriosclerosis.
  • An increase in LDL-C is one of the major risk factors for arteriosclerotic diseases, and differential quantification of LDL-C is clinically useful.
  • Examples of conventional LDL-C quantification methods include ultracentrifugation, electrophoresis, and calculation methods based on the Friedewald equation.
  • the ultracentrifugation method is a method of measuring the amount of cholesterol after separating LDL using an ultracentrifuge using the difference in specific gravity of lipoprotein (Non-patent Document 1).
  • the operation is complicated and has drawbacks in terms of speed and simplicity.
  • Examples of the electrophoresis method include a method of separating agarose gel and the like using a difference in charge of lipoprotein and a method of separating a polyacrylamide gel and using a difference in particle size of lipoprotein.
  • the electrophoresis method has poor quantitativeness and has problems in terms of convenience and economy.
  • TC total cholesterol
  • HDL-C cholesterol
  • HDL high-density lipoprotein
  • T-TG total triglyceride
  • LDL-C in a sample comprising a first step of eliminating cholesterol in HDL, very low density lipoprotein (hereinafter referred to as VLDL) and chylomicron, and then a second step of quantifying residual cholesterol in the sample Quantification method (Patent Document 1).
  • a surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether and an enzyme for measuring cholesterol are added to serum to preferentially react cholesterol in HDL and VLDL among lipoproteins. And then determining the reaction amount of the remaining cholesterol, LDL-C quantification method (Patent Document 2).
  • a method of selectively measuring LDL-C among lipoproteins by adding a polyoxyethylene derivative, a polyoxyethylene-polyoxypropylene copolymer, and a cholesterol measuring enzyme to a sample (Patent Document 3).
  • LpX is an abnormal lipoprotein that has the same specific gravity as LDL that appears during cholestasis and does not exist at all in healthy people, and its chemical composition is almost free cholesterol, which is one of the constituent components, It does not contain apo B and is very different from normal LDL. Therefore, it is considered that cholesterol in LpX, which is an abnormal lipoprotein, should not be measured (Non-patent Document 3).
  • An object of the present invention is to provide a method, a reagent, and a kit for simply and accurately measuring LDL-C in a specimen.
  • the present inventors have determined that polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, and If necessary, the sample is reacted with cholesterol esterase and cholesterol oxidase in the presence of at least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfates and arylsulfonic acid derivatives.
  • the present invention relates to the following (1) to (22).
  • Specimen, cholesterol esterase and cholesterol oxidase in the presence of polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion A method for measuring cholesterol in a low-density lipoprotein in a specimen, which comprises reacting and measuring hydrogen peroxide produced by the reaction.
  • the concentration of cholesterol oxidase in the reaction solution is 0.25 to 1.0 kU / L, and the concentration of polyoxyethylene dodecylamine is 0.06 to 0.3 mmol / L. .
  • the concentration of cholesterol oxidase in the second reagent is 2.0 to 8.0 kU / L, and the concentration of polyoxyethylene octadecylamine in the second reagent is 0.075 to 0.3 mmol / L.
  • the kit according to the description (21) Further, at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate salt and an aryl sulfonic acid derivative is contained in either or both of the first reagent and the second reagent, (15) to (15) 20) The kit according to any one of the above. (22) The kit according to any one of (15) to (21), further comprising a reagent for measuring hydrogen peroxide in one or both of the first reagent and the second reagent.
  • the present invention provides a method, a reagent, and a kit for measuring LDL-C in a sample simply and accurately.
  • the gel filtration chromatogram of a LpX specimen is shown.
  • the horizontal axis represents the fraction number, and the vertical axis represents the measured value of cholesterol concentration.
  • ⁇ is total cholesterol (TC) concentration
  • ⁇ is free cholesterol (FC) concentration
  • * is cholesterol concentration measured using the kit of Example 5
  • ⁇ is kit of Example 6. Represents the cholesterol concentration in the measurement used.
  • the method for measuring LDL-C in a specimen according to the present invention is a method that does not require a lipoprotein fractionation operation by a physical method such as centrifugation.
  • LDL-C in a sample is measured without eliminating cholesterol in lipoproteins other than LDL in the sample.
  • this is a method for measuring LDL-C in a sample without measuring cholesterol in lipoproteins other than LDL in the sample.
  • the measurement method of the present invention comprises a sample, cholesterol esterase and cholesterol oxidase, polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion. And hydrogen peroxide produced by the reaction is measured.
  • the measurement method of the present invention comprises a sample, cholesterol ester hydrolase and cholesterol oxidase, polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine. And reacting in the presence of at least one substance selected from the group consisting of a polyanion, a polyoxyethylene polycyclic ether sulfate ester salt and an aryl sulfonic acid derivative, and measuring hydrogen peroxide produced by the reaction It is a method.
  • the measurement method of the present invention it is possible to accurately measure LDL-C without measuring cholesterol in LpX, which is an abnormal lipoprotein.
  • the measurement method of the present invention includes: [1] Presence of polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion by reacting the sample with cholesterol ester hydrolase and cholesterol oxidase The steps performed below; [2] A step of measuring hydrogen peroxide generated in the step [1]; [3] A calibration curve prepared in advance using a known concentration of LDL-C and representing the relationship between the LDL-C concentration and the amount of information derived from the hydrogen peroxide, and the measurement value in [2] above Correlating; and [4] A step of determining the LDL-C concentration in the specimen.
  • the reaction [1] can also be carried out in the presence of at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate ester salt and an arylsulfonic acid derivative. That is, the reaction of the specimen with cholesterol ester hydrolase and cholesterol oxidase is performed by polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, and It can also be performed in the presence of at least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfates and arylsulfonic acid derivatives.
  • the steps [1] and [2] are preferably performed in an aqueous medium.
  • an aqueous medium the below-mentioned aqueous medium etc. are mentioned, for example.
  • hydrogen peroxide produced by the reaction of the sample with cholesterol ester hydrolase and cholesterol oxidase is measured using, for example, a hydrogen peroxide electrode or a reagent for measuring hydrogen peroxide described below. Can do.
  • Specimens used in the measurement method of the present invention include, for example, whole blood, plasma, serum and the like, and plasma and serum are preferable.
  • the cholesterol ester hydrolase in the present invention is not particularly limited as long as it has an ability to hydrolyze cholesterol ester.
  • cholesterol esterase derived from animals, plants or microorganisms, lipoprotein lipase, genetic engineering Cholesterol esterase, lipoprotein lipase and the like produced by the technique can also be used.
  • cholesterol ester hydrolase chemically modified cholesterol ester hydrolase can be used in addition to unmodified cholesterol ester hydrolase. Moreover, a commercial item can also be used as a cholesterol ester hydrolase.
  • cholesterol ester hydrolases As commercially available cholesterol ester hydrolases, cholesterol esterase (COE-311; manufactured by Toyobo Co., Ltd.), lipoprotein lipase (LPL-311; manufactured by Toyobo Co., Ltd.), cholesterol esterase III (CHEIII; manufactured by Amano Pharmaceutical Co., Ltd.) Etc.
  • COE-311 cholesterol esterase (COE-311; manufactured by Toyobo Co., Ltd.)
  • LPL-311 lipoprotein lipase
  • CHEIII cholesterol esterase III
  • two or more kinds of cholesterol ester hydrolases can be used in combination.
  • Examples of a group (chemical modification group) that modifies the enzyme in chemical modification of cholesterol ester hydrolase include, for example, a group mainly composed of polyethylene glycol, a group mainly composed of polypropylene glycol, and a co-polymerization of polypropylene glycol and polyethylene glycol.
  • Examples include a group having a combination, a group containing a water-soluble polysaccharide, a sulfopropyl group, a sulfobutyl group, a polyurethane group, a group having a chelating function, and the like, and a group having polyethylene glycol as a main component is preferable.
  • Examples of the water-soluble polysaccharide include dextran, pullulan, and soluble starch.
  • a reagent (chemical modifier) for chemically modifying cholesterol ester hydrolase As a reagent (chemical modifier) for chemically modifying cholesterol ester hydrolase, the above-mentioned chemical modification group and a functional group or structure capable of reacting with the amino group, carboxyl group, sulfhydryl group, etc. of the enzyme are used.
  • the compound etc. to have together are mentioned.
  • functional groups or structures capable of reacting with amino groups in enzymes include carboxyl groups, active ester groups (N-hydroxysuccinimide groups, etc.), acid anhydrides, acid chlorides, aldehydes, epoxide groups, 1,3- Examples thereof include propane sultone and 1,4-butane sultone.
  • the functional group or structure capable of reacting with the carboxyl group in the enzyme include an amino group.
  • groups or structures reactive with sulfhydryl groups in enzymes include maleimide groups, disulfides, ⁇ -haloesters ( ⁇ -iodoest
  • Chemical modifiers include: Sunbright VFM-4101, Sunbright ME-050AS, Sunbright DE-030AS (all of which are NOF Corporations) having a group mainly composed of polyethylene glycol and an N-hydroxysuccinimide group.
  • Sanbright AKM series for example, Sunbright AKM-1510, etc.
  • Sunbright ADM series for example, Sunbright ADM series
  • Sunbright ACM series both made by NOF Corporation having a polyalkylene glycol-based group and an acid anhydride structure.
  • EPOX-3400 having a group mainly composed of polyethylene glycol and epoxide group, M-EPOX-5000 (both manufactured by Sheawater Polymers), diethylenetriamine having a chelate function and an acid anhydride structure -N, N, N ', N' ', N' '- And pentahydrodiacetic acid (DTPADTanhydride; manufactured by Dojindo Laboratories).
  • the chemical modification of cholesterol ester hydrolase can be performed, for example, by the following method, but is not limited to this method.
  • a pH 8.0 or higher buffer solution for example, HEPES buffer solution
  • 0.01 to 500-fold molar amount of a chemical modifier is added at 0 to 55 ° C. for 5 minutes to 5 minutes. Stir for hours.
  • chemically modified cholesterol ester hydrolase is not only the reaction solution itself, but also the one obtained by removing unreacted chemical modifiers etc. with an ultrafiltration membrane as necessary You can also
  • the concentration of the cholesterol esterase in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but the concentration in the reaction solution is usually 0.001 to 800 kU. / L, preferably 0.01 to 300 kU / L.
  • the cholesterol oxidase in the present invention is not particularly limited as long as it is an enzyme having the ability to oxidize cholesterol to generate hydrogen peroxide.
  • cholesterol oxidase derived from animals, plants, or microorganisms, as well as genetic engineering techniques. Cholesterol oxidase (CHODI; manufactured by Kyowa Hakko Kogyo Co., Ltd.), cholesterol oxidase (CHODI; manufactured by Kikkoman Corp.), cholesterol oxidase (CHO-CE; manufactured by Kikkoman Corp.), cholesterol oxidase ( Commercial products such as COO-321 (manufactured by Toyobo Co., Ltd.) can also be used. In the present invention, two or more kinds of cholesterol oxidases can be used in combination.
  • the cholesterol oxidase may be an unmodified enzyme or a chemically modified enzyme.
  • a chemically modified cholesterol oxidase can be produced, for example, by the aforementioned chemical modification method using the aforementioned chemical modifier.
  • the concentration of cholesterol oxidase in the reaction solution in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but polyoxyethylene dodecyl is used as polyoxyethylene alkylamine.
  • cholesterol oxidase is preferably 0.25 to 1.0 kU / L with respect to 0.06 to 0.3 mmol / L of polyoxyethylene dodecylamine in the reaction solution.
  • cholesterol oxidase is preferably 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of polyoxyethylene octadecylamine in the reaction solution.
  • the enzyme activity (U) of the cholesterol esterase for example, an activity value determined by an enzyme manufacturer can be used.
  • the enzyme activity (U) of cholesterol oxidase is defined as the amount of enzyme that reacts with 1 ⁇ mol of cholesterol per minute by the reaction of the following enzyme activity measurement reagent and cholesterol oxidase at 37 ° C.
  • ⁇ Reagent for enzyme activity measurement > Phosphate buffer solution (pH 7.15) 0.15 mol / L
  • alkyl in polyoxyethylene / polyoxyalkylene alkyl aryl ether examples include octyl, nonyl, decyl, dodecyl, etc., and aryl in POE / POA alkyl aryl ether is And phenyl.
  • the POE / POA polymerization mode in the POE / POA alkylaryl ether is not particularly limited, and examples thereof include block polymerization type and random polymerization type polymerization modes. Examples of the block polymerization type include a diblock copolymer, a triblock copolymer, and a tetrablock copolymer.
  • POE / POA alkyl aryl ether examples include polyoxypropylene other than polyoxyethylene, polyoxybutylene, and the like.
  • Specific examples of the POE / POA alkyl aryl ether include Emulgen L40 and the like (manufactured by Kao Corporation), Acroneses KP189R, Acroneses KP189R-40, Acroneses NP-189R (above, NOF Corporation) and the like.
  • the concentration of the POE / POA alkylaryl ether in the reaction solution in the measurement method of the present invention is not particularly limited as long as it is a concentration that enables measurement of LDL-C of the present invention. Is 0.001 to 200 g / L, preferably 0.01 to 50 g / L.
  • POE / POA condensate polyoxyethylene / polyoxyalkylene condensate
  • block polymerization type and random polymerization type polymerization types are available.
  • block polymerization type include a diblock copolymer, a triblock copolymer, and a tetrablock copolymer.
  • polyoxyalkylene (POA) in the POE / POA condensate include polyoxypropylene other than polyoxyethylene, polyoxybutylene, and the like.
  • the molecular weight of polyoxyalkylene (POA) is 500 to 6000, preferably 1500 to 4000.
  • the molecular weight of the POE / POA condensate is 500 to 12000, preferably 1500 to 8000.
  • POE / POA condensate examples include Pluronic L-121, Pluronic P-103, Pluronic F-108 (above, manufactured by ADEKA), Pronon B-204, Acroneses B-208 (above, manufactured by NOF Corporation). Etc.
  • the concentration of the POE / POA condensate in the reaction solution in the measurement method of the present invention is not particularly limited as long as it is a concentration that enables measurement of LDL-C of the present invention.
  • the concentration in the reaction solution is It is 0.001 to 200 g / L, preferably 0.01 to 50 g / L.
  • Examples of the alkyl in polyoxyethylene alkylamine include, for example, 6 to 30 carbon atoms such as hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl ( Myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triaconsil, etc.
  • POE alkylamine examples include, for example, 6 to 30 carbon atoms such as hexyl, heptyl, octy
  • alkyls are preferred.
  • the alkyl having 10 or more carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, heneicosyl, docosyl (behenyl), Examples include tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triaconsil and the like.
  • alkyl having 12 carbon atoms [dodecyl (lauryl)] and alkyl having 18 carbon atoms [octadecyl (stearyl)] are particularly preferable.
  • the degree of polymerization of the polyoxyethylene of the POE alkylamine is 1 to 40, preferably 2 to 20.
  • POE alkylamine examples include, for example, Naimine L-201 (oxyethylene dodecylamine; manufactured by NOF Corporation), Naimine L-202, Naimine L-207, Naimine L-215 (above, polyoxyethylene dodecyl) Amine; manufactured by NOF Corporation), Nymeen S-202, Nymeen S-204, Nymeen S-210, Nymeen S-215, Nymeen S-220 (above, polyoxyethylene octadecylamine; made by NOF Corporation), Nymeen T2- 202, Nymeen T2-210, Nymeen T2-230 [above, polyoxyethylene alkyl (tallow) amine; manufactured by NOF Corporation], Nymeen F-202, Nymeen F-203, Nymeen F-205, Nymeen F-210, Nymeen F-215 [above, polyoxyethylene Alkyl (coconut oil) amine; manufactured by NOF Corporation], BROWNON L-202, BROWNON L
  • the concentration of the POE alkylamine in the reaction solution in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but in the case of POE dodecylamine, it is usually 0.06. In the case of POE octadecylamine, it is usually 0.02 to 0.075 mmol / L.
  • Examples of the polyanion include dextran sulfate or a salt thereof, heparin or a salt thereof, phosphotungstic acid or a salt thereof, sulfated cyclodextrin or a salt thereof, a sulfated oligosaccharide or a salt thereof, and the like. 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.
  • Examples of sulfated oligosaccharides include sulfated agarose, sulfated trehalose, and chondroitin sulfate.
  • Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, magnesium salt and the like. In the present invention, two or more polyanions may be used.
  • the concentration of the polyanion in the reaction solution in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and usually 0.005 to 100 g / L. And preferably 0.05 to 10 g / L.
  • At least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfate (hereinafter abbreviated as POE polycyclic ether sulfate) and arylsulfonic acid derivatives is used.
  • POE polycyclic ether sulfate polyoxyethylene polycyclic ether sulfate
  • arylsulfonic acid derivatives arylsulfonic acid derivatives.
  • LDL-C in a sample containing a very small amount of LDL (hereinafter referred to as a low LDL sample), such as serum derived from a patient with low LDLemia.
  • a low LDL sample such as serum derived from a patient with low LDLemia.
  • the measurement of LDL-C using a low LDL sample is easily affected by chylomicrons and interfering substances in the sample, and in the conventional method, the LDL-C concentration determined by the measurement may be a negative value. It was.
  • Low LDL specimens contain a small amount of LDL, and LDL contains cholesterol, so low LDL specimens contain a small amount of LDL-C. Therefore, the LDL-C concentration determined by measurement is inherently a positive value and never a negative value.
  • LDL-C is accurately measured even in such low LDL samples. can do.
  • Examples of the POE polycyclic ether sulfate ester salt include polyoxyethylene polycyclic phenyl ether sulfate ester salt and polyoxyethylene naphthyl ether sulfate ester salt.
  • Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, magnesium salt and the like.
  • Specific examples (commercially available products) of POE polycyclic ether sulfate ester salts include, for example, New Coal 707SF, New Coal B4-SN (manufactured by Nippon Emulsifier Co., Ltd.) and the like.
  • aryl sulfonic acid derivative examples include naphthalene sulfonic acid formalin condensate. Specific examples (commercially available) of the aryl sulfonic acid derivatives include Disrol SH (manufactured by Nippon Emulsifier Co., Ltd.), Demol RN, Demol MS, Demol SN-B (manufactured by Kao Corporation), and the like.
  • the concentration of each of the POE polycyclic ether sulfate ester salt and the arylsulfonic acid derivative in the reaction solution in the LDL-C measurement method of the present invention is not particularly limited as long as it allows the measurement of LDL-C of the present invention. Usually, it is 0.005 to 7.5 g / L, and preferably 0.01 to 6 g / L.
  • the aqueous medium used in the present invention is not particularly limited as long as it is an aqueous medium that enables the LDL-C measurement method of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution. preferable.
  • the pH in the LDL-C measurement method of the present invention may be any pH as long as it enables the LDL-C measurement method of the present invention, and examples thereof include pH 4 to 10.
  • a buffer solution is used as the aqueous medium, it is desirable to use a buffering agent corresponding to the set pH.
  • 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) iminodiacetic acid (ADA) Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) ), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfone Acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane Sulfonic acid (HEPE
  • the concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L.
  • the reaction temperature in the LDL-C measurement method of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, but is preferably 10 to 50 ° C., more preferably 30 to 40 ° C. preferable.
  • the reaction temperature set by a general-purpose automatic analyzer is usually 37 ° C.
  • the reaction time in the LDL-C measurement method of the present invention is not particularly limited as long as the reaction time enables the LDL-C measurement method of the present invention, but it is preferably 1 to 60 minutes, and preferably 2 to 30 minutes. More preferred.
  • LDL-C can be measured by measuring the amount of hydrogen peroxide generated by the reaction.
  • the amount of generated hydrogen peroxide can be measured using, for example, a hydrogen peroxide electrode or a hydrogen peroxide measuring reagent.
  • the reagent for measuring hydrogen peroxide is a reagent for converting the generated hydrogen peroxide into a detectable substance. Examples of detectable substances include dyes and luminescence, and dyes are preferred.
  • the detectable substance is a dye
  • the hydrogen peroxide measurement reagent contains an oxidation coloring type chromogen and a peroxidation active substance such as peroxidase. Examples of the oxidative coloring type chromogen include the following oxidative coloring type chromogen.
  • the detectable substance is luminescence
  • the hydrogen peroxide measuring reagent contains a chemiluminescent substance. Examples of the chemiluminescent substance include luminol, isoluminol, lucigenin, and acridinium ester.
  • hydrogen peroxide is oxidized in the presence of the peroxidative active substance. It can measure by producing
  • hydrogen peroxide can be measured by reacting with the chemiluminescent substance to generate photons and measuring the generated photons. .
  • oxidative coloring chromogen examples include a leuco chromogen and an oxidative coupling coloring chromogen.
  • a leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase.
  • the oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase.
  • a peroxide active substance such as hydrogen peroxide and peroxidase.
  • the combination of the two compounds include a combination of a coupler and an aniline, a combination of a coupler and a phenol.
  • couplers examples include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone.
  • anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (
  • phenols examples include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.
  • the concentration of the peroxide active substance is not particularly limited as long as it is a concentration suitable for the measurement, but when peroxidase is used as the peroxide active substance, 1 to 100 kU / L is preferable.
  • the concentration of the oxidative coloring type chromogen is not particularly limited as long as it is a concentration suitable for the measurement of hydrogen peroxide, but is preferably 0.01 to 10 g / L.
  • the reagent for LDL-C measurement of the present invention is a reagent containing POE / POA alkylaryl ether, POE / POA condensate, POE alkylamine, polyanion, cholesterol ester hydrolase, and cholesterol oxidase.
  • the LDL-C measurement reagent of the present invention can further contain at least one substance selected from the group consisting of POE polycyclic ether sulfate salts and arylsulfonic acid derivatives.
  • the LDL-C measurement reagent of the present invention can be used in the LDL-C measurement method of the present invention.
  • the LDL-C measurement reagent of the present invention can take the form of a kit suitable for storage, distribution and use. Examples of the LDL-C measurement kit of the present invention include a two-reagent kit, a three-reagent kit, and the like, and a two-reagent kit comprising a first reagent and a second reagent is preferred.
  • the LDL-C measurement reagent and measurement kit of the present invention may be lyophilized or dissolved in an aqueous medium.
  • the reagent is used after being dissolved in an aqueous medium.
  • the aqueous medium include the aforementioned aqueous medium.
  • An ether sulfate ester salt, an aryl sulfonic acid derivative, and a reagent for measuring hydrogen peroxide can be used.
  • a two-reagent LDL-C measurement kit comprising a first reagent and a second reagent
  • cholesterol esterase is included in either or both of the first reagent and the second reagent.
  • Cholesterol oxidase is not contained in the first reagent, but is contained in the second reagent.
  • the POE / POA aryl ether may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the second reagent is preferred.
  • the POE / POA condensate may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the second reagent is preferred.
  • POE alkylamine is included in the second reagent.
  • the polyanion is included in the first reagent.
  • the POE polycyclic ether sulfate ester salt may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the first reagent is preferred.
  • the aryl sulfonic acid derivative may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the first reagent is preferred.
  • the reagent for measuring hydrogen peroxide may be contained in one or both of the first reagent and the second reagent, but when the reagent contains an oxidative coupling type chromogen, the oxidative coupling type chromogen is included. It is preferable that the two compounds, ie, coupler and aniline, or coupler and phenol are contained in separate reagents.
  • the concentration of cholesterol esterase in the LDL-C measurement reagent of the present invention is usually 0.001 to 800 kU / L, preferably 0.01 to 300 kU / L.
  • the content of cholesterol esterase is usually 0.001 to 800 kU / L, preferably 0.01 when dissolved in an aqueous medium. The content is ⁇ 300 kU / L.
  • the concentration of cholesterol esterase in the LDL-C measurement kit of the present invention is usually 0.004 to 3200 kU / L, preferably 0.04 to 1200 kU / L.
  • the content of cholesterol esterase is usually 0.004 to 3200 kU / L, preferably 0.04 when dissolved in an aqueous medium. The content is ⁇ 1200 kU / L.
  • the concentration of the POE • POA alkylaryl ether in the LDL-C measurement reagent of the present invention is not particularly limited as long as it allows the LDL-C measurement method of the present invention, and is usually 0.001 to 200 g / L. And preferably 0.01 to 50 g / L.
  • the content of the POE ⁇ POA alkylaryl ether is usually 0.001 to 200 g / L in a state of being dissolved in an aqueous medium, preferably 0. The content is from 01 to 50 g / L.
  • the concentration of the POE / POA alkyl aryl ether in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the LDL-C measurement method of the present invention, and is usually 0.004 to 800 g / L. Preferably, it is 0.04 to 200 g / L.
  • the content of the POE • POA alkylaryl ether is usually 0.004 to 800 g / L in a state of being dissolved in an aqueous medium, preferably 0.8. The content is from 04 to 200 g / L.
  • the concentration of the POE / POA condensate in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and is usually 0.001 to 200 g / L. Yes, preferably 0.01 to 50 g / L.
  • the content of the POE / POA condensate is usually 0.001 to 200 g / L, preferably 0.01, when dissolved in an aqueous medium. The content is ⁇ 50 g / L.
  • the concentration of the POE / POA condensate in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, and is usually 0.004 to 800 g / L. Yes, preferably 0.04 to 200 g / L.
  • the content of the POE / POA condensate is usually 0.004 to 800 g / L, preferably 0.04 when dissolved in an aqueous medium. The content is ⁇ 200 g / L.
  • the concentration of the polyanion in the LDL-C measurement reagent of the present invention is not particularly limited as long as it enables the LDL-C measurement method of the present invention, and is usually 0.005 to 100 g / L, preferably 0.05 to 10 g / L.
  • the polyanion content is usually 0.005 to 100 g / L, preferably 0.05 to 10 g / L when dissolved in an aqueous medium. Content.
  • the concentration of the polyanion in the first reagent in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, and usually 0.005 to 150 g / L. And preferably 0.05 to 15 g / L.
  • the content of the polyanion in the first reagent is usually 0.005 to 150 g / L in a state dissolved in an aqueous medium, and preferably is 0.1. The content is from 05 to 15 g / L.
  • the concentrations of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative in the LDL-C measurement reagent of the present invention are not particularly limited as long as the concentration enables the LDL-C measurement method of the present invention. Usually, it is 0.005 to 7.5 g / L, preferably 0.01 to 6 g / L.
  • the content of each of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative is usually 0.005 to 7 in a state dissolved in an aqueous medium. The content is 0.5 g / L, preferably 0.01 to 6 g / L.
  • the concentrations of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative in the LDL-C measurement kit of the present invention are not particularly limited as long as the concentration enables the LDL-C measurement method of the present invention. Usually, it is 0.007 to 10 g / L, and preferably 0.015 to 8 g / L.
  • the content of each of the POE polycyclic ether sulfate ester and the aryl sulfonic acid derivative is usually 0.007 to 10 g when dissolved in an aqueous medium. / L, preferably 0.015 to 8 g / L.
  • the cholesterol oxidase concentration in the LDL-C measurement reagent of the present invention is usually 0.25 to 1.0 kU / L relative to POE dodecylamine 0.06 to 0.3 mmol / L in the LDL-C measurement reagent. 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of POE octadecylamine in the reagent for LDL-C measurement.
  • the cholesterol oxidase concentration in the LDL-C measurement reagent in the lyophilized state the cholesterol oxidase concentration is usually 0.06 to 0.3 mmol of POE dodecylamine when dissolved in an aqueous medium.
  • the content is 0.25 to 1.0 kU / L with respect to / L, and the content is 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of POE octadecylamine. is there.
  • the concentration of cholesterol oxidase in the second reagent in the LDL-C measurement kit of the present invention is usually 1.0 to 4.4 with respect to 0.25 to 1.25 mmol / L of POE dodecylamine in the second reagent. 0 kU / L, and 2.0 to 8.0 kU / L with respect to 0.075 to 0.3 mmol / L of POE octadecylamine in the second reagent.
  • the content of cholesterol oxidase in the second reagent is usually such that the concentration of cholesterol oxidase dissolved in an aqueous medium is POE dodecylamine.
  • the content is 1.0 to 4.0 kU / L with respect to 25 to 1.25 mmol / L, and is 2.0 to 8.0 kU / L with respect to 0.075 to 0.3 mmol / L of POE octadecylamine.
  • the content of L is 1.0 to 4.0 kU / L with respect to 25 to 1.25 mmol / L, and is 2.0 to 8.0 kU / L with respect to 0.075 to 0.3 mmol / L of POE octadecylamine.
  • the LDL-C measurement reagent and measurement kit of the present invention may contain an aqueous medium, a stabilizer, an antiseptic, 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 ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride and the like.
  • the preservative include sodium azide and antibiotics.
  • Examples of the influence substance eliminating 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.
  • Reagent 1 A reagent containing POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, polyanion, cholesterol ester hydrolase, and cholesterol oxidase.
  • ⁇ Reagent 2 At least one substance selected from the group consisting of POE ⁇ POA alkyl aryl ethers, POE ⁇ POA condensates, POE alkyl amines, polyanions, POE polycyclic ether sulfate salts and aryl sulfonic acid derivatives, cholesterol ester hydrolase, and A reagent containing cholesterol oxidase.
  • a reagent comprising a POE / POA alkyl aryl ether, a POE / POA condensate, a POE alkyl amine, a polyanion, a cholesterol ester hydrolase, a cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
  • Reagent 4 POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, polyanion, POE polycyclic ether sulfate ester and at least one substance selected from the group consisting of aryl sulfonic acid derivatives, cholesterol ester hydrolase, cholesterol oxidation
  • a reagent containing an enzyme and a reagent for measuring hydrogen peroxide A reagent containing an enzyme and a reagent for measuring hydrogen peroxide.
  • First reagent At least one substance selected from the group consisting of polyanions, POE polycyclic ether sulfates and aryl sulfonic acid derivatives.
  • Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, cholesterol ester hydrolysate Degradation enzyme, cholesterol oxidase kit 5
  • First Reagent At least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE Alkylamine, cholesterol oxidase kit 6
  • First Reagent At least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, cholesterol ester hydrolase second rea
  • Kit 7 First reagent Polyanion, Reagent for hydrogen peroxide measurement Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, hydrogen peroxide measurement reagent, cholesterol esterase, cholesterol oxidase kit 8 First reagent Polyanion, Reagent for measuring hydrogen peroxide, Cholesterol ester hydrolase Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, Reagent for measuring hydrogen peroxide, Cholesterol oxidase kit 9 First reagent Polyanion, hydrogen peroxide measurement reagent, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, hydrogen peroxide measurement reagent, cholesterol ester hydrolase, Cholesterol oxidase
  • First reagent at least one substance selected from the group consisting of polyanions, POE polycyclic ether sulfates and aryl sulfonic acid derivatives; reagent for measuring hydrogen peroxide; second reagent; POE / POA alkyl aryl ether; POE / POA condensate; POE alkylamine, hydrogen peroxide measurement reagent, cholesterol ester hydrolase, cholesterol oxidase kit 11
  • First reagent at least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, hydrogen peroxide measuring reagent, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkylamine, hydrogen peroxide measuring reagent, cholesterol oxidase kit 12
  • First reagent at least one substance selected from the group consisting of polyanion, POE
  • Enzyme peroxidase (Toyobo Co., Ltd.) CHODI (cholesterol oxidase; manufactured by Kikkoman Corporation) LPL-311 (Cholesterol ester hydrolase; manufactured by Toyobo Co., Ltd.)
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L Peroxidase 10 kU / L
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L
  • Akroneses KP189R 14 g / L Pluronic L-121 7 g / L Braunon L-205 0.25 to 1.75 mmol / L LPL-311 3 kU / L CHODI 1.0-8.0 kU / L
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L Peroxidase 10 kU / L
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L
  • Akroneses KP189R 14 g / L Pluronic L-121 7 g / L LPL-311 3 kU / L CHODI 1.0-8.0 kU / L
  • LDL-C in 50 samples of human serum was measured by the following procedure.
  • (1) Preparation of a calibration curve As standard solutions, physiological saline (LDL-C concentration: 0.0 mg / dL) and serum (LDL-C concentration: 140 mg / dL) were used as kits for Example 1 and Comparative Example.
  • a calibration curve indicating the relationship between the LDL-C concentration and the “absorbance” was prepared using each kit and using a Hitachi 7170S automatic analyzer.
  • “absorbance” represents a value obtained by subtracting E1 from E2 based on two absorbances (E1 and E2) measured in the following reaction.
  • Standard solution (3 ⁇ L) and first reagent (0.15 mL) were added to the reaction cell and heated at 37 ° C. for 5 minutes, and the absorbance (E1) of the reaction solution was measured at a main wavelength of 600 nm and a subwavelength of 700 nm, Then, a second reagent (0.05 mL) was added to the reaction solution, and the mixture was further heated at 37 ° C. for 5 minutes, and the absorbance (E2) of the reaction solution was measured at a main wavelength of 600 nm and a sub wavelength of 700 nm.
  • LDL-C concentration in human serum sample The LDL-C concentration in each sample was determined from the “absorbance” measured in (2) and the calibration curve prepared in (1).
  • a kit a commercially available LDL-C measurement kit, Determiner L LDL-C (manufactured by Kyowa Medex Co., Ltd.) was used, and 50 samples of the same human serum were used as samples. LDL-C in the specimen was measured.
  • Table 1 shows the correlation coefficient between the measurement using the kits of Example 1 and Comparative Example and the measurement using the Determiner L LDL-C.
  • the CHODI in the second reagent was 1.0 to 4.0 kU / L
  • the Brownian L-205 in the second reagent was 0.25 to 1.25 mmol / L.
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L Peroxidase 10 kU / L
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L
  • Akroneses KP189R 14 g / L Pluronic L-121 7 g / L Naimine S-204 0.075-0.375 mmol / L LPL-311 3 kU / L CHODI 1.0-8.0 kU / L
  • Example 3 is the same as the method described in Example 2, except that the kit of Example 3 is used instead of the kit of Example 1 and a sample (50 samples) different from the sample of Example 1 is used as the sample.
  • the correlation coefficient between the measurement using this kit and the measurement using Determiner L LDL-C was calculated. The results are shown in Table 2.
  • the CHODI in the second reagent was 2.0 to 8.0 kU / L
  • the Niimine S-204 in the second reagent was 0.075 to 0.3 mmol / L.
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L Peroxidase 10 kU / L
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L
  • Akroneses KP189R 14 g / L Pluronic L-121 7 g / L Braunon L-205 0.75 mmol / L LPL-311 3 kU / L CHODI 3 kU / L
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L Peroxidase 10 kU / L
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L
  • Akroneses KP189R 14 g / L Pluronic L-121 7 g / L Naimine S-204 0.15 mmol / L LPL-311 3 kU / L CHODI 4 kU / L
  • LpX specimen serum derived from a patient with liver disease containing a large amount of LpX, which is one of abnormal lipoproteins
  • LpX is characterized in that the constituent cholesterol is almost only free cholesterol.
  • the peak of the LpX fraction appears at the same position as the peak of the VLDL fraction. Therefore, first, a supernatant fraction having a specific gravity smaller than 1.006 was separated from the LpX specimen by ultracentrifugation. That is, a sample diluted with physiological saline is added to a tube for ultracentrifugation, this tube is set in a Hitachi rotor 50.4TI rotor, ultracentrifugation is performed at 29,000 rpm for 18.5 hours, and centrifugation is completed. Thereafter, the supernatant (containing chylomicron and VLDL) separated using a tube slicer was removed to obtain a fraction containing LpX.
  • the fraction containing LpX obtained above was subjected to gel filtration column chromatography under the following conditions, and for each of the obtained fractions, using a total cholesterol measurement kit and a free cholesterol measurement kit, The total cholesterol concentration and free cholesterol concentration in each fraction were measured. Similarly, for each fraction, the cholesterol concentration in each fraction was measured using the respective kits of Examples 5 and 6. The result (chromatogram) is shown in FIG.
  • Coloring reagent (1) Total cholesterol (TC) measurement kit: Determiner L TC II (manufactured by Kyowa Medex) Color reaction temperature: 37 ° C Detection wavelength: Main wavelength 600 nm; Sub wavelength 800 nm Sample volume: 4 ⁇ L (2) Free cholesterol (FC) measurement kit: Determiner L FC (manufactured by Kyowa Medex) Color reaction temperature: 37 ° C Detection wavelength: Main wavelength 600 nm; Sub wavelength 700 nm Sample volume: 4 ⁇ L
  • the elution pattern (chromatogram) in this gel filtration column chromatography is shown in FIG.
  • the fraction corresponds to LpX. That is, it was confirmed that the serum derived from the liver disease patient contained a large amount of LpX, which is one of abnormal lipoproteins.
  • the fraction eluted next to LpX in the chromatogram of FIG. 1 is a fraction containing LpY (an abnormal lipoprotein) and a small amount of LDL.
  • First reagent MOPS (pH 7.0) 20 mmol / L Dextran sulfate sodium 0.75 g / L Sodium sulfate 2 g / L DOSE 0.3 g / L POE polycyclic ether sulfate ester or aryl sulfonic acid derivative (see Table 3 for types and concentrations)
  • Second reagent MOPS (pH 7.0) 20 mmol / L 4-Aminoantipyrine 0.5 g / L Peroxidase 20 kU / L Akroneses KP189R 14 g / L Pluronic L-121 7 g / L Braunon L-205 0.75 mmol / L LPL-311 3 kU / L CHODI 4 kU / L
  • the present invention provides a measurement method, measurement reagent, and measurement kit for LDL-C useful for diagnosis of metabolic syndrome, arteriosclerosis, and the like.

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Abstract

Provided is a method for measuring low-density lipoprotein cholesterol in a specimen, characterized in that a specimen is reacted with a cholesterol ester hydrolase and a cholesterol oxidase in the presence of a polyoxyethylene/polyoxyalkylene alkyl-aryl ether, polyoxyethylene/polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanions, and the hydrogen peroxide generated by said reaction is measured. As a result of this configuration, a method for accurately and simply measuring low-density lipoprotein cholesterol in a specimen is provided.

Description

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Method for measuring cholesterol in low density lipoprotein, reagent for measurement and kit for measurement
　本発明は、検体中に含まれる低密度リポ蛋白（以下、ＬＤＬという）中のコレステロール（以下、ＬＤＬ－Ｃという）の測定方法、測定用試薬及び測定用キットに関する。 The present invention relates to a method for measuring cholesterol (hereinafter referred to as LDL-C) in a low density lipoprotein (hereinafter referred to as LDL) contained in a specimen, a reagent for measurement, and a kit for measurement.
　ＬＤＬは、末梢細胞にコレステロールを供給する役割を有し、冠動脈硬化症をはじめとする各種動脈硬化症の直接的因子である。ＬＤＬ－Ｃの増加は動脈硬化性疾患の主要な危険因子の１つであり、ＬＤＬ－Ｃを分別定量することは臨床上有用である。 LDL has a role of supplying cholesterol to peripheral cells, and is a direct factor of various arteriosclerosis including coronary arteriosclerosis. An increase in LDL-C is one of the major risk factors for arteriosclerotic diseases, and differential quantification of LDL-C is clinically useful.
　従来のＬＤＬ－Ｃの定量方法としては、超遠心法、電気泳動法、フリードワルド（Friedewald）式による演算方法などが挙げられる。超遠心法は、リポ蛋白の比重の差を利用し、超遠心分離機を用いてＬＤＬを分離した後、そのコレステロール量を測定する方法である（非特許文献１）が、超遠心法による分離操作は煩雑で、迅速性、簡便性などの面で欠点がある。電気泳動法としては、リポ蛋白の電荷の差を利用し、アガロースゲルなどを支持体として分離する方法やリポ蛋白の粒子サイズの差を利用し、ポリアクリルアミドゲルを支持体として分離する方法などがあるが、電気泳動法は定量性に乏しく、簡便性、経済性などの面で問題がある。フリードワルド式による演算方法では、総コレステロール（以下、ＴＣという）、高密度リポ蛋白（以下、ＨＤＬという）中のコレステロール（以下、ＨＤＬ－Ｃという）及び総トリグリセライド（以下、Ｔ－ＴＧという）の測定値から、次の計算式に従いＬＤＬ－Ｃ量を算出する（非特許文献２）が、この方法は、血清中のＴ－ＴＧの含有量や食事の影響を受けるため、正確性に問題がある。 Examples of conventional LDL-C quantification methods include ultracentrifugation, electrophoresis, and calculation methods based on the Friedewald equation. The ultracentrifugation method is a method of measuring the amount of cholesterol after separating LDL using an ultracentrifuge using the difference in specific gravity of lipoprotein (Non-patent Document 1). The operation is complicated and has drawbacks in terms of speed and simplicity. Examples of the electrophoresis method include a method of separating agarose gel and the like using a difference in charge of lipoprotein and a method of separating a polyacrylamide gel and using a difference in particle size of lipoprotein. However, the electrophoresis method has poor quantitativeness and has problems in terms of convenience and economy. According to the Friedwald calculation method, total cholesterol (hereinafter referred to as TC), cholesterol (hereinafter referred to as HDL-C) in high-density lipoprotein (hereinafter referred to as HDL), and total triglyceride (hereinafter referred to as T-TG). The amount of LDL-C is calculated from the measured value according to the following formula (Non-patent Document 2). However, this method is affected by the content of T-TG in the serum and the diet, so there is a problem in accuracy. is there.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　また近年、超遠心法などの分離操作を必要とせず、汎用の自動分析機装置に搭載可能なＬＤＬ－Ｃの定量方法も報告されており、この様なＬＤＬ－Ｃの定量方法として、例えば以下の方法が知られている。 In recent years, a method for quantifying LDL-C that does not require a separation operation such as ultracentrifugation and can be mounted on a general-purpose automatic analyzer has been reported. The method is known.
　ＬＤＬ以外のリポ蛋白に作用する界面活性剤の存在下において、コレステロールエステル加水分解酵素及びコレステロール酸化酵素（以下、コレステロール測定酵素という）を作用させ、生じた過酸化水素を消去することにより、試料中のＨＤＬ、超低密度リポ蛋白（以下、ＶＬＤＬという）及びカイロミクロン中のコレステロールを消去する第１工程と、次いで、試料中の残存コレステロールを定量する第２工程とからなる試料中のＬＤＬ－Ｃの定量方法（特許文献１）。 In the presence of a surfactant that acts on lipoproteins other than LDL, cholesterol ester hydrolase and cholesterol oxidase (hereinafter referred to as cholesterol measuring enzyme) are allowed to act, and the generated hydrogen peroxide is eliminated. LDL-C in a sample comprising a first step of eliminating cholesterol in HDL, very low density lipoprotein (hereinafter referred to as VLDL) and chylomicron, and then a second step of quantifying residual cholesterol in the sample Quantification method (Patent Document 1).
　血清に対し、ポリオキシエチレンアルキレンフェニルエーテル及びポリオキシエチレンアルキレントリベンジルフェニルエーテルから選ばれる界面活性剤、並びにコレステロール測定酵素を添加し、リポ蛋白質のうちＨＤＬ中及びＶＬＤＬ中のコレステロールを優先的に反応させた後に、残りのコレステロールの反応量を測定するＬＤＬ－Ｃの定量方法（特許文献２）。 A surfactant selected from polyoxyethylene alkylene phenyl ether and polyoxyethylene alkylene tribenzyl phenyl ether and an enzyme for measuring cholesterol are added to serum to preferentially react cholesterol in HDL and VLDL among lipoproteins. And then determining the reaction amount of the remaining cholesterol, LDL-C quantification method (Patent Document 2).
　試料に対し、ポリオキシエチレン誘導体とポリオキシエチレン－ポリオキシプロピレン共重合体、並びにコレステロール測定酵素を添加し、リポ蛋白質のうちＬＤＬ－Ｃを選択的に測定する方法（特許文献３）。 A method of selectively measuring LDL-C among lipoproteins by adding a polyoxyethylene derivative, a polyoxyethylene-polyoxypropylene copolymer, and a cholesterol measuring enzyme to a sample (Patent Document 3).
　試料に対し、ジメチル－α－シクロデキストリン又は／及びポリ－β－シクロデキストリンの存在下で測定する、ＬＤＬ－Ｃの測定法（特許文献４）。 A method for measuring LDL-C in which a sample is measured in the presence of dimethyl-α-cyclodextrin and / or poly-β-cyclodextrin (Patent Document 4).
　カイロミクロン、ＨＤＬ、ＬＤＬ、ＶＬＤＬのいずれか１種以上を含む試料中のコレステロールを直接、選択的に測定する方法において、リン脂質、リン脂質類似基を含有する化合物の存在下、試料中のＬＤＬ－Ｃを定量する方法（特許文献５）。 In a method for directly and selectively measuring cholesterol in a sample containing any one or more of chylomicron, HDL, LDL, and VLDL, LDL in the sample in the presence of a compound containing a phospholipid or a phospholipid analog group A method for quantifying -C (Patent Document 5).
　ＬｐＸは、胆汁うっ滞時に出現するＬＤＬと同じ比重をもつ、健常人では全く存在しない異常リポ蛋白であり、化学組成も、構成成分の１つであるコレステロールがほとんど遊離型コレステロールであり、また、アポＢを含まず、正常ＬＤＬとは大きく異なる。従って、異常リポ蛋白であるＬｐＸ中のコレステロールを測定すべきではない、と考えられている（非特許文献３）。 LpX is an abnormal lipoprotein that has the same specific gravity as LDL that appears during cholestasis and does not exist at all in healthy people, and its chemical composition is almost free cholesterol, which is one of the constituent components, It does not contain apo B and is very different from normal LDL. Therefore, it is considered that cholesterol in LpX, which is an abnormal lipoprotein, should not be measured (Non-patent Document 3).
特開平１０－３８８８８号公報Japanese Patent Laid-Open No. 10-38888 特開平９－３１３２００号公報JP-A-9-313200 ＷＯ００／１７３８８パンフレットWO00 / 17388 pamphlet 特開平１１－３０６１７号公報Japanese Patent Laid-Open No. 11-30617 特開２００２－２０２３１４号公報JP 2002-202314 A
　本発明の目的は、検体中のＬＤＬ－Ｃを簡便かつ正確に測定するための方法、試薬及びキットを提供することにある。 An object of the present invention is to provide a method, a reagent, and a kit for simply and accurately measuring LDL-C in a specimen.
　本発明者らはＬＤＬ－Ｃの測定方法について鋭意検討を重ねた結果、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン、ポリアニオン、および、必要に応じて、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下で、検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素とを反応させることで、ＬＤＬ以外のリポ蛋白質中のコレステロールを消去することなく、また、リポ蛋白質の物理的な分画操作を行うことなく、検体中のＬＤＬ－Ｃを簡便かつ正確に測定できることを見出し、本発明を完成させた。すなわち、本発明は、下記（１）～（２２）に関する。 As a result of intensive studies on the measurement method of LDL-C, the present inventors have determined that polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, and If necessary, the sample is reacted with cholesterol esterase and cholesterol oxidase in the presence of at least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfates and arylsulfonic acid derivatives. Thus, it has been found that LDL-C in a sample can be measured easily and accurately without erasing cholesterol in lipoproteins other than LDL and without performing physical fractionation of lipoproteins. Completed the invention. That is, the present invention relates to the following (1) to (22).
（１）　検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素とを、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン及びポリアニオンの存在下に反応させ、該反応で生成する過酸化水素を測定することを特徴とする、検体中の低密度リポ蛋白中のコレステロールの測定方法。
（２）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである（１）記載の測定方法。
（３）　反応液中のコレステロール酸化酵素の濃度が０．２５～１．０ｋＵ／Ｌであり、ポリオキシエチレンドデシルアミンの濃度が０．０６～０．３ｍｍｏｌ／Ｌである（２）記載の方法。
（４）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである（１）記載の測定方法。
（５）　反応液中のコレステロール酸化酵素の濃度が０．５～２．０ｋＵ／Ｌのコレステロール酸化酵素であり、ポリオキシエチレンオクタデシルアミンの濃度が０．０２～０．０７５ｍｍｏｌ／Ｌである（４）記載の方法。
（６）　検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素との反応が、さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下に行われる、（１）～（５）のいずれかに記載の方法。
（７）　過酸化水素の測定が、過酸化水素測定用試薬を用いて行われる（１）～（６）のいずれかに記載の測定方法。 (1) Specimen, cholesterol esterase and cholesterol oxidase in the presence of polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion A method for measuring cholesterol in a low-density lipoprotein in a specimen, which comprises reacting and measuring hydrogen peroxide produced by the reaction.
(2) The measuring method according to (1), wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
(3) The method according to (2), wherein the concentration of cholesterol oxidase in the reaction solution is 0.25 to 1.0 kU / L, and the concentration of polyoxyethylene dodecylamine is 0.06 to 0.3 mmol / L. .
(4) The measuring method according to (1), wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
(5) A cholesterol oxidase concentration of 0.5 to 2.0 kU / L in the reaction solution, and a polyoxyethylene octadecylamine concentration of 0.02 to 0.075 mmol / L (4 ) Described method.
(6) In the presence of at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate salt and an arylsulfonic acid derivative, the reaction of the sample with cholesterol ester hydrolase and cholesterol oxidase The method according to any one of (1) to (5), which is performed.
(7) The measurement method according to any one of (1) to (6), wherein the measurement of hydrogen peroxide is performed using a reagent for measuring hydrogen peroxide.
（８）　ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン、ポリアニオン、コレステロールエステル加水分解酵素、及び、コレステロール酸化酵素を含有することを特徴とする、検体中の低密度リポ蛋白中のコレステロール測定用試薬。
（９）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである（８）記載の試薬。
（１０）　試薬中のコレステロール酸化酵素の濃度が０．２５～１．０ｋＵ／Ｌであり、ポリオキシエチレンドデシルアミンの濃度が０．０６～０．３ｍｍｏｌ／Ｌである（９）記載の試薬。
（１１）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである（８）記載の試薬。
（１２）　試薬中のコレステロール酸化酵素の濃度が０．５～２．０ｋＵ／Ｌであり、ポリオキシエチレンオクタデシルアミンの濃度が０．０２～０．０７５ｍｍｏｌ／Ｌである（１１）記載の試薬。
（１３）　さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を含有する、（８）～（１２）のいずれかに記載の試薬。
（１４）　さらに、過酸化水素測定用試薬を含む（８）～（１３）のいずれかに記載の試薬。 (8) It contains polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, cholesterol ester hydrolase, and cholesterol oxidase A reagent for measuring cholesterol in low-density lipoprotein in a specimen.
(9) The reagent according to (8), wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
(10) The reagent according to (9), wherein the concentration of cholesterol oxidase in the reagent is 0.25 to 1.0 kU / L, and the concentration of polyoxyethylene dodecylamine is 0.06 to 0.3 mmol / L.
(11) The reagent according to (8), wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
(12) The reagent according to (11), wherein the concentration of cholesterol oxidase in the reagent is 0.5 to 2.0 kU / L, and the concentration of polyoxyethylene octadecylamine is 0.02 to 0.075 mmol / L.
(13) The reagent according to any one of (8) to (12), further comprising at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate ester salt and an arylsulfonic acid derivative.
(14) The reagent according to any one of (8) to (13), further comprising a reagent for measuring hydrogen peroxide.
（１５）　ポリアニオンを含む第一試薬、コレステロール酸化酵素及びポリオキシエチレンアルキルアミンを含む第二試薬を含有し、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、及び、コレステロールエステル加水分解酵素のそれぞれを第一試薬、第二試薬のいずれか又は両方に含有することを特徴とする、検体中の低密度リポ蛋白中のコレステロール測定用キット。
（１６）　ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、及び、ポリオキシエチレン・ポリオキシアルキレン縮合物が第二試薬に含まれる（１５）記載のキット。
（１７）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである（１５）又は（１６）記載のキット。
（１８）　第二試薬中のコレステロール酸化酵素の濃度が１．０～４．０ｋＵ／Ｌであり、第二試薬中のポリオキシエチレンドデシルアミンの濃度が０．２５～１．２５ｍｍｏｌ／Ｌである（１７）記載のキット。
（１９）　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである（１５）又は（１６）記載のキット。
（２０）　第二試薬中のコレステロール酸化酵素の濃度が２．０～８．０ｋＵ／Ｌであり、第二試薬中のポリオキシエチレンオクタデシルアミンの濃度が０．０７５～０．３ｍｍｏｌ／Ｌである（１９）記載のキット。
（２１）　さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を第一試薬、第二試薬のいずれか又は両方に含む、（１５）～（２０）のいずれかに記載のキット。
（２２）　さらに、過酸化水素測定用試薬を第一試薬、第二試薬のいずれか又は両方に含有する（１５）～（２１）のいずれかに記載のキット。 (15) containing a first reagent containing a polyanion, a second reagent containing cholesterol oxidase and polyoxyethylene alkylamine, a polyoxyethylene-polyoxyalkylene alkylaryl ether, a polyoxyethylene-polyoxyalkylene condensate, and A kit for measuring cholesterol in low-density lipoprotein in a specimen, wherein each of cholesterol ester hydrolase is contained in either or both of the first reagent and the second reagent.
(16) The kit according to (15), wherein polyoxyethylene / polyoxyalkylene alkylaryl ether and polyoxyethylene / polyoxyalkylene condensate are contained in the second reagent.
(17) The kit according to (15) or (16), wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
(18) The concentration of cholesterol oxidase in the second reagent is 1.0 to 4.0 kU / L, and the concentration of polyoxyethylene dodecylamine in the second reagent is 0.25 to 1.25 mmol / L. (17) The kit according to the above.
(19) The kit according to (15) or (16), wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
(20) The concentration of cholesterol oxidase in the second reagent is 2.0 to 8.0 kU / L, and the concentration of polyoxyethylene octadecylamine in the second reagent is 0.075 to 0.3 mmol / L. (19) The kit according to the description.
(21) Further, at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate salt and an aryl sulfonic acid derivative is contained in either or both of the first reagent and the second reagent, (15) to (15) 20) The kit according to any one of the above.
(22) The kit according to any one of (15) to (21), further comprising a reagent for measuring hydrogen peroxide in one or both of the first reagent and the second reagent.
　本発明により、検体中のＬＤＬ－Ｃを簡便かつ正確に測定するための方法、試薬及びキットが提供される。 The present invention provides a method, a reagent, and a kit for measuring LDL-C in a sample simply and accurately.
ＬｐＸ検体のゲルろ過クロマトグラムを示す。横軸は、フラクション番号を、縦軸は、コレステロール濃度の測定値を表す。○は、総コレステロール（ＴＣ）濃度を、▲は、遊離型コレステロール（ＦＣ）濃度を、＊は、実施例５のキットを用いた測定でのコレステロール濃度を、◇は、実施例６のキットを用いた測定でのコレステロール濃度を表す。The gel filtration chromatogram of a LpX specimen is shown. The horizontal axis represents the fraction number, and the vertical axis represents the measured value of cholesterol concentration. ○ is total cholesterol (TC) concentration, ▲ is free cholesterol (FC) concentration, * is cholesterol concentration measured using the kit of Example 5, and ◇ is kit of Example 6. Represents the cholesterol concentration in the measurement used.
　本発明による検体中のＬＤＬ－Ｃの測定法は、遠心分離などの物理的方法によるリポ蛋白の分画操作を必要としない方法である。また、ＬＤＬ－Ｃの測定に先立って検体中のＬＤＬ以外のリポ蛋白中のコレステロールを消去することなく、検体中のＬＤＬ－Ｃを測定する方法である。またＬＤＬ－Ｃの測定に先立って、検体中のＬＤＬ以外のリポ蛋白中のコレステロールを測定することなく、検体中のＬＤＬ－Ｃを測定する方法である。 The method for measuring LDL-C in a specimen according to the present invention is a method that does not require a lipoprotein fractionation operation by a physical method such as centrifugation. In addition, prior to measurement of LDL-C, LDL-C in a sample is measured without eliminating cholesterol in lipoproteins other than LDL in the sample. In addition, prior to the measurement of LDL-C, this is a method for measuring LDL-C in a sample without measuring cholesterol in lipoproteins other than LDL in the sample.
　本発明の測定方法は、検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素とを、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン及びポリアニオンの存在下に反応させ、該反応で生成する過酸化水素を測定することを特徴とする方法である。 The measurement method of the present invention comprises a sample, cholesterol esterase and cholesterol oxidase, polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion. And hydrogen peroxide produced by the reaction is measured.
　さらに、本発明の測定方法は、検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素とを、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン、ポリアニオン、及び、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下に反応させ、該反応で生成する過酸化水素を測定することを特徴とする方法である。 Furthermore, the measurement method of the present invention comprises a sample, cholesterol ester hydrolase and cholesterol oxidase, polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine. And reacting in the presence of at least one substance selected from the group consisting of a polyanion, a polyoxyethylene polycyclic ether sulfate ester salt and an aryl sulfonic acid derivative, and measuring hydrogen peroxide produced by the reaction It is a method.
　本発明の測定方法によれば、異常リポ蛋白であるＬｐＸ中のコレステロールを測定せず、正確なＬＤＬ－Ｃの測定が可能となる。 According to the measurement method of the present invention, it is possible to accurately measure LDL-C without measuring cholesterol in LpX, which is an abnormal lipoprotein.
　本発明の測定方法は、
［１］検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素との反応を、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン及びポリアニオンの存在下に行う工程；
［２］上記［１］の工程で生成する過酸化水素を測定する工程；
［３］予め、既知濃度のＬＤＬ－Ｃを用いて作成された、ＬＤＬ－Ｃ濃度と、該過酸化水素由来の情報量との関係を表す検量線と、上記［２］での測定値とを相関付ける工程；及び、
［４］検体中のＬＤＬ－Ｃ濃度を決定する工程
を含む。ここで、上記［１］の反応は、さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下に行うこともできる。すなわち、検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素との反応は、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン、ポリアニオン、及び、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下に行うこともできる。 The measurement method of the present invention includes:
[1] Presence of polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion by reacting the sample with cholesterol ester hydrolase and cholesterol oxidase The steps performed below;
[2] A step of measuring hydrogen peroxide generated in the step [1];
[3] A calibration curve prepared in advance using a known concentration of LDL-C and representing the relationship between the LDL-C concentration and the amount of information derived from the hydrogen peroxide, and the measurement value in [2] above Correlating; and
[4] A step of determining the LDL-C concentration in the specimen. Here, the reaction [1] can also be carried out in the presence of at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate ester salt and an arylsulfonic acid derivative. That is, the reaction of the specimen with cholesterol ester hydrolase and cholesterol oxidase is performed by polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, and It can also be performed in the presence of at least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfates and arylsulfonic acid derivatives.
　上記［１］工程及び［２］工程は、水性媒体中で行われることが好ましい。水性媒体としては、例えば後述の水性媒体等が挙げられる。 The steps [1] and [2] are preferably performed in an aqueous medium. As an aqueous medium, the below-mentioned aqueous medium etc. are mentioned, for example.
　本発明の測定方法において、検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素との反応で生成する過酸化水素は、例えば過酸化水素電極や後述の過酸化水素測定用試薬を用いて測定することができる。 In the measurement method of the present invention, hydrogen peroxide produced by the reaction of the sample with cholesterol ester hydrolase and cholesterol oxidase is measured using, for example, a hydrogen peroxide electrode or a reagent for measuring hydrogen peroxide described below. Can do.
　本発明の測定方法において用いられる検体としては、例えば全血、血漿、血清等が挙げられ、血漿及び血清が好ましい。 Specimens used in the measurement method of the present invention include, for example, whole blood, plasma, serum and the like, and plasma and serum are preferable.
　本発明におけるコレステロールエステル加水分解酵素としては、コレステロールエステルを加水分解する能力を有する酵素であれば特に制限はなく、例えば動物、植物又は微生物由来のコレステロールエステラーゼ、リポプロテインリパーゼの他、遺伝子工学的な手法により製造されるコレステロールエステラーゼ、リポプロテインリパーゼ等も用いることができる。 The cholesterol ester hydrolase in the present invention is not particularly limited as long as it has an ability to hydrolyze cholesterol ester. For example, cholesterol esterase derived from animals, plants or microorganisms, lipoprotein lipase, genetic engineering Cholesterol esterase, lipoprotein lipase and the like produced by the technique can also be used.
　コレステロールエステル加水分解酵素としては、無修飾のコレステロールエステル加水分解酵素の他、化学的に修飾されたコレステロールエステル加水分解酵素も使用することができる。また、コレステロールエステル加水分解酵素としては市販品を使用することもできる。 As the cholesterol ester hydrolase, chemically modified cholesterol ester hydrolase can be used in addition to unmodified cholesterol ester hydrolase. Moreover, a commercial item can also be used as a cholesterol ester hydrolase.
　市販されているコレステロールエステル加水分解酵素としては、コレステロールエステラーゼ（ＣＯＥ－３１１；東洋紡績社製）、リポプロテインリパーゼ（ＬＰＬ－３１１；東洋紡績社製）、コレステロールエステラーゼIII（ＣＨＥIII；天野製薬社製）等が挙げられる。また、本発明においては、２種類以上のコレステロールエステル加水分解酵素を組み合わせて用いることもできる。 As commercially available cholesterol ester hydrolases, cholesterol esterase (COE-311; manufactured by Toyobo Co., Ltd.), lipoprotein lipase (LPL-311; manufactured by Toyobo Co., Ltd.), cholesterol esterase III (CHEIII; manufactured by Amano Pharmaceutical Co., Ltd.) Etc. In the present invention, two or more kinds of cholesterol ester hydrolases can be used in combination.
　コレステロールエステル加水分解酵素の化学修飾において当該酵素を修飾する基（化学修飾基）としては、例えばポリエチレングリコールを主成分とする基、ポリプロピレングリコールを主成分とする基、ポリプロピレングリコールとポリエチレングリコールの共重合体を有する基、水溶性多糖類を含有する基、スルホプロピル基、スルホブチル基、ポリウレタン基、キレート機能を有する基等が挙げられるが、ポリエチレングリコールを主成分とする基が好ましい。水溶性多糖類としては、例えばデキストラン、プルラン、可溶性デンプン等が挙げられる。 Examples of a group (chemical modification group) that modifies the enzyme in chemical modification of cholesterol ester hydrolase include, for example, a group mainly composed of polyethylene glycol, a group mainly composed of polypropylene glycol, and a co-polymerization of polypropylene glycol and polyethylene glycol. Examples include a group having a combination, a group containing a water-soluble polysaccharide, a sulfopropyl group, a sulfobutyl group, a polyurethane group, a group having a chelating function, and the like, and a group having polyethylene glycol as a main component is preferable. Examples of the water-soluble polysaccharide include dextran, pullulan, and soluble starch.
　コレステロールエステル加水分解酵素を化学的に修飾するための試薬（化学修飾剤）としては、上記の化学修飾基と、酵素のアミノ基、カルボキシル基、スルフヒドリル基等と反応し得る官能基又は構造とを併せ持つ化合物等が挙げられる。酵素中のアミノ基と反応し得る官能基又は構造としては、例えばカルボキシル基、活性エステル基（Ｎ－ヒドロキシサクシンイミド基等）、酸無水物、酸塩化物、アルデヒド、エポキシド基、１，３－プロパンスルトン、１，４－ブタンスルトン等が挙げられる。酵素中のカルボキシル基と反応し得る官能基又は構造としては、例えばアミノ基等が挙げられる。酵素中のスルフヒドリル基と反応性がある基又は構造としては、例えばマレイミド基、ジスルフィド、α－ハロエステル（α－ヨードエステル等）等が挙げられる。 As a reagent (chemical modifier) for chemically modifying cholesterol ester hydrolase, the above-mentioned chemical modification group and a functional group or structure capable of reacting with the amino group, carboxyl group, sulfhydryl group, etc. of the enzyme are used. The compound etc. to have together are mentioned. Examples of functional groups or structures capable of reacting with amino groups in enzymes include carboxyl groups, active ester groups (N-hydroxysuccinimide groups, etc.), acid anhydrides, acid chlorides, aldehydes, epoxide groups, 1,3- Examples thereof include 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 groups or structures reactive with sulfhydryl groups in enzymes include maleimide groups, disulfides, α-haloesters (α-iodoesters, etc.) and the like.
　化学修飾剤として、市販品を使用することもできる。市販されている化学修飾剤としては、ポリエチレングリコールを主成分とする基とＮ－ヒドロキシサクシンイミド基とを有するサンブライトＶＦＭ－４１０１、サンブライトＭＥ－０５０ＡＳ、サンブライトＤＥ－０３０ＡＳ（いずれも日油社製）、ポリアルキレングリコールを主成分とする基と酸無水物構造とを有するサンブライトＡＫＭシリーズ（例えば、サンブライトＡＫＭ－１５１０等）、サンブライトＡＤＭシリーズ、サンブライトＡＣＭシリーズ（いずれも日油社製）、ポリエチレングリコールを主成分とする基とエポキシド基とを有するＥＰＯＸ－３４００、Ｍ－ＥＰＯＸ－５０００（いずれもSheawater Polymers社製）、キレート機能を有する基と酸無水物構造とを有するジエチレントリアミン－Ｎ，Ｎ，Ｎ’，Ｎ’’，Ｎ’’－ペンタ無水二酢酸（DTPA anhydride；同仁化学研究所社製）等が挙げられる。 Commercial products can also be used as chemical modifiers. Commercially available chemical modifiers include: Sunbright VFM-4101, Sunbright ME-050AS, Sunbright DE-030AS (all of which are NOF Corporations) having a group mainly composed of polyethylene glycol and an N-hydroxysuccinimide group. Sanbright AKM series (for example, Sunbright AKM-1510, etc.), Sunbright ADM series, and Sunbright ACM series (both made by NOF Corporation) having a polyalkylene glycol-based group and an acid anhydride structure. Manufactured by Co., Ltd.), EPOX-3400 having a group mainly composed of polyethylene glycol and epoxide group, M-EPOX-5000 (both manufactured by Sheawater Polymers), diethylenetriamine having a chelate function and an acid anhydride structure -N, N, N ', N' ', N' '- And pentahydrodiacetic acid (DTPADTanhydride; manufactured by Dojindo Laboratories).
　コレステロールエステル加水分解酵素の化学修飾は、例えば以下の方法で行うことができるが、本方法に限定されるものではない。まず、コレステロールエステル加水分解酵素をｐＨ８．０以上の緩衝液（例えばＨＥＰＥＳ緩衝液）に溶解し、０～５５℃で０．０１～５００倍モル量の化学修飾剤を添加し、５分間～５時間攪拌する。酵素反応においては、化学的に修飾されたコレステロールエステル加水分解酵素として、この反応液そのもののみならず、必要に応じて限外濾過膜等により未反応の化学修飾剤等を除去したものも、使用することもできる。 The chemical modification of cholesterol ester hydrolase can be performed, for example, by the following method, but is not limited to this method. First, cholesterol esterase is dissolved in a pH 8.0 or higher buffer solution (for example, HEPES buffer solution), and 0.01 to 500-fold molar amount of a chemical modifier is added at 0 to 55 ° C. for 5 minutes to 5 minutes. Stir for hours. In the enzymatic reaction, chemically modified cholesterol ester hydrolase is not only the reaction solution itself, but also the one obtained by removing unreacted chemical modifiers etc. with an ultrafiltration membrane as necessary You can also
　本発明の測定方法におけるコレステロールエステル加水分解酵素の濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はないが、反応液中の濃度は通常０．００１～８００ｋＵ／Ｌであり、０．０１～３００ｋＵ／Ｌが好ましい。 The concentration of the cholesterol esterase in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but the concentration in the reaction solution is usually 0.001 to 800 kU. / L, preferably 0.01 to 300 kU / L.
　本発明におけるコレステロール酸化酵素としては、コレステロールを酸化して過酸化水素を生成する能力を有する酵素であれば特に制限はなく、例えば動物、植物又は微生物由来のコレステロールオキシダーゼの他、遺伝子工学的な手法により製造されるコレステロールオキシダーゼ等も用いることができ、コレステロールオキシダーゼ（ＣＨＯＤＩ；協和発酵工業社製）、コレステロールオキシダーゼ（ＣＨＯＤＩ；キッコーマン社製）、コレステロールオキシダーゼ（ＣＨＯ－ＣＥ；キッコーマン社製）、コレステロールオキシダーゼ（ＣＯＯ－３２１；東洋紡績社製）等の市販品を用いることもできる。また、本発明においては、２種類以上のコレステロール酸化酵素を組み合わせて用いることもできる。 The cholesterol oxidase in the present invention is not particularly limited as long as it is an enzyme having the ability to oxidize cholesterol to generate hydrogen peroxide. For example, cholesterol oxidase derived from animals, plants, or microorganisms, as well as genetic engineering techniques. Cholesterol oxidase (CHODI; manufactured by Kyowa Hakko Kogyo Co., Ltd.), cholesterol oxidase (CHODI; manufactured by Kikkoman Corp.), cholesterol oxidase (CHO-CE; manufactured by Kikkoman Corp.), cholesterol oxidase ( Commercial products such as COO-321 (manufactured by Toyobo Co., Ltd.) can also be used. In the present invention, two or more kinds of cholesterol oxidases can be used in combination.
　コレステロール酸化酵素は、無修飾の酵素であっても、化学的に修飾された酵素であってもよい。化学的に修飾されたコレステロール酸化酵素は、例えば前述の化学修飾剤を用いて、前述の化学修飾方法により作製することができる。 The cholesterol oxidase may be an unmodified enzyme or a chemically modified enzyme. A chemically modified cholesterol oxidase can be produced, for example, by the aforementioned chemical modification method using the aforementioned chemical modifier.
　本発明の測定方法における反応液中のコレステロール酸化酵素の濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はないが、ポリオキシエチレンアルキルアミンとしてポリオキシエチレンドデシルアミンを用いる場合は、反応液中のポリオキシエチレンドデシルアミン０．０６～０．３ｍｍｏｌ／Ｌに対して、コレステロール酸化酵素０．２５～１．０ｋＵ／Ｌが好ましく、ポリオキシエチレンアルキルアミンとしてポリオキシエチレンオクタデシルアミンを用いる場合は、反応液中のポリオキシエチレンオクタデシルアミン０．０２～０．０７５ｍｍｏｌ／Ｌに対して、コレステロール酸化酵素０．５～２．０ｋＵ／Ｌが好ましい。 The concentration of cholesterol oxidase in the reaction solution in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but polyoxyethylene dodecyl is used as polyoxyethylene alkylamine. In the case of using an amine, cholesterol oxidase is preferably 0.25 to 1.0 kU / L with respect to 0.06 to 0.3 mmol / L of polyoxyethylene dodecylamine in the reaction solution. When oxyethylene octadecylamine is used, cholesterol oxidase is preferably 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of polyoxyethylene octadecylamine in the reaction solution.
　本発明において、コレステロールエステル加水分解酵素の酵素活性（Ｕ）は、例えば酵素メーカーで値付けされた活性値を用いることができる。 In the present invention, as the enzyme activity (U) of the cholesterol esterase, for example, an activity value determined by an enzyme manufacturer can be used.
　本発明において、コレステロール酸化酵素の酵素活性（Ｕ）は、以下の酵素活性測定用試薬とコレステロール酸化酵素との３７℃での反応により、１分あたりコレステロール１μｍｏｌが反応する酵素量と定義される。
＜酵素活性測定用試薬＞
　リン酸緩衝液（ｐＨ７．１５）　　　　　　　　　０．１５　ｍｏｌ／Ｌ
　ポリオキシエチレンオクチルフェニルエーテル　　１０　ｇ／Ｌ
　２－プロパノール　　　　　　　　　　　　　　　２．５％
　４－アミノアンチピリン　　　　　　　　　　　　０．２　ｇ／Ｌ
　フェノール　　　　　　　　　　　　　　　　　　１　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　　　　　　　　　２６　ｋＵ／Ｌ
　コレステロール　　　　　　　　　　　　　　　　０．１６　ｇ／Ｌ In the present invention, the enzyme activity (U) of cholesterol oxidase is defined as the amount of enzyme that reacts with 1 μmol of cholesterol per minute by the reaction of the following enzyme activity measurement reagent and cholesterol oxidase at 37 ° C.
<Reagent for enzyme activity measurement>
Phosphate buffer solution (pH 7.15) 0.15 mol / L
Polyoxyethylene octyl phenyl ether 10 g / L
2-propanol 2.5%
4-Aminoantipyrine 0.2 g / L
Phenol 1 g / L
Peroxidase 26 kU / L
Cholesterol 0.16 g / L
　ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル（以下、ＰＯＥ・ＰＯＡアルキルアリールエーテルと略記する）におけるアルキルとしては例えばオクチル、ノニル、デシル、ドデシルなどが挙げられ、ＰＯＥ・ＰＯＡアルキルアリールエーテルにおけるアリールとしてはフェニル等が挙げられる。ＰＯＥ・ＰＯＡアルキルアリールエーテルにおけるＰＯＥ・ＰＯＡの重合様式としてはとくに制限はなく、例えば、ブロック重合型、ランダム重合型の重合様式のものが挙げられる。ブロック重合型としては、例えばジブロックコポリマー、トリブロックコポリマー、テトラブロックコポリマー等が挙げられる。 Examples of alkyl in polyoxyethylene / polyoxyalkylene alkyl aryl ether (hereinafter abbreviated as POE / POA alkyl aryl ether) include octyl, nonyl, decyl, dodecyl, etc., and aryl in POE / POA alkyl aryl ether is And phenyl. The POE / POA polymerization mode in the POE / POA alkylaryl ether is not particularly limited, and examples thereof include block polymerization type and random polymerization type polymerization modes. Examples of the block polymerization type include a diblock copolymer, a triblock copolymer, and a tetrablock copolymer.
　ＰＯＥ・ＰＯＡアルキルアリールエーテルにおけるポリオキシアルキレン（ＰＯＡ）としては、ポリオキシエチレン以外のポリオキシプロピレン、ポリオキシブチレン等が挙げられる。ＰＯＥ・ＰＯＡアルキルアリールエーテルの具体例としては、エマルゲンＬ４０等（花王社製）、アクロネセスＫＰ１８９Ｒ、アクロネセスＫＰ１８９Ｒ－４０、アクロネセスＮＰ－１８９Ｒ（以上、日油社製）等が挙げられる。 Examples of polyoxyalkylene (POA) in POE / POA alkyl aryl ether include polyoxypropylene other than polyoxyethylene, polyoxybutylene, and the like. Specific examples of the POE / POA alkyl aryl ether include Emulgen L40 and the like (manufactured by Kao Corporation), Acroneses KP189R, Acroneses KP189R-40, Acroneses NP-189R (above, NOF Corporation) and the like.
　本発明の測定方法における反応液中のＰＯＥ・ＰＯＡアルキルアリールエーテルの濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はないが、通常、反応液中の濃度は０．００１～２００ｇ／Ｌであり、好ましくは０．０１～５０ｇ／Ｌである。 The concentration of the POE / POA alkylaryl ether in the reaction solution in the measurement method of the present invention is not particularly limited as long as it is a concentration that enables measurement of LDL-C of the present invention. Is 0.001 to 200 g / L, preferably 0.01 to 50 g / L.
　ポリオキシエチレン・ポリオキシアルキレン縮合物（以下、ＰＯＥ・ＰＯＡ縮合物と略記する）におけるＰＯＥ・ＰＯＡの重合様式としてはとくに制限はなく、例えば、ブロック重合型、ランダム重合型の重合様式のものが挙げられる。ブロック重合型としては、例えばジブロックコポリマー、トリブロックコポリマー、テトラブロックコポリマー等が挙げられる。ＰＯＥ・ＰＯＡ縮合物におけるポリオキシアルキレン（ＰＯＡ）としては、ポリオキシエチレン以外のポリオキシプロピレン、ポリオキシブチレン等が挙げられる。ポリオキシアルキレン（ＰＯＡ）の分子量としては、５００～６０００であり、好ましくは１５００～４０００である。ＰＯＥ・ＰＯＡ縮合物の分子量としては、５００～１２０００であり、好ましくは１５００～８０００である。 There is no particular limitation on the polymerization mode of POE / POA in the polyoxyethylene / polyoxyalkylene condensate (hereinafter abbreviated as POE / POA condensate). For example, block polymerization type and random polymerization type polymerization types are available. Can be mentioned. Examples of the block polymerization type include a diblock copolymer, a triblock copolymer, and a tetrablock copolymer. Examples of polyoxyalkylene (POA) in the POE / POA condensate include polyoxypropylene other than polyoxyethylene, polyoxybutylene, and the like. The molecular weight of polyoxyalkylene (POA) is 500 to 6000, preferably 1500 to 4000. The molecular weight of the POE / POA condensate is 500 to 12000, preferably 1500 to 8000.
　ＰＯＥ・ＰＯＡ縮合物の具体例としては、プルロニックＬ－１２１、プルロニックＰ－１０３、プルロニックＦ－１０８（以上、ＡＤＥＫＡ社製）、プロノンＢ－２０４、アクロネセスＢ－２０８（以上、日油社製）等が挙げられる。 Specific examples of the POE / POA condensate include Pluronic L-121, Pluronic P-103, Pluronic F-108 (above, manufactured by ADEKA), Pronon B-204, Acroneses B-208 (above, manufactured by NOF Corporation). Etc.
　本発明の測定方法における反応液中のＰＯＥ・ＰＯＡ縮合物の濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はないが、通常、反応液中の濃度は０．００１～２００ｇ／Ｌであり、好ましくは０．０１～５０ｇ／Ｌである。 The concentration of the POE / POA condensate in the reaction solution in the measurement method of the present invention is not particularly limited as long as it is a concentration that enables measurement of LDL-C of the present invention. Usually, the concentration in the reaction solution is It is 0.001 to 200 g / L, preferably 0.01 to 50 g / L.
　ポリオキシエチレンアルキルアミン（以下、ＰＯＥアルキルアミンと略記する）におけるアルキルとしては、例えば炭素数６～３０の、例えばヘキシル、ヘプチル、オクチル、ノニル、デシル、ウンデシル、ドデシル（ラウリル）、トリデシル、テトラデシル（ミリスチル）、ペンタデシル、ヘキサデシル（セチル）、ヘプタデシル、オクタデシル（ステアリル）、ノナデシル、イコシル、ヘネイコシル、ドコシル（ベヘニル）、トリコシル、テトラコシル、ペンタコシル、ヘキサコシル、ヘプタコシル、オクタコシル、ノナコシル、トリアコンシル等が挙げられ、炭素数１０以上のアルキルが好ましい。炭素１０以上のアルキルとしては、例えばデシル、ウンデシル、ドデシル（ラウリル）、トリデシル、テトラデシル（ミリスチル）、ペンタデシル、ヘキサデシル（セチル）、ヘプタデシル、オクタデシル（ステアリル）、ノナデシル、イコシル、ヘネイコシル、ドコシル（ベヘニル）、トリコシル、テトラコシル、ペンタコシル、ヘキサコシル、ヘプタコシル、オクタコシル、ノナコシル、トリアコンシル等が挙げられる。中でも、炭素数１２のアルキル［ドデシル（ラウリル）］と炭素数１８のアルキル［オクタデシル（ステアリル）］が特に好ましい。ＰＯＥアルキルアミンのポリオキシエチレンのオキシエチレンの重合度としては、１～４０であり、好ましくは、２～２０である。 Examples of the alkyl in polyoxyethylene alkylamine (hereinafter abbreviated as POE alkylamine) include, for example, 6 to 30 carbon atoms such as hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl ( Myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, heneicosyl, docosyl (behenyl), tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triaconsil, etc. Ten or more alkyls are preferred. Examples of the alkyl having 10 or more carbon atoms include decyl, undecyl, dodecyl (lauryl), tridecyl, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), nonadecyl, icosyl, heneicosyl, docosyl (behenyl), Examples include tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triaconsil and the like. Among them, alkyl having 12 carbon atoms [dodecyl (lauryl)] and alkyl having 18 carbon atoms [octadecyl (stearyl)] are particularly preferable. The degree of polymerization of the polyoxyethylene of the POE alkylamine is 1 to 40, preferably 2 to 20.
　ＰＯＥアルキルアミンの具体例（製品）としては、例えばナイミーンＬ－２０１（オキシエチレンドデシルアミン；日油社製）、ナイミーンＬ－２０２、ナイミーンＬ－２０７、ナイミーンＬ－２１５（以上、ポリオキシエチレンドデシルアミン；日油社製）、ナイミーンＳ－２０２、ナイミーンＳ－２０４、ナイミーンＳ－２１０、ナイミーンＳ－２１５、ナイミーンＳ－２２０（以上、ポリオキシエチレンオクタデシルアミン；日油社製）、ナイミーンＴ２－２０２、ナイミーンＴ２－２１０、ナイミーンＴ２－２３０［以上、ポリオキシエチレンアルキル（牛脂）アミン；日油社製］、ナイミーンＦ－２０２、ナイミーンＦ－２０３、ナイミーンＦ－２０５、ナイミーンＦ－２１０、ナイミーンＦ－２１５［以上、ポリオキシエチレンアルキル（ヤシ油）アミン；日油社製］、ブラウノンＬ－２０２、ブラウノンＬ－２０５、ブラウノンＬ－２０７、ブラウノンＬ－２１０、ブラウノンＬ－２３０（以上、ポリオキシエチレンドデシルアミン；青木油脂社製）、ブラウノンＳ－２０７、ブラウノンＳ－２１０、ブラウノンＳ－２１５、ブラウノンＳ－２２０、ブラウノンＳ－２３０（以上、ポリオキシエチレンオクタデシルアミン；青木油脂社製）、ブラウノンＳ－２０５Ｔ、ブラウノンＳ－２０８Ｔ、ブラウノンＳ－２１０Ｔ、ブラウノンＳ－２１５Ｔ、ブラウノンＳ－２３０Ｔ［以上、ポリオキシエチレンアルキル（牛脂）アミン；青木油脂社製］、ニューコールＯＤ４２０（ポリオキシエチレンオクタデシルアミン；日本乳化剤社製）、パイオニンＤ３１０４（ポリオキシエチレンドデシルアミン；竹本油脂社製）、パイオニンＤ３１１０（ポリオキシエチレンドデシルアミン；竹本油脂社製）、パイオニンＤ３６０５［ポリオキシエチレンアルキル（大豆）アミン；竹本油脂社製］、パイオニンＤ３６１５Ｔ［ポリオキシエチレンアルキル（牛脂）アミン；竹本油脂社製］等が挙げられる。 Specific examples (products) of POE alkylamine include, for example, Naimine L-201 (oxyethylene dodecylamine; manufactured by NOF Corporation), Naimine L-202, Naimine L-207, Naimine L-215 (above, polyoxyethylene dodecyl) Amine; manufactured by NOF Corporation), Nymeen S-202, Nymeen S-204, Nymeen S-210, Nymeen S-215, Nymeen S-220 (above, polyoxyethylene octadecylamine; made by NOF Corporation), Nymeen T2- 202, Nymeen T2-210, Nymeen T2-230 [above, polyoxyethylene alkyl (tallow) amine; manufactured by NOF Corporation], Nymeen F-202, Nymeen F-203, Nymeen F-205, Nymeen F-210, Nymeen F-215 [above, polyoxyethylene Alkyl (coconut oil) amine; manufactured by NOF Corporation], BROWNON L-202, BROWNON L-205, BROWNON L-207, BROWNON L-210, BROWNON L-230 (above, polyoxyethylene dodecylamine; manufactured by Aoki Yushi Co., Ltd.) ), Brownon S-207, Brownon S-210, Brownon S-215, Brownon S-220, Brownon S-230 (above, polyoxyethylene octadecylamine; manufactured by Aoki Yushi), Brownon S-205T, Brownon S-208T BROWNON S-210T, BROWNON S-215T, BROWNON S-230T [above, polyoxyethylene alkyl (beef tallow) amine; manufactured by Aoki Yushi Co., Ltd.], New Coal OD420 (polyoxyethylene octadecylamine; manufactured by Nippon Emulsifier Co., Ltd.), pionine D3104 (Poly Xylethylenedodecylamine; manufactured by Takemoto Yushi Co., Ltd.), Pionein D3110 (polyoxyethylene dodecylamine; manufactured by Takemoto Yushi Co., Ltd.), pionein D3605 [polyoxyethylene alkyl (soybean) amine; manufactured by Takemoto Yushi Co., Ltd.], Pionein D3615T [polyoxyethylene Alkyl (beef tallow) amine; manufactured by Takemoto Yushi Co., Ltd.].
　本発明の測定方法における反応液中のＰＯＥアルキルアミンの濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はないが、ＰＯＥドデシルアミンの場合は通常０．０６～０．３ｍｍｏｌ／Ｌであり、ＰＯＥオクタデシルアミンの場合は通常０．０２～０．０７５ｍｍｏｌ／Ｌである。 The concentration of the POE alkylamine in the reaction solution in the measurement method of the present invention is not particularly limited as long as it allows measurement of LDL-C of the present invention, but in the case of POE dodecylamine, it is usually 0.06. In the case of POE octadecylamine, it is usually 0.02 to 0.075 mmol / L.
　ポリアニオンとしては、例えばデキストラン硫酸もしくはその塩、ヘパリンもしくはその塩、リンタングステン酸又はその塩、硫酸化シクロデキストリン又はその塩、硫酸化オリゴ糖又はその塩等が挙げられるが、デキストラン硫酸もしくはその塩が好ましい。デキストラン硫酸としては、例えば分子量が４万、８万、２０万、５０万、１００万、２００万等のデキストラン硫酸等が挙げられる。硫酸化オリゴ糖としては、例えば硫酸化アガロース、硫酸化トレハロース、コンドロイチン硫酸等が挙げられる。塩としては、例えばナトリウム塩、カリウム塩、リチウム塩、アンモニウム塩、マグネシウム塩等が挙げられる。また、本発明においては、ポリアニオンを２種以上用いてもよい。 Examples of the polyanion include dextran sulfate or a salt thereof, heparin or a salt thereof, phosphotungstic acid or a salt thereof, sulfated cyclodextrin or a salt thereof, a sulfated oligosaccharide or a salt thereof, and the like. 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. Examples of sulfated oligosaccharides include sulfated agarose, sulfated trehalose, and chondroitin sulfate. Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, magnesium salt and the like. In the present invention, two or more polyanions may be used.
　本発明のＬＤＬ－Ｃの測定方法における反応液中のポリアニオンの濃度としては、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００５～１００ｇ／Ｌであり、好ましくは０．０５～１０ｇ／Ｌである。 The concentration of the polyanion in the reaction solution in the LDL-C measurement method of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and usually 0.005 to 100 g / L. And preferably 0.05 to 10 g / L.
　本発明の測定方法において、さらに、ポリオキシエチレン多環エーテル硫酸エステル塩（以下、ＰＯＥ多環エーテル硫酸エステル塩と略記する）およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を用いることにより、低ＬＤＬ血症患者由来の血清等の、ＬＤＬが極僅かしか含まれない検体（以下、低ＬＤＬ検体という）中のＬＤＬ－Ｃの測定に好適に使用することができる。低ＬＤＬ検体を用いるＬＤＬ－Ｃの測定は、検体中のカイロミクロンや干渉物質等の影響を受け易く、従来の方法では、測定により決定されたＬＤＬ－Ｃ濃度が負の値となる場合があった。低ＬＤＬ検体には、ＬＤＬが僅かに含まれ、ＬＤＬ中にはコレステロールが含まれることから、低ＬＤＬ検体は僅かではあるがＬＤＬ－Ｃを含む。従って、測定により決定されるＬＤＬ－Ｃ濃度は、本来、正の値であり、負の値とはなることはない。本発明の測定方法において、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を用いることにより、このような低ＬＤＬ検体においても、ＬＤＬ－Ｃを正確に測定することができる。 In the measurement method of the present invention, at least one substance selected from the group consisting of polyoxyethylene polycyclic ether sulfate (hereinafter abbreviated as POE polycyclic ether sulfate) and arylsulfonic acid derivatives is used. Thus, it can be suitably used for the measurement of LDL-C in a sample containing a very small amount of LDL (hereinafter referred to as a low LDL sample), such as serum derived from a patient with low LDLemia. The measurement of LDL-C using a low LDL sample is easily affected by chylomicrons and interfering substances in the sample, and in the conventional method, the LDL-C concentration determined by the measurement may be a negative value. It was. Low LDL specimens contain a small amount of LDL, and LDL contains cholesterol, so low LDL specimens contain a small amount of LDL-C. Therefore, the LDL-C concentration determined by measurement is inherently a positive value and never a negative value. In the measurement method of the present invention, by using at least one substance selected from the group consisting of POE polycyclic ether sulfates and aryl sulfonic acid derivatives, LDL-C is accurately measured even in such low LDL samples. can do.
　ＰＯＥ多環エーテル硫酸エステル塩としては、例えばポリオキシエチレン多環フェニルエーテル硫酸エステル塩、ポリオキシエチレンナフチルエーテル硫酸エステル塩等が挙げられる。塩としては、例えばナトリウム塩、カリウム塩、リチウム塩、アンモニウム塩、マグネシウム塩等が挙げられる。ＰＯＥ多環エーテル硫酸エステル塩の具体例（市販品）としては、例えばニューコール７０７ＳＦ、ニューコールＢ４－ＳＮ（以上、日本乳化剤社製）等が挙げられる。 Examples of the POE polycyclic ether sulfate ester salt include polyoxyethylene polycyclic phenyl ether sulfate ester salt and polyoxyethylene naphthyl ether sulfate ester salt. Examples of the salt include sodium salt, potassium salt, lithium salt, ammonium salt, magnesium salt and the like. Specific examples (commercially available products) of POE polycyclic ether sulfate ester salts include, for example, New Coal 707SF, New Coal B4-SN (manufactured by Nippon Emulsifier Co., Ltd.) and the like.
　アリールスルホン酸誘導体としては、例えばナフタレンスルホン酸ホルマリン縮合物等が挙げられる。アリールスルホン酸誘導体の具体例（市販品）としては、例えばディスロールＳＨ（日本乳化剤社製）、デモールＲＮ、デモールＭＳ、デモールＳＮ－Ｂ（以上、花王社製）等が挙げられる。 Examples of the aryl sulfonic acid derivative include naphthalene sulfonic acid formalin condensate. Specific examples (commercially available) of the aryl sulfonic acid derivatives include Disrol SH (manufactured by Nippon Emulsifier Co., Ltd.), Demol RN, Demol MS, Demol SN-B (manufactured by Kao Corporation), and the like.
　本発明のＬＤＬ－Ｃ測定方法における反応液中のＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体それぞれの濃度としては、本発明のＬＤＬ－Ｃの測定を可能とする濃度であれば特に制限はなく、通常、０．００５～７．５ｇ／Ｌであり、好ましくは、０．０１～６ｇ／Ｌである。 The concentration of each of the POE polycyclic ether sulfate ester salt and the arylsulfonic acid derivative in the reaction solution in the LDL-C measurement method of the present invention is not particularly limited as long as it allows the measurement of LDL-C of the present invention. Usually, it is 0.005 to 7.5 g / L, and preferably 0.01 to 6 g / L.
　本発明において用いられる水性媒体は、本発明のＬＤＬ－Ｃの測定方法を可能とする水性媒体であれば特に制限はなく、例えば脱イオン水、蒸留水、緩衝液等が挙げられ、緩衝液が好ましい。 The aqueous medium used in the present invention is not particularly limited as long as it is an aqueous medium that enables the LDL-C measurement method of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution. preferable.
　本発明のＬＤＬ－Ｃの測定方法におけるｐＨは、本発明のＬＤＬ－Ｃの測定方法を可能とするｐＨであればいずれでもよいが、例えばｐＨ４～１０が挙げられる。水性媒体として緩衝液を用いる場合には、設定するｐＨに応じた緩衝剤を用いることが望ましい。緩衝液に用いる緩衝剤としては、例えばトリス（ヒドロキシメチル）アミノメタン緩衝剤、リン酸緩衝剤、ホウ酸緩衝剤、グッドの緩衝剤等が挙げられる。 The pH in the LDL-C measurement method of the present invention may be any pH as long as it enables the LDL-C measurement method of the present invention, and examples thereof include pH 4 to 10. When a buffer solution is used as the aqueous medium, it is desirable to use a buffering agent corresponding to the set pH. Examples of the buffer used in the buffer include tris (hydroxymethyl) aminomethane buffer, phosphate buffer, borate buffer, Good's buffer, and the like.
　グッドの緩衝剤としては、例えば２－モルホリノエタンスルホン酸（ＭＥＳ）、ビス（２－ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Ｂｉｓ－Ｔｒｉｓ）、Ｎ－（２－アセトアミド）イミノ二酢酸（ＡＤＡ）、ピペラジン－Ｎ，Ｎ’－ビス（２－エタンスルホン酸）（ＰＩＰＥＳ）、Ｎ－（２－アセトアミド）－２－アミノエタンスルホン酸（ＡＣＥＳ）、３－モルホリノ－２－ヒドロキシプロパンスルホン酸（ＭＯＰＳＯ）、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）－２－アミノエタンスルホン酸（ＢＥＳ）、３－モルホリノプロパンスルホン酸（ＭＯＰＳ）、Ｎ－〔トリス（ヒドロキシメチル）メチル〕－２－アミノエタンスルホン酸（ＴＥＳ）、２－〔４－（２－ヒドロキシエチル）－１－ピペラジニル〕エタンスルホン酸（ＨＥＰＥＳ）、３－〔Ｎ，Ｎ－ビス（２－ヒドロキシエチル）アミノ〕－２－ヒドロキシプロパンスルホン酸（ＤＩＰＳＯ）、Ｎ－〔トリス（ヒドロキシメチル）メチル〕－２－ヒドロキシ－３－アミノプロパンスルホン酸（ＴＡＰＳＯ）、ピペラジン－Ｎ，Ｎ’－ビス（２－ヒドロキシプロパンスルホン酸）（ＰＯＰＳＯ）、３－〔４－（２－ヒドロキシエチル）－１－ピペラジニル〕－２－ヒドロキシプロパンスルホン酸（ＨＥＰＰＳＯ）、３－〔４－（２－ヒドロキシエチル）－１－ピペラジニル〕プロパンスルホン酸〔（Ｈ）ＥＰＰＳ〕、Ｎ－〔トリス（ヒドロキシメチル）メチル〕グリシン（Ｔｒｉｃｉｎｅ）、Ｎ，Ｎ－ビス（２－ヒドロキシエチル）グリシン（Ｂｉｃｉｎｅ）、Ｎ－トリス（ヒドロキシメチル）メチル－３－アミノプロパンスルホン酸（ＴＡＰＳ）、Ｎ－シクロヘキシル－２－アミノエタンスルホン酸（ＣＨＥＳ）、Ｎ－シクロヘキシル－３－アミノ－２－ヒドロキシプロパンスルホン酸（ＣＡＰＳＯ）、Ｎ－シクロヘキシル－３－アミノプロパンスルホン酸（ＣＡＰＳ）等が挙げられる。 Good buffering agents include, for example, 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminodiacetic acid (ADA) Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) ), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] -2-aminoethanesulfone Acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethane Sulfonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), N- [tris (hydroxymethyl) methyl] -2-hydroxy-3- Aminopropanesulfonic acid (TAPSO), piperazine-N, N′-bis (2-hydroxypropanesulfonic acid) (POPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] -2-hydroxypropanesulfone Acid (HEPPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid [(H) EPPS], N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N— Bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) Tyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3- Examples include aminopropane sulfonic acid (CAPS).
　緩衝液の濃度は測定に適した濃度であれば特に制限はされないが、０．００１～２．０ｍｏｌ／Ｌが好ましく、０．００５～１．０ｍｏｌ／Ｌがより好ましい。 The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L.
　本発明のＬＤＬ－Ｃの測定方法における反応温度は、本発明のＬＤＬ－Ｃの測定方法を可能とする温度であれば特に制限はないが、１０～５０℃が好ましく、３０～４０℃がより好ましい。汎用の自動分析装置で設定される反応温度は通常３７℃である。 The reaction temperature in the LDL-C measurement method of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, but is preferably 10 to 50 ° C., more preferably 30 to 40 ° C. preferable. The reaction temperature set by a general-purpose automatic analyzer is usually 37 ° C.
　本発明のＬＤＬ－Ｃの測定方法における反応時間は、本発明のＬＤＬ－Ｃの測定方法を可能とする反応時間であれば特に制限はないが、１～６０分間が好ましく、２～３０分間がより好ましい。 The reaction time in the LDL-C measurement method of the present invention is not particularly limited as long as the reaction time enables the LDL-C measurement method of the present invention, but it is preferably 1 to 60 minutes, and preferably 2 to 30 minutes. More preferred.
　本発明のＬＤＬ－Ｃの測定方法において、ＬＤＬ－Ｃの測定は、反応により生成した過酸化水素の量を測定することにより行うことができる。 In the LDL-C measurement method of the present invention, LDL-C can be measured by measuring the amount of hydrogen peroxide generated by the reaction.
　生成した過酸化水素の量は、例えば過酸化水素電極や過酸化水素測定用試薬を用いて測定することができる。過酸化水素測定用試薬は、生成した過酸化水素を検出可能な物質へ変換するための試薬である。検出可能な物質としては、例えば色素、発光等が挙げられるが、色素が好ましい。検出可能な物質が色素の場合には、過酸化水素測定用試薬は、酸化発色型色原体及びペルオキシダーゼ等の過酸化活性物質を含有する。酸化発色型色原体としては、例えば後述の酸化発色型色原体が挙げられる。検出可能な物質が発光の場合には、過酸化水素測定用試薬は、化学発光物質を含有する。化学発光物質としては、例えばルミノール、イソルミノール、ルシゲニン、アクリジニウムエステル等が挙げられる。 The amount of generated hydrogen peroxide can be measured using, for example, a hydrogen peroxide electrode or a hydrogen peroxide measuring reagent. The reagent for measuring hydrogen peroxide is a reagent for converting the generated hydrogen peroxide into a detectable substance. Examples of detectable substances include dyes and luminescence, and dyes are preferred. When the detectable substance is a dye, the hydrogen peroxide measurement reagent contains an oxidation coloring type chromogen and a peroxidation active substance such as peroxidase. Examples of the oxidative coloring type chromogen include the following oxidative coloring type chromogen. When the detectable substance is luminescence, the hydrogen peroxide measuring reagent contains a chemiluminescent substance. Examples of the chemiluminescent substance include luminol, isoluminol, lucigenin, and acridinium ester.
　過酸化水素測定用試薬として、酸化発色型色原体及びペルオキシダーゼ等の過酸化活性物質を含有する試薬を用いる場合には、過酸化水素は、過酸化活性物質の存在下に酸化発色型色原体と反応して色素を生成し、生成した色素を測定することにより、測定することができる。また、化学発光物質を含有する過酸化水素測定用試薬を用いる場合には、過酸化水素は、化学発光物質と反応してフォトンを生じ、生じたフォトンを測定することにより、測定することができる。 In the case of using a reagent containing an oxidative coloring type chromogen and a peroxidase active substance such as peroxidase as the hydrogen peroxide measurement reagent, hydrogen peroxide is oxidized in the presence of the peroxidative active substance. It can measure by producing | generating a pigment | dye by reacting with a body and measuring the produced | generated pigment | dye. When a reagent for measuring hydrogen peroxide containing a chemiluminescent substance is used, hydrogen peroxide can be measured by reacting with the chemiluminescent substance to generate photons and measuring the generated photons. .
　酸化発色型色原体としては、例えばロイコ型色原体、酸化カップリング発色型色原体等が挙げられる。ロイコ型色原体は、過酸化水素及びペルオキシダーゼ等の過酸化活性物質の存在下、単独で色素へ変換される物質である。具体的には、テトラメチルベンジジン、ｏ－フェニレンジアミン、１０－Ｎ－カルボキシメチルカルバモイル－３，７－ビス（ジメチルアミノ）－１０Ｈ－フェノチアジン（ＣＣＡＰ）、１０－Ｎ－メチルカルバモイル－３，７－ビス（ジメチルアミノ）－１０Ｈ－フェノチアジン（ＭＣＤＰ）、Ｎ－（カルボキシメチルアミノカルボニル）－４，４’－ビス（ジメチルアミノ）ジフェニルアミン ナトリウム塩（ＤＡ－６４）、１０－Ｎ－（カルボキシメチルアミノカルボニル）－３，７－ビス（ジメチルアミノ）－１０Ｈ－フェノチアジン ナトリウム塩（ＤＡ－６７）、４，４’－ビス（ジメチルアミノ）ジフェニルアミン、ビス〔３－ビス（４－クロロフェニル）メチル－４－ジメチルアミノフェニル〕アミン（ＢＣＭＡ）等が挙げられる。 Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen. A leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (DA-64), 10-N- (carboxymethylaminocarbonyl) ) -3,7-bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67), 4,4′-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethyl Aminophenyl] amine (BCMA), etc. It is.
　酸化カップリング発色型色原体は、過酸化水素及びペルオキシダーゼ等の過酸化活性物質の存在下、２つの化合物が酸化的カップリングして色素を生成する物質である。２つの化合物の組み合わせとしては、カプラーとアニリン類との組み合わせ、カプラーとフェノール類との組み合わせ等が挙げられる。 The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline, a combination of a coupler and a phenol.
　カプラーとしては、例えば４－アミノアンチピリン（４－ＡＡ）、３－メチル－２－ベンゾチアゾリノンヒドラゾン等が挙げられる。 Examples of couplers include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone.
　アニリン類としては、Ｎ－（３－スルホプロピル）アニリン、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（ＴＯＯＳ）、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３，５－ジメチルアニリン（ＭＡＯＳ）、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（ＤＡＯＳ）、Ｎ－エチル－Ｎ－（３－スルホプロピル）－３－メチルアニリン（ＴＯＰＳ）、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（ＨＤＡＯＳ）、Ｎ，Ｎ－ジメチル－３－メチルアニリン、Ｎ，Ｎ－ジ（３－スルホプロピル）－３，５－ジメトキシアニリン、Ｎ－エチル－Ｎ－（３－スルホプロピル）－３－メトキシアニリン、Ｎ－エチル－Ｎ－（３－スルホプロピル）アニリン、Ｎ－エチル－Ｎ－（３－スルホプロピル）－３，５－ジメトキシアニリン、Ｎ－（３－スルホプロピル）－３，５－ジメトキシアニリン、Ｎ－エチル－Ｎ－（３－スルホプロピル）－３，５－ジメチルアニリン、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－３－メトキシアニリン、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）アニリン、Ｎ－エチル－Ｎ－（３－メチルフェニル）－Ｎ’－サクシニルエチレンジアミン（ＥＭＳＥ）、Ｎ－（３，５－ジメトキシフェニル）－Ｎ’－サクシニルエチレンジアミン（ＤＯＳＥ）、Ｎ－エチル－Ｎ－（３－メチルフェニル）－Ｎ’－アセチルエチレンジアミン、Ｎ－エチル－Ｎ－（２－ヒドロキシ－３－スルホプロピル）－４－フルオロ－３，５－ジメトキシアニリン（Ｆ－ＤＡＯＳ）、Ｎ－［２－（サクシニルアミノ）エチル］－２－メトキシ－５－メチルアニリン（ＭＡＳＥ）、Ｎ－エチル－Ｎ－［２－（サクシニルアミノ）エチル］－２－メトキシ－５－メチルアニリン（Ｅｔ－ＭＡＳＥ）等が挙げられる。 Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy -3-Sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl N- (3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl- N- (3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3 -Sulfopropyl) aniline, N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (EMSE), N- (3,5-dimethoxyphenyl) -N'-succinylethylenediamine (DOSE), N- Ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (2-hydroxy-3-sulfopropyl)- -Fluoro-3,5-dimethoxyaniline (F-DAOS), N- [2- (succinylamino) ethyl] -2-methoxy-5-methylaniline (MASE), N-ethyl-N- [2- (succinyl) Amino) ethyl] -2-methoxy-5-methylaniline (Et-MASE) and the like.
　フェノール類としては、フェノール、４－クロロフェノール、３－メチルフェノール、３－ヒドロキシ－２，４，６－トリヨード安息香酸（ＨＴＩＢ）等が挙げられる。 Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.
　過酸化水素の測定において、過酸化活性物質の濃度は、測定に適した濃度であれば特に制限はないが、過酸化活性物質としてペルオキシダーゼを用いる場合は、１～１００ｋＵ／Ｌが好ましい。また、酸化発色型色原体の濃度は、過酸化水素の測定に適した濃度であれば特に制限はないが、０．０１～１０ｇ／Ｌが好ましい。 In the measurement of hydrogen peroxide, the concentration of the peroxide active substance is not particularly limited as long as it is a concentration suitable for the measurement, but when peroxidase is used as the peroxide active substance, 1 to 100 kU / L is preferable. The concentration of the oxidative coloring type chromogen is not particularly limited as long as it is a concentration suitable for the measurement of hydrogen peroxide, but is preferably 0.01 to 10 g / L.
　（ＬＤＬ－Ｃ測定用試薬・測定用キット）
　本発明のＬＤＬ－Ｃ測定用試薬は、ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、コレステロールエステル加水分解酵素、及び、コレステロール酸化酵素を含有する試薬である。本発明のＬＤＬ－Ｃ測定用試薬は、さらに、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を含むことができる。 (LDL-C measurement reagent / measurement kit)
The reagent for LDL-C measurement of the present invention is a reagent containing POE / POA alkylaryl ether, POE / POA condensate, POE alkylamine, polyanion, cholesterol ester hydrolase, and cholesterol oxidase. The LDL-C measurement reagent of the present invention can further contain at least one substance selected from the group consisting of POE polycyclic ether sulfate salts and arylsulfonic acid derivatives.
　本発明のＬＤＬ－Ｃ測定用試薬は、本発明のＬＤＬ－Ｃ測定方法に用いることができる。本発明のＬＤＬ－Ｃ測定用試薬は、保存、流通及び使用に適したキットの形態を取ることができる。本発明のＬＤＬ－Ｃ測定用キットとしては、例えば２試薬系のキット、３試薬系のキット等が挙げられるが、第一試薬と第二試薬とからなる２試薬系のキットが好ましい。 The LDL-C measurement reagent of the present invention can be used in the LDL-C measurement method of the present invention. The LDL-C measurement reagent of the present invention can take the form of a kit suitable for storage, distribution and use. Examples of the LDL-C measurement kit of the present invention include a two-reagent kit, a three-reagent kit, and the like, and a two-reagent kit comprising a first reagent and a second reagent is preferred.
　本発明のＬＤＬ－Ｃ測定用試薬及び測定用キットは、凍結乾燥された状態でも、水性媒体に溶解された状態でもよい。凍結乾燥された状態の試薬又はキットを用いて検体中のＬＤＬ－Ｃを測定する場合には、当該試薬は水性媒体に溶解して使用される。該水性媒体としては、例えば前述の水性媒体等が挙げられる。 The LDL-C measurement reagent and measurement kit of the present invention may be lyophilized or dissolved in an aqueous medium. When LDL-C in a specimen is measured using a lyophilized reagent or kit, the reagent is used after being dissolved in an aqueous medium. Examples of the aqueous medium include the aforementioned aqueous medium.
　本発明のＬＤＬ－Ｃ測定用試薬及び測定用キットにおいては、前述のコレステロールエステル加水分解酵素、コレステロール酸化酵素、ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩、アリールスルホン酸誘導体、及び、過酸化水素測定用試薬を用いることができる。 In the LDL-C measuring reagent and measuring kit of the present invention, the aforementioned cholesterol ester hydrolase, cholesterol oxidase, POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, polyanion, POE polycycle An ether sulfate ester salt, an aryl sulfonic acid derivative, and a reagent for measuring hydrogen peroxide can be used.
　第一試薬と第二試薬とからなる２試薬系のＬＤＬ－Ｃ測定用キットにおいては、コレステロールエステル加水分解酵素は、第一試薬、第二試薬のいずれか又は両方に含まれる。コレステロール酸化酵素は第一試薬に含まれず、第二試薬に含まれる。ＰＯＥ・ＰＯＡアリールエーテルは、第一試薬、第二試薬のいずれか又は両方に含まれてよいが、第二試薬に含まれる態様が好ましい。ＰＯＥ・ＰＯＡ縮合物は、第一試薬、第二試薬のいずれか又は両方に含まれてよいが、第二試薬に含まれる態様が好ましい。ＰＯＥアルキルアミンは、第二試薬に含まれる。ポリアニオンは第一試薬に含まれる。 In a two-reagent LDL-C measurement kit comprising a first reagent and a second reagent, cholesterol esterase is included in either or both of the first reagent and the second reagent. Cholesterol oxidase is not contained in the first reagent, but is contained in the second reagent. The POE / POA aryl ether may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the second reagent is preferred. The POE / POA condensate may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the second reagent is preferred. POE alkylamine is included in the second reagent. The polyanion is included in the first reagent.
　ＰＯＥ多環エーテル硫酸エステル塩は、第一試薬、第二試薬のいずれか又は両方に含まれてよいが、第一試薬に含まれる態様が好ましい。 The POE polycyclic ether sulfate ester salt may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the first reagent is preferred.
　アリールスルホン酸誘導体は、第一試薬、第二試薬のいずれか又は両方に含まれてよいが、第一試薬に含まれる態様が好ましい。 The aryl sulfonic acid derivative may be contained in either or both of the first reagent and the second reagent, but the embodiment contained in the first reagent is preferred.
　過酸化水素測定用試薬は、第一試薬、第二試薬のいずれか又は両方に含有されてもよいが、当該試薬が酸化カップリング型色原体を含む場合には、酸化カップリング型色原体の２つの化合物、すなわち、カプラーとアニリン類、又は、カプラーとフェノール類はそれぞれ別々の試薬に含まれる態様が好ましい。 The reagent for measuring hydrogen peroxide may be contained in one or both of the first reagent and the second reagent, but when the reagent contains an oxidative coupling type chromogen, the oxidative coupling type chromogen is included. It is preferable that the two compounds, ie, coupler and aniline, or coupler and phenol are contained in separate reagents.
　本発明のＬＤＬ－Ｃ測定用試薬におけるコレステロールエステル加水分解酵素の濃度は、通常０．００１～８００ｋＵ／Ｌであり、好ましくは０．０１～３００ｋＵ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、コレステロールエステル加水分解酵素の含量は、通常、水性媒体で溶解された状態での濃度が０．００１～８００ｋＵ／Ｌ、好ましくは０．０１～３００ｋＵ／Ｌとなる含量である。 The concentration of cholesterol esterase in the LDL-C measurement reagent of the present invention is usually 0.001 to 800 kU / L, preferably 0.01 to 300 kU / L. In the lyophilized reagent for LDL-C measurement, the content of cholesterol esterase is usually 0.001 to 800 kU / L, preferably 0.01 when dissolved in an aqueous medium. The content is ˜300 kU / L.
　本発明のＬＤＬ－Ｃ測定用キットにおけるコレステロールエステル加水分解酵素の濃度は、通常０．００４～３２００ｋＵ／Ｌであり、好ましくは０．０４～１２００ｋＵ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、コレステロールエステル加水分解酵素の含量は、通常、水性媒体で溶解された状態での濃度が０．００４～３２００ｋＵ／Ｌ、好ましくは０．０４～１２００ｋＵ／Ｌとなる含量である。 The concentration of cholesterol esterase in the LDL-C measurement kit of the present invention is usually 0.004 to 3200 kU / L, preferably 0.04 to 1200 kU / L. In the LDL-C measurement kit in a lyophilized state, the content of cholesterol esterase is usually 0.004 to 3200 kU / L, preferably 0.04 when dissolved in an aqueous medium. The content is ˜1200 kU / L.
　本発明のＬＤＬ－Ｃ測定用試薬におけるＰＯＥ・ＰＯＡアルキルアリールエーテルの濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００１～２００ｇ／Ｌであり、好ましくは０．０１～５０ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、ＰＯＥ・ＰＯＡアルキルアリールエーテルの含量は、通常、水性媒体で溶解された状態での濃度が０．００１～２００ｇ／Ｌ、好ましくは０．０１～５０ｇ／Ｌとなる含量である。 The concentration of the POE • POA alkylaryl ether in the LDL-C measurement reagent of the present invention is not particularly limited as long as it allows the LDL-C measurement method of the present invention, and is usually 0.001 to 200 g / L. And preferably 0.01 to 50 g / L. In the reagent for LDL-C measurement in a lyophilized state, the content of the POE · POA alkylaryl ether is usually 0.001 to 200 g / L in a state of being dissolved in an aqueous medium, preferably 0. The content is from 01 to 50 g / L.
　本発明のＬＤＬ－Ｃ測定用キットにおけるＰＯＥ・ＰＯＡアルキルアリールエーテルの濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００４～８００ｇ／Ｌであり、好ましくは０．０４～２００ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、ＰＯＥ・ＰＯＡアルキルアリールエーテルの含量は、通常、水性媒体で溶解された状態での濃度が０．００４～８００ｇ／Ｌ、好ましくは０．０４～２００ｇ／Ｌとなる含量である。 The concentration of the POE / POA alkyl aryl ether in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the LDL-C measurement method of the present invention, and is usually 0.004 to 800 g / L. Preferably, it is 0.04 to 200 g / L. In the LDL-C measurement kit in a lyophilized state, the content of the POE • POA alkylaryl ether is usually 0.004 to 800 g / L in a state of being dissolved in an aqueous medium, preferably 0.8. The content is from 04 to 200 g / L.
　本発明のＬＤＬ－Ｃ測定用試薬におけるＰＯＥ・ＰＯＡ縮合物の濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００１～２００ｇ／Ｌであり、好ましくは０．０１～５０ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、ＰＯＥ・ＰＯＡ縮合物の含量は、通常、水性媒体で溶解された状態での濃度が０．００１～２００ｇ／Ｌ、好ましくは０．０１～５０ｇ／Ｌとなる含量である。 The concentration of the POE / POA condensate in the LDL-C measurement reagent of the present invention is not particularly limited as long as it is a concentration that enables the LDL-C measurement method of the present invention, and is usually 0.001 to 200 g / L. Yes, preferably 0.01 to 50 g / L. In the reagent for LDL-C measurement in a lyophilized state, the content of the POE / POA condensate is usually 0.001 to 200 g / L, preferably 0.01, when dissolved in an aqueous medium. The content is ˜50 g / L.
　本発明のＬＤＬ－Ｃ測定用キットにおけるＰＯＥ・ＰＯＡ縮合物の濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００４～８００ｇ／Ｌであり、好ましくは０．０４～２００ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、ＰＯＥ・ＰＯＡ縮合物の含量は、通常、水性媒体で溶解された状態での濃度が０．００４～８００ｇ／Ｌ、好ましくは０．０４～２００ｇ／Ｌとなる含量である。 The concentration of the POE / POA condensate in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, and is usually 0.004 to 800 g / L. Yes, preferably 0.04 to 200 g / L. In the LDL-C measurement kit in a freeze-dried state, the content of the POE / POA condensate is usually 0.004 to 800 g / L, preferably 0.04 when dissolved in an aqueous medium. The content is ˜200 g / L.
　本発明のＬＤＬ－Ｃ測定用試薬におけるポリアニオンの濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００５～１００ｇ／Ｌであり、好ましくは０．０５～１０ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、ポリアニオンの含量は、通常、水性媒体で溶解された状態での濃度が０．００５～１００ｇ／Ｌ、好ましくは０．０５～１０ｇ／Ｌとなる含量である。 The concentration of the polyanion in the LDL-C measurement reagent of the present invention is not particularly limited as long as it enables the LDL-C measurement method of the present invention, and is usually 0.005 to 100 g / L, preferably 0.05 to 10 g / L. In the LDL-C measurement reagent in a lyophilized state, the polyanion content is usually 0.005 to 100 g / L, preferably 0.05 to 10 g / L when dissolved in an aqueous medium. Content.
　本発明のＬＤＬ－Ｃ測定用キットにおけるポリアニオンの第一試薬中の濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００５～１５０ｇ／Ｌであり、好ましくは０．０５～１５ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、ポリアニオンの第一試薬中の含量は、通常、水性媒体で溶解された状態での濃度が０．００５～１５０ｇ／Ｌ、好ましくは０．０５～１５ｇ／Ｌとなる含量である。 The concentration of the polyanion in the first reagent in the LDL-C measurement kit of the present invention is not particularly limited as long as it enables the measurement method of LDL-C of the present invention, and usually 0.005 to 150 g / L. And preferably 0.05 to 15 g / L. In the LDL-C measurement kit in a lyophilized state, the content of the polyanion in the first reagent is usually 0.005 to 150 g / L in a state dissolved in an aqueous medium, and preferably is 0.1. The content is from 05 to 15 g / L.
　本発明のＬＤＬ－Ｃ測定用試薬における、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体それぞれの濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常、０．００５～７．５ｇ／Ｌであり、好ましくは０．０１～６ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体それぞれの含量は、通常、水性媒体で溶解された状態での濃度が０．００５～７．５ｇ／Ｌ、好ましくは０．０１～６ｇ／Ｌとなる含量である。 The concentrations of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative in the LDL-C measurement reagent of the present invention are not particularly limited as long as the concentration enables the LDL-C measurement method of the present invention. Usually, it is 0.005 to 7.5 g / L, preferably 0.01 to 6 g / L. In the LDL-C measurement reagent in the lyophilized state, the content of each of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative is usually 0.005 to 7 in a state dissolved in an aqueous medium. The content is 0.5 g / L, preferably 0.01 to 6 g / L.
　本発明のＬＤＬ－Ｃ測定用キットにおける、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体それぞれの濃度は、本発明のＬＤＬ－Ｃの測定方法を可能とする濃度であれば特に制限はなく、通常０．００７～１０ｇ／Ｌであり、好ましくは０．０１５～８ｇ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体それぞれの含量は、通常、水性媒体で溶解された状態での濃度が０．００７～１０ｇ／Ｌ、好ましくは０．０１５～８ｇ／Ｌとなる含量である。 The concentrations of the POE polycyclic ether sulfate ester salt and the aryl sulfonic acid derivative in the LDL-C measurement kit of the present invention are not particularly limited as long as the concentration enables the LDL-C measurement method of the present invention. Usually, it is 0.007 to 10 g / L, and preferably 0.015 to 8 g / L. In the LDL-C measurement kit in the lyophilized state, the content of each of the POE polycyclic ether sulfate ester and the aryl sulfonic acid derivative is usually 0.007 to 10 g when dissolved in an aqueous medium. / L, preferably 0.015 to 8 g / L.
　本発明のＬＤＬ－Ｃ測定用試薬におけるコレステロール酸化酵素濃度は、通常ＬＤＬ－Ｃ測定用試薬中のＰＯＥドデシルアミン０．０６～０．３ｍｍｏｌ／Ｌに対して、０．２５～１．０ｋＵ／Ｌであり、ＬＤＬ－Ｃ測定用試薬中のＰＯＥオクタデシルアミン０．０２～０．０７５ｍｍｏｌ／Ｌに対して、０．５～２．０ｋＵ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用試薬においては、コレステロール酸化酵素濃度の含量は、通常、水性媒体で溶解された状態でのコレステロール酸化酵素濃度が、ＰＯＥドデシルアミン０．０６～０．３ｍｍｏｌ／Ｌに対して、０．２５～１．０ｋＵ／Ｌとなる含量であり、ＰＯＥオクタデシルアミン０．０２～０．０７５ｍｍｏｌ／Ｌに対して、０．５～２．０ｋＵ／Ｌとなる含量である。 The cholesterol oxidase concentration in the LDL-C measurement reagent of the present invention is usually 0.25 to 1.0 kU / L relative to POE dodecylamine 0.06 to 0.3 mmol / L in the LDL-C measurement reagent. 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of POE octadecylamine in the reagent for LDL-C measurement. In the LDL-C measurement reagent in the lyophilized state, the cholesterol oxidase concentration is usually 0.06 to 0.3 mmol of POE dodecylamine when dissolved in an aqueous medium. The content is 0.25 to 1.0 kU / L with respect to / L, and the content is 0.5 to 2.0 kU / L with respect to 0.02 to 0.075 mmol / L of POE octadecylamine. is there.
　本発明のＬＤＬ－Ｃ測定用キットにおけるコレステロール酸化酵素の第二試薬中の濃度は、通常第二試薬中のＰＯＥドデシルアミン０．２５～１．２５ｍｍｏｌ／Ｌに対して、１．０～４．０ｋＵ／Ｌであり、第二試薬中のＰＯＥオクタデシルアミン０．０７５～０．３ｍｍｏｌ／Ｌに対して、２．０～８．０ｋＵ／Ｌである。凍結乾燥された状態のＬＤＬ－Ｃ測定用キットにおいては、コレステロール酸化酵素の第二試薬中の含量は、通常、水性媒体で溶解された状態でのコレステロール酸化酵素の濃度が、ＰＯＥドデシルアミン０．２５～１．２５ｍｍｏｌ／Ｌに対して、１．０～４．０ｋＵ／Ｌとなる含量であり、ＰＯＥオクタデシルアミン０．０７５～０．３ｍｍｏｌ／Ｌに対して、２．０～８．０ｋＵ／Ｌとなる含量である。 The concentration of cholesterol oxidase in the second reagent in the LDL-C measurement kit of the present invention is usually 1.0 to 4.4 with respect to 0.25 to 1.25 mmol / L of POE dodecylamine in the second reagent. 0 kU / L, and 2.0 to 8.0 kU / L with respect to 0.075 to 0.3 mmol / L of POE octadecylamine in the second reagent. In the LDL-C measurement kit in a lyophilized state, the content of cholesterol oxidase in the second reagent is usually such that the concentration of cholesterol oxidase dissolved in an aqueous medium is POE dodecylamine. The content is 1.0 to 4.0 kU / L with respect to 25 to 1.25 mmol / L, and is 2.0 to 8.0 kU / L with respect to 0.075 to 0.3 mmol / L of POE octadecylamine. The content of L.
　本発明のＬＤＬ－Ｃ測定用試薬及び測定用キットには、必要に応じて、水性媒体、安定化剤、防腐剤、影響物質消去剤、反応促進剤等が含有されてもよい。水性媒体としては、例えば前述の水性媒体等が挙げられる。安定化剤としては、例えばエチレンジアミン四酢酸（ＥＤＴＡ）、シュークロース、塩化カルシウム等が挙げられる。防腐剤としては、例えばアジ化ナトリウム、抗生物質等が挙げられる。影響物質消去剤としては、例えばアスコルビン酸の影響を消去するためのアスコルビン酸オキシダーゼ等が挙げられる。反応促進剤としては、例えばコリパーゼ、ホスホリパーゼ等の酵素、硫酸ナトリウム、塩化ナトリウム等の塩類等が挙げられる。 The LDL-C measurement reagent and measurement kit of the present invention may contain an aqueous medium, a stabilizer, an antiseptic, an influential substance erasing agent, a reaction accelerator and the like, if necessary. As an aqueous medium, the above-mentioned aqueous medium etc. are mentioned, for example. Examples of the stabilizer include ethylenediaminetetraacetic acid (EDTA), sucrose, calcium chloride and the like. Examples of the preservative include sodium azide and antibiotics. Examples of the influence substance eliminating agent include ascorbate oxidase for eliminating the influence of ascorbic acid. Examples of the reaction accelerator include enzymes such as colipase and phospholipase, and salts such as sodium sulfate and sodium chloride.
　以下に、本発明のＬＤＬ－Ｃ測定用試薬の具体的態様を記すが、本発明のＬＤＬ－Ｃ測定用試薬はこれらに限定されない。
・試薬１
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、コレステロールエステル加水分解酵素、及び、コレステロール酸化酵素を含有する試薬。
・試薬２
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、コレステロールエステル加水分解酵素、及び、コレステロール酸化酵素を含有する試薬。 Specific embodiments of the reagent for LDL-C measurement of the present invention are described below, but the reagent for LDL-C measurement of the present invention is not limited to these.
・ Reagent 1
A reagent containing POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, polyanion, cholesterol ester hydrolase, and cholesterol oxidase.
・ Reagent 2
At least one substance selected from the group consisting of POE · POA alkyl aryl ethers, POE · POA condensates, POE alkyl amines, polyanions, POE polycyclic ether sulfate salts and aryl sulfonic acid derivatives, cholesterol ester hydrolase, and A reagent containing cholesterol oxidase.
・試薬３
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、コレステロールエステル加水分解酵素、コレステロール酸化酵素、及び、過酸化水素測定用試薬を含有する試薬。
・試薬４
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、コレステロールエステル加水分解酵素、コレステロール酸化酵素、及び、過酸化水素測定用試薬を含有する試薬。 ・ Reagent 3
A reagent comprising a POE / POA alkyl aryl ether, a POE / POA condensate, a POE alkyl amine, a polyanion, a cholesterol ester hydrolase, a cholesterol oxidase, and a reagent for measuring hydrogen peroxide.
・ Reagent 4
POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, polyanion, POE polycyclic ether sulfate ester and at least one substance selected from the group consisting of aryl sulfonic acid derivatives, cholesterol ester hydrolase, cholesterol oxidation A reagent containing an enzyme and a reagent for measuring hydrogen peroxide.
　以下に、本発明のＬＤＬ－Ｃ測定用キットの具体的態様を記すが、本発明のＬＤＬ－Ｃ測定用キットはこれらに限定されない。
・キット１
第一試薬
　ポリアニオン
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロールエステル加水分解酵素、コレステロール酸化酵素
・キット２
第一試薬
　ポリアニオン、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロール酸化酵素
・キット３
第一試薬
　ポリアニオン、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロールエステル加水分解酵素、コレステロール酸化酵素 Specific embodiments of the LDL-C measurement kit of the present invention are described below, but the LDL-C measurement kit of the present invention is not limited thereto.
・ Kit 1
First Reagent Polyanion Second Reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, cholesterol ester hydrolase, cholesterol oxidase / kit 2
First Reagent Polyanion, Cholesterol Ester Hydrolase Second Reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkylamine, cholesterol oxidase / kit 3
1st reagent Polyanion, cholesterol ester hydrolase 2nd reagent POE / POA alkylaryl ether, POE / POA condensate, POE alkylamine, cholesterol ester hydrolase, cholesterol oxidase
・キット４
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロールエステル加水分解酵素、コレステロール酸化酵素
・キット５
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロール酸化酵素
・キット６
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、コレステロールエステル加水分解酵素、コレステロール酸化酵素 ・ Kit 4
First reagent At least one substance selected from the group consisting of polyanions, POE polycyclic ether sulfates and aryl sulfonic acid derivatives. Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, cholesterol ester hydrolysate Degradation enzyme, cholesterol oxidase kit 5
First Reagent: At least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE Alkylamine, cholesterol oxidase kit 6
First Reagent: At least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE Alkylamine, cholesterol ester hydrolase, cholesterol oxidase
・キット７
第一試薬
　ポリアニオン、過酸化水素測定用試薬
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素、コレステロール酸化酵素
・キット８
第一試薬
　ポリアニオン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロール酸化酵素
・キット９
第一試薬
　ポリアニオン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素、コレステロール酸化酵素 ・ Kit 7
First reagent Polyanion, Reagent for hydrogen peroxide measurement Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, hydrogen peroxide measurement reagent, cholesterol esterase, cholesterol oxidase kit 8
First reagent Polyanion, Reagent for measuring hydrogen peroxide, Cholesterol ester hydrolase Second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, Reagent for measuring hydrogen peroxide, Cholesterol oxidase kit 9
First reagent Polyanion, hydrogen peroxide measurement reagent, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkyl amine, hydrogen peroxide measurement reagent, cholesterol ester hydrolase, Cholesterol oxidase
・キット１０
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、過酸化水素測定用試薬
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素、コレステロール酸化酵素
・キット１１
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、過酸化水素測定用試薬、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロール酸化酵素
・キット１２
第一試薬
　ポリアニオン、ＰＯＥ多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質、過酸化水素測定用試薬、コレステロールエステル加水分解酵素
第二試薬
　ＰＯＥ・ＰＯＡアルキルアリールエーテル、ＰＯＥ・ＰＯＡ縮合物、ＰＯＥアルキルアミン、過酸化水素測定用試薬、コレステロールエステル加水分解酵素、コレステロール酸化酵素 ・ Kit 10
First reagent: at least one substance selected from the group consisting of polyanions, POE polycyclic ether sulfates and aryl sulfonic acid derivatives; reagent for measuring hydrogen peroxide; second reagent; POE / POA alkyl aryl ether; POE / POA condensate; POE alkylamine, hydrogen peroxide measurement reagent, cholesterol ester hydrolase, cholesterol oxidase kit 11
First reagent: at least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, hydrogen peroxide measuring reagent, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkylamine, hydrogen peroxide measuring reagent, cholesterol oxidase kit 12
First reagent: at least one substance selected from the group consisting of polyanion, POE polycyclic ether sulfate salt and aryl sulfonic acid derivative, hydrogen peroxide measuring reagent, cholesterol ester hydrolase second reagent POE / POA alkyl aryl ether, POE / POA condensate, POE alkylamine, hydrogen peroxide measuring reagent, cholesterol ester hydrolase, cholesterol oxidase
　以下、実施例により本発明をより詳細に説明するが、これらは本発明の範囲を何ら限定するものではない。尚、本実施例及び比較例においては、下記メーカーの試薬、酵素及び界面活性剤を使用した。 Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the scope of the present invention. In the examples and comparative examples, reagents, enzymes and surfactants from the following manufacturers were used.
試薬
　ＭＯＰＳ（同仁化学研究所社製）
　ＤＯＳＥ（ダイトーケミックス社製）
　硫酸ナトリウム（関東化学社製）
　デキストラン硫酸ナトリウム（分子量５０万）（アマシャム社製）
　４－アミノアンチピリン（埼京化成社製） Reagent MOPS (manufactured by Dojindo Laboratories)
DOSE (manufactured by Daitokemix)
Sodium sulfate (manufactured by Kanto Chemical)
Dextran sulfate sodium (molecular weight 500,000) (Amersham)
4-aminoantipyrine (manufactured by Saikyo Kasei Co., Ltd.)
酵素
　ペルオキシダーゼ（東洋紡績社製）
　ＣＨＯＤＩ（コレステロール酸化酵素；キッコーマン社製）
　ＬＰＬ－３１１（コレステロールエステル加水分解酵素；東洋紡績社製） Enzyme peroxidase (Toyobo Co., Ltd.)
CHODI (cholesterol oxidase; manufactured by Kikkoman Corporation)
LPL-311 (Cholesterol ester hydrolase; manufactured by Toyobo Co., Ltd.)
界面活性剤
　ブラウノンＬ－２０５（ポリオキシエチレンドデシルアミン；青木油脂社製）
　ナイミーンＳ－２０４（ポリオキシエチレンオクタデシルアミン；日油社製）
　アクロネセスＫＰ１８９Ｒ（ＰＯＥ・ＰＯＡアルキルアリールエーテル；日油社製）
　プルロニックＬ－１２１（ＰＯＥ・ＰＯＡ縮合物；ＡＤＥＫＡ社製）
　ニューコール７０７ＳＦ（ＰＯＥ多環エーテル硫酸エステル塩）；日本乳化剤社製）
　ニューコールＢ４－ＳＮ（ＰＯＥ多環エーテル硫酸エステル塩；日本乳化剤社製）
　ディスロールＳＨ（アリールスルホン酸誘導体；日本乳化剤社製）
　デモールＲＮ（アリールスルホン酸誘導体；花王社製）
　デモールＭＳ（アリールスルホン酸誘導体；花王社製）
　デモールＳＮ－Ｂ（アリールスルホン酸誘導体；花王社製） Surfactant Braunon L-205 (Polyoxyethylene dodecylamine; manufactured by Aoki Yushi Co., Ltd.)
Naimine S-204 (polyoxyethylene octadecylamine; manufactured by NOF Corporation)
Akroneses KP189R (POE / POA alkyl aryl ether; manufactured by NOF Corporation)
Pluronic L-121 (POE / POA condensate; manufactured by ADEKA)
New Coal 707SF (POE polycyclic ether sulfate ester); manufactured by Nippon Emulsifier Co., Ltd.)
New Coal B4-SN (POE polycyclic ether sulfate ester; manufactured by Nippon Emulsifier Co., Ltd.)
Disroll SH (aryl sulfonic acid derivative; manufactured by Nippon Emulsifier Co., Ltd.)
Demol RN (aryl sulfonic acid derivative; manufactured by Kao Corporation)
Demol MS (aryl sulfonic acid derivative; manufactured by Kao Corporation)
Demol SN-B (aryl sulfonic acid derivative; manufactured by Kao Corporation)
　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　　　０．３　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　　　７　ｇ／Ｌ
　ブラウノンＬ－２０５　　　　　　　０．２５～１．７５　ｍｍｏｌ／Ｌ
　ＬＰＬ－３１１　　　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　　　１．０～８．０　ｋＵ／Ｌ An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
Braunon L-205 0.25 to 1.75 mmol / L
LPL-311 3 kU / L
CHODI 1.0-8.0 kU / L
［比較例］　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　　　０．３　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　　　７　ｇ／Ｌ
　ＬＰＬ－３１１　　　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　　　１．０～８．０　ｋＵ／Ｌ Comparative Example An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
LPL-311 3 kU / L
CHODI 1.0-8.0 kU / L
　実施例１及び比較例の各キットを用いて、ヒト血清５０検体中のＬＤＬ－Ｃを以下の手順により測定した。
（１）検量線の作成
　標準液として、生理食塩水（ＬＤＬ－Ｃ濃度：０．０ｍｇ／ｄＬ）及び血清（ＬＤＬ－Ｃ濃度：１４０ｍｇ／ｄＬ）を、キットとして、実施例１及び比較例の各キットを用いて、それぞれ日立７１７０Ｓ形自動分析装置により、ＬＤＬ－Ｃ濃度と「吸光度」との間の関係を示す検量線を作成した。
　ここでの「吸光度」とは、以下の反応で測定された２つの吸光度（Ｅ１及びＥ２）を基に、Ｅ２からＥ１を差し引くことにより得られた値を表す。
　反応セルへ標準液（３μＬ）と第一試薬（０．１５ｍＬ）とを添加し３７℃で５分間加温し、反応液の吸光度（Ｅ１）を主波長６００ｎｍ、副波長７００ｎｍで測定し、次いで、この反応液に第二試薬（０．０５ｍＬ）を添加しさらに３７℃で５分間加温し、反応液の吸光度（Ｅ２）を主波長６００ｎｍ、副波長７００ｎｍで測定した。 Using each kit of Example 1 and Comparative Example, LDL-C in 50 samples of human serum was measured by the following procedure.
(1) Preparation of a calibration curve As standard solutions, physiological saline (LDL-C concentration: 0.0 mg / dL) and serum (LDL-C concentration: 140 mg / dL) were used as kits for Example 1 and Comparative Example. A calibration curve indicating the relationship between the LDL-C concentration and the “absorbance” was prepared using each kit and using a Hitachi 7170S automatic analyzer.
Here, “absorbance” represents a value obtained by subtracting E1 from E2 based on two absorbances (E1 and E2) measured in the following reaction.
Standard solution (3 μL) and first reagent (0.15 mL) were added to the reaction cell and heated at 37 ° C. for 5 minutes, and the absorbance (E1) of the reaction solution was measured at a main wavelength of 600 nm and a subwavelength of 700 nm, Then, a second reagent (0.05 mL) was added to the reaction solution, and the mixture was further heated at 37 ° C. for 5 minutes, and the absorbance (E2) of the reaction solution was measured at a main wavelength of 600 nm and a sub wavelength of 700 nm.
（２）ヒト血清検体と実施例１及び比較例の各キットとの反応による当該検体における「吸光度」の測定
　（１）の検量線の作成において用いた標準液の代わりにヒト血清検体を用いる以外は（１）の「吸光度」の算出方法と同様の方法により、当該検体に対する「吸光度」を測定した。 (2) Measurement of “absorbance” in the sample by reaction of the human serum sample with each of the kits of Example 1 and Comparative Example, except that the human serum sample is used instead of the standard solution used in the preparation of the calibration curve in (1) The “absorbance” for the sample was measured by the same method as the “absorbance” calculation method in (1).
（３）ヒト血清検体中のＬＤＬ－Ｃ濃度の決定
　（２）で測定出した「吸光度」と、（１）で作成した検量線とから、各検体中のＬＤＬ－Ｃ濃度を決定した。
　次いで、キットとして、市販のＬＤＬ－Ｃ測定用キットであるデタミナーＬ　ＬＤＬ－Ｃ（協和メデックス社製）を用いて、検体として同じヒト血清５０検体を用いて、上記と同様の手順により、それぞれの検体中のＬＤＬ－Ｃを測定した。 (3) Determination of LDL-C concentration in human serum sample The LDL-C concentration in each sample was determined from the “absorbance” measured in (2) and the calibration curve prepared in (1).
Next, as a kit, a commercially available LDL-C measurement kit, Determiner L LDL-C (manufactured by Kyowa Medex Co., Ltd.) was used, and 50 samples of the same human serum were used as samples. LDL-C in the specimen was measured.
　実施例１及び比較例の各キットを用いた測定と、デタミナーＬ　ＬＤＬ－Ｃを用いた測定との間の相関係数を第１表に示す。 Table 1 shows the correlation coefficient between the measurement using the kits of Example 1 and Comparative Example and the measurement using the Determiner L LDL-C.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　第１表から明らかな様に、第二試薬中のＣＨＯＤＩが１．０～４．０ｋＵ／Ｌ、第二試薬中のブラウノンＬ－２０５が０．２５～１．２５ｍｍｏｌ／Ｌの実施例１のキットを用いる測定において、市販キットを用いる測定との間に良好な相関が得られることが判明した。 As is apparent from Table 1, the CHODI in the second reagent was 1.0 to 4.0 kU / L, and the Brownian L-205 in the second reagent was 0.25 to 1.25 mmol / L. In the measurement using the kit, it was found that a good correlation was obtained with the measurement using the commercially available kit.
　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　０．３　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　７　ｇ／Ｌ
　ナイミーンＳ－２０４　　　　　０．０７５～０．３７５　ｍｍｏｌ／Ｌ
　ＬＰＬ－３１１　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　１．０～８．０　ｋＵ／Ｌ An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
Naimine S-204 0.075-0.375 mmol / L
LPL-311 3 kU / L
CHODI 1.0-8.0 kU / L
　実施例１のキットの代わりに実施例３のキットを用い、検体として実施例１の検体とは異なる検体（５０検体）を用いる以外は、実施例２に記載の方法と同様に、実施例３のキットを用いる測定と、デタミナーＬ　ＬＤＬ－Ｃを用いる測定との間の相関係数を算出した。その結果を第２表に示す。 Example 3 is the same as the method described in Example 2, except that the kit of Example 3 is used instead of the kit of Example 1 and a sample (50 samples) different from the sample of Example 1 is used as the sample. The correlation coefficient between the measurement using this kit and the measurement using Determiner L LDL-C was calculated. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　第２表から明らかな様に、第二試薬中のＣＨＯＤＩが２．０～８．０ｋＵ／Ｌ、第二試薬中のナイミーンＳ－２０４が０．０７５～０．３ｍｍｏｌ／Ｌの実施例３のキットを用いる測定において、市販キットを用いる測定との間に良好な相関が得られることが判明した。 As is apparent from Table 2, the CHODI in the second reagent was 2.0 to 8.0 kU / L, and the Niimine S-204 in the second reagent was 0.075 to 0.3 mmol / L. In the measurement using the kit, it was found that a good correlation was obtained with the measurement using the commercially available kit.
　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　０．３　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　７　ｇ／Ｌ
　ブラウノンＬ－２０５　　　　　０．７５　ｍｍｏｌ／Ｌ
　ＬＰＬ－３１１　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　３　ｋＵ／Ｌ An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
Braunon L-205 0.75 mmol / L
LPL-311 3 kU / L
CHODI 3 kU / L
　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　０．３　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　７　ｇ／Ｌ
　ナイミーンＳ－２０４　　　　　０．１５　ｍｍｏｌ／Ｌ
　ＬＰＬ－３１１　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　４　ｋＵ／Ｌ An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
Naimine S-204 0.15 mmol / L
LPL-311 3 kU / L
CHODI 4 kU / L
［試験例］
　異常リポ蛋白の１つであるＬｐＸを多量に含む肝疾患患者由来の血清［以下、ＬｐＸ検体と記す］を用いて、本発明のＬＤＬ－Ｃ測定方法において、ＬｐＸ中のコレステロールに対する反応性が抑制されることを以下の方法により示した。 [Test example]
Reactivity to cholesterol in LpX is suppressed in the LDL-C measurement method of the present invention by using serum derived from a patient with liver disease containing a large amount of LpX, which is one of abnormal lipoproteins (hereinafter referred to as LpX specimen). It was shown by the following method.
　ＬｐＸは、その構成成分であるコレステロールが、ほとんど遊離型コレステロールのみであることを特徴とする。ＬｐＸ検体そのものを下記に示す方法でゲルろ過クロマトグラフィーに付すと、ＬｐＸ画分のピークがＶＬＤＬ画分のピークと同じ位置に出現する。そこで、先ず、ＬｐＸ検体を超遠心分離により、比重が１．００６よりも小さい上清画分を分離した。すなわち、超遠心分離用チューブに生理食塩水で希釈した試料を添加し、このチューブを日立ローター５０．４ＴＩローターにセットし、２９，０００ｒｐｍで１８．５時間、超遠心分離を行い、遠心分離終了後、チューブスライサーを用いて分離した上清（カイロミクロン及びＶＬＤＬを含む）を除き、ＬｐＸを含む画分を得た。 LpX is characterized in that the constituent cholesterol is almost only free cholesterol. When the LpX specimen itself is subjected to gel filtration chromatography by the method shown below, the peak of the LpX fraction appears at the same position as the peak of the VLDL fraction. Therefore, first, a supernatant fraction having a specific gravity smaller than 1.006 was separated from the LpX specimen by ultracentrifugation. That is, a sample diluted with physiological saline is added to a tube for ultracentrifugation, this tube is set in a Hitachi rotor 50.4TI rotor, ultracentrifugation is performed at 29,000 rpm for 18.5 hours, and centrifugation is completed. Thereafter, the supernatant (containing chylomicron and VLDL) separated using a tube slicer was removed to obtain a fraction containing LpX.
　上記で得られたＬｐＸを含む画分を、以下の条件により、ゲルろ過カラムクロマトグラフィーに付して、得られた各フラクションについて、総コレステロール測定用キット及び遊離型コレステロール測定用キットを用いて、各フラクション中の総コレステロール濃度及び遊離型コレステロール濃度を測定した。同様に、各フラクションについて、各フラクション中のコレステロール濃度を、実施例５及び６それぞれのキットを用いて測定した。その結果（クロマトグラム）を図１に示す。 The fraction containing LpX obtained above was subjected to gel filtration column chromatography under the following conditions, and for each of the obtained fractions, using a total cholesterol measurement kit and a free cholesterol measurement kit, The total cholesterol concentration and free cholesterol concentration in each fraction were measured. Similarly, for each fraction, the cholesterol concentration in each fraction was measured using the respective kits of Examples 5 and 6. The result (chromatogram) is shown in FIG.
＜ゲルろ過カラムクロマトグラフィー＞
　システム：ＢＩＯＣＡＤ７００Ｅ（PreSeptive Biosystems社製）
　カラム：ＳｕｐｅｒｏｓｅＨＲ６カラム（ファルマシア社製）
　溶出溶媒：ＥＤＴＡ（１ｍｍｏｌ／Ｌ）を含有する０．１５ｍｏｌ／Ｌ塩化ナトリウム水溶液（ｐＨ７．４）
　流速：０．３ｍＬ／分 <Gel filtration column chromatography>
System: BIOCAD700E (manufactured by PreSeptive Biosystems)
Column: Superose HR6 column (Pharmacia)
Elution solvent: 0.15 mol / L sodium chloride aqueous solution (pH 7.4) containing EDTA (1 mmol / L)
Flow rate: 0.3 mL / min
＜濃度測定＞
　発色試薬：
　(1)総コレステロール（ＴＣ）測定用キット：デタミナーＬ　ＴＣ　ＩＩ（協和メデックス社製）
　発色反応温度：３７℃
　検出波長：主波長６００ｎｍ；副波長８００ｎｍ
　サンプル量：４μＬ
　(2)遊離型コレステロール（ＦＣ）測定用キット：デタミナーＬ　ＦＣ（協和メデックス社製）
　発色反応温度：３７℃
　検出波長：主波長６００ｎｍ；副波長７００ｎｍ
　サンプル量：４μＬ <Density measurement>
Coloring reagent:
(1) Total cholesterol (TC) measurement kit: Determiner L TC II (manufactured by Kyowa Medex)
Color reaction temperature: 37 ° C
Detection wavelength: Main wavelength 600 nm; Sub wavelength 800 nm
Sample volume: 4 μL
(2) Free cholesterol (FC) measurement kit: Determiner L FC (manufactured by Kyowa Medex)
Color reaction temperature: 37 ° C
Detection wavelength: Main wavelength 600 nm; Sub wavelength 700 nm
Sample volume: 4 μL
　本ゲルろ過カラムクロマトグラフィーにおける溶出パターン（クロマトグラム）を図１に示す。図１に示されているように、最初に溶出されるフラクションにおいては、総コレステロールが遊離型コレステロールとほぼ同濃度であり、また、アポＢ測定用キットを用いた同様の測定により、当該フラクションにはアポＢが含まれないことが確認されていることから、当該フラクションがＬｐＸに該当することを確認した。すなわち、当該肝疾患患者由来の血清が異常リポ蛋白の１つであるＬｐＸを多量に含むことを確認した。尚、図１のクロマトグラムにおいてＬｐＸに次いで溶出されるフラクションは、ＬｐＹ（異常リポ蛋白の１つ）と微量のＬＤＬを含むフラクションであることが確認された。 The elution pattern (chromatogram) in this gel filtration column chromatography is shown in FIG. As shown in FIG. 1, in the first eluted fraction, the total cholesterol is almost the same concentration as the free cholesterol, and the same measurement using the Apo B measurement kit results in the fraction being eluted. Since it was confirmed that Apo B is not contained, it was confirmed that the fraction corresponds to LpX. That is, it was confirmed that the serum derived from the liver disease patient contained a large amount of LpX, which is one of abnormal lipoproteins. In addition, it was confirmed that the fraction eluted next to LpX in the chromatogram of FIG. 1 is a fraction containing LpY (an abnormal lipoprotein) and a small amount of LDL.
　一方、実施例５及び６のキットを用いて、本ゲルろ過カラムクロマトグラフィーの各フラクション中のコレステロール濃度を測定した結果、図１に示す通り、ＬｐＸ中のコレステロールとは反応しないことが分かった。従って、本発明のＬＤＬ－Ｃ測定用キットは、ＬｐＸ中のコレステロールと反応せず、本発明のＬＤＬ－Ｃ測定方法は、ＬｐＸ中のコレステロールに対する反応性が抑制された方法であることが判明した。 On the other hand, as a result of measuring the cholesterol concentration in each fraction of the present gel filtration column chromatography using the kits of Examples 5 and 6, it was found that it did not react with cholesterol in LpX as shown in FIG. Therefore, it was found that the LDL-C measurement kit of the present invention did not react with cholesterol in LpX, and the LDL-C measurement method of the present invention was a method in which reactivity to cholesterol in LpX was suppressed. .
　以下の第一試薬及び第二試薬からなるＬＤＬ－Ｃ測定用キットを調製した。
第一試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　デキストラン硫酸ナトリウム　　０．７５　ｇ／Ｌ
　硫酸ナトリウム　　　　　　　　２　ｇ／Ｌ
　ＤＯＳＥ　　　　　　　　　　　０．３　ｇ／Ｌ
　ＰＯＥ多環エーテル硫酸エステル塩またはアリールスルホン酸誘導体（その種類と濃度は第３表参照）
　ペルオキシダーゼ　　　　　　　１０　ｋＵ／Ｌ
第二試薬
　ＭＯＰＳ（ｐＨ７．０）　　　　２０　ｍｍｏｌ／Ｌ
　４－アミノアンチピリン　　　　０．５　ｇ／Ｌ
　ペルオキシダーゼ　　　　　　　２０　ｋＵ／Ｌ
　アクロネセスＫＰ１８９Ｒ　　　１４　ｇ／Ｌ
　プルロニックＬ－１２１　　　　７　ｇ／Ｌ
　ブラウノンＬ－２０５　　　　　０．７５　ｍｍｏｌ／Ｌ
　ＬＰＬ－３１１　　　　　　　　３　ｋＵ／Ｌ
　ＣＨＯＤＩ　　　　　　　　　　４　ｋＵ／Ｌ An LDL-C measurement kit comprising the following first reagent and second reagent was prepared.
First reagent MOPS (pH 7.0) 20 mmol / L
Dextran sulfate sodium 0.75 g / L
Sodium sulfate 2 g / L
DOSE 0.3 g / L
POE polycyclic ether sulfate ester or aryl sulfonic acid derivative (see Table 3 for types and concentrations)
Peroxidase 10 kU / L
Second reagent MOPS (pH 7.0) 20 mmol / L
4-Aminoantipyrine 0.5 g / L
Peroxidase 20 kU / L
Akroneses KP189R 14 g / L
Pluronic L-121 7 g / L
Braunon L-205 0.75 mmol / L
LPL-311 3 kU / L
CHODI 4 kU / L
［試験例］
　検体として、低ＬＤＬ血症患者由来の血清を用い、キットとして、実施例７のキットを用いる以外は、実施例２に記載の方法と同様の方法により、当該検体中のＬＤＬ－Ｃ濃度を決定した。その結果を第３表に示す。 [Test example]
The concentration of LDL-C in the sample is determined by the same method as described in Example 2 except that serum from a hypo LDL blood patient is used as a sample and the kit of Example 7 is used as a kit. did. The results are shown in Table 3.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　第３表から明らかなように、ＰＯＥ多環エーテル硫酸エステル塩、アリールスルホン酸誘導体のいずれをも含まない実施例１のキットを用いた測定においては、ＬＤＬ－Ｃ濃度が負の値となったが、ＰＯＥ多環エーテル硫酸エステル塩、アリールスルホン酸誘導体のいずれかを含む実施例７のキットを用いた測定においては、ＬＤＬ－Ｃ濃度が正の値となり、正確なＬＤＬ－Ｃの測定が可能となることが示された。 As is apparent from Table 3, in the measurement using the kit of Example 1 containing neither the POE polycyclic ether sulfate ester salt nor the arylsulfonic acid derivative, the LDL-C concentration was a negative value. However, in the measurement using the kit of Example 7 containing either a POE polycyclic ether sulfate salt or an aryl sulfonic acid derivative, the LDL-C concentration becomes a positive value, and accurate LDL-C measurement is possible. It was shown that
　本発明により、メタボリックシンドロームや動脈硬化等の診断に有用なＬＤＬ－Ｃの測定方法、測定用試薬及び測定用キットが提供される。
  The present invention provides a measurement method, measurement reagent, and measurement kit for LDL-C useful for diagnosis of metabolic syndrome, arteriosclerosis, and the like.

Claims (22)

  1. 　検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素とを、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン及びポリアニオンの存在下に反応させ、該反応で生成する過酸化水素を測定することを特徴とする、検体中の低密度リポ蛋白中のコレステロールの測定方法。 The sample is reacted with cholesterol esterase and cholesterol oxidase in the presence of polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine and polyanion, A method for measuring cholesterol in a low-density lipoprotein in a specimen, which comprises measuring hydrogen peroxide produced by the reaction.
  2. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである請求項１記載の測定方法。 The measuring method according to claim 1, wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
  3. 　反応液中のコレステロール酸化酵素の濃度が０．２５～１．０ｋＵ／Ｌであり、ポリオキシエチレンドデシルアミンの濃度が０．０６～０．３ｍｍｏｌ／Ｌである請求項２記載の方法。 The method according to claim 2, wherein the concentration of cholesterol oxidase in the reaction solution is 0.25 to 1.0 kU / L, and the concentration of polyoxyethylene dodecylamine is 0.06 to 0.3 mmol / L.
  4. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである請求項１記載の測定方法。 The measuring method according to claim 1, wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
  5. 　反応液中のコレステロール酸化酵素の濃度が０．５～２．０ｋＵ／Ｌのコレステロール酸化酵素であり、ポリオキシエチレンオクタデシルアミンの濃度が０．０２～０．０７５ｍｍｏｌ／Ｌである請求項４記載の方法。 The cholesterol oxidase concentration in the reaction solution is 0.5 to 2.0 kU / L, and the polyoxyethylene octadecylamine concentration is 0.02 to 0.075 mmol / L. Method.
  6. 　検体と、コレステロールエステル加水分解酵素及びコレステロール酸化酵素との反応が、さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質の存在下に行われる、請求項１～５のいずれかに記載の方法。 The reaction of the specimen with cholesterol ester hydrolase and cholesterol oxidase is further performed in the presence of at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate salt and an aryl sulfonic acid derivative. The method according to any one of claims 1 to 5.
  7. 　過酸化水素の測定が、過酸化水素測定用試薬を用いて行われる請求項１～６のいずれかに記載の測定方法。 The measurement method according to any one of claims 1 to 6, wherein the measurement of hydrogen peroxide is performed using a reagent for measuring hydrogen peroxide.
  8. 　ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、ポリオキシエチレンアルキルアミン、ポリアニオン、コレステロールエステル加水分解酵素、及び、コレステロール酸化酵素を含有することを特徴とする、検体中の低密度リポ蛋白中のコレステロール測定用試薬。 Sample containing polyoxyethylene / polyoxyalkylene alkylaryl ether, polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkylamine, polyanion, cholesterol esterase, and cholesterol oxidase A reagent for measuring cholesterol in low density lipoprotein.
  9. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである請求項８記載の試薬。 The reagent according to claim 8, wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
  10. 　試薬中のコレステロール酸化酵素の濃度が０．２５～１．０ｋＵ／Ｌであり、ポリオキシエチレンドデシルアミンの濃度が０．０６～０．３ｍｍｏｌ／Ｌである請求項９記載の試薬。 The reagent according to claim 9, wherein the concentration of cholesterol oxidase in the reagent is 0.25 to 1.0 kU / L, and the concentration of polyoxyethylene dodecylamine is 0.06 to 0.3 mmol / L.
  11. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである請求項８記載の試薬。 The reagent according to claim 8, wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
  12. 　試薬中のコレステロール酸化酵素の濃度が０．５～２．０ｋＵ／Ｌであり、ポリオキシエチレンオクタデシルアミンの濃度が０．０２～０．０７５ｍｍｏｌ／Ｌである請求項１１記載の試薬。 The reagent according to claim 11, wherein the concentration of cholesterol oxidase in the reagent is 0.5 to 2.0 kU / L, and the concentration of polyoxyethylene octadecylamine is 0.02 to 0.075 mmol / L.
  13. 　さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を含有する、請求項８～１２のいずれかに記載の試薬。 The reagent according to any one of claims 8 to 12, further comprising at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate ester salt and an arylsulfonic acid derivative.
  14. 　さらに、過酸化水素測定用試薬を含む請求項８～１３のいずれかに記載の試薬。 The reagent according to any one of claims 8 to 13, further comprising a reagent for measuring hydrogen peroxide.
  15. 　ポリアニオンを含む第一試薬、コレステロール酸化酵素及びポリオキシエチレンアルキルアミンを含む第二試薬を含有し、ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、ポリオキシエチレン・ポリオキシアルキレン縮合物、及び、コレステロールエステル加水分解酵素のそれぞれを第一試薬、第二試薬のいずれか又は両方に含有することを特徴とする、検体中の低密度リポ蛋白中のコレステロール測定用キット。 A polyoxyethylene polyoxyalkylene alkyl aryl ether, a polyoxyethylene polyoxyalkylene alkyl aryl ether, a polyoxyethylene polyoxyalkylene condensate containing a first reagent containing a polyanion, a cholesterol oxidase and a polyoxyethylene alkylamine, and a cholesterol ester A kit for measuring cholesterol in low-density lipoprotein in a specimen, wherein each of the hydrolases is contained in one or both of the first reagent and the second reagent.
  16. 　ポリオキシエチレン・ポリオキシアルキレンアルキルアリールエーテル、及び、ポリオキシエチレン・ポリオキシアルキレン縮合物が第二試薬に含まれる請求項１５記載のキット。 The kit according to claim 15, wherein the second reagent contains polyoxyethylene / polyoxyalkylene alkylaryl ether and polyoxyethylene / polyoxyalkylene condensate.
  17. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンドデシルアミンである請求項１５又は１６記載のキット。 The kit according to claim 15 or 16, wherein the polyoxyethylene alkylamine is polyoxyethylene dodecylamine.
  18. 　第二試薬中のコレステロール酸化酵素の濃度が１．０～４．０ｋＵ／Ｌであり、第二試薬中のポリオキシエチレンドデシルアミンの濃度が０．２５～１．２５ｍｍｏｌ／Ｌである請求項１７記載のキット。 The concentration of cholesterol oxidase in the second reagent is 1.0 to 4.0 kU / L, and the concentration of polyoxyethylene dodecylamine in the second reagent is 0.25 to 1.25 mmol / L. The described kit.
  19. 　ポリオキシエチレンアルキルアミンが、ポリオキシエチレンオクタデシルアミンである請求項１５又は１６記載のキット。 The kit according to claim 15 or 16, wherein the polyoxyethylene alkylamine is polyoxyethylene octadecylamine.
  20. 　第二試薬中のコレステロール酸化酵素の濃度が２．０～８．０ｋＵ／Ｌであり、第二試薬中のポリオキシエチレンオクタデシルアミンの濃度が０．０７５～０．３ｍｍｏｌ／Ｌである請求項１９記載のキット。 20. The concentration of cholesterol oxidase in the second reagent is 2.0 to 8.0 kU / L, and the concentration of polyoxyethylene octadecylamine in the second reagent is 0.075 to 0.3 mmol / L. The described kit.
  21. 　さらに、ポリオキシエチレン多環エーテル硫酸エステル塩およびアリールスルホン酸誘導体からなる群より選ばれる少なくとも１つの物質を第一試薬、第二試薬のいずれか又は両方に含む、請求項１５～２０のいずれかに記載のキット。 21. The method according to claim 15, further comprising at least one substance selected from the group consisting of a polyoxyethylene polycyclic ether sulfate ester salt and an arylsulfonic acid derivative in either or both of the first reagent and the second reagent. The kit according to 1.
  22. 　さらに、過酸化水素測定用試薬を第一試薬、第二試薬のいずれか又は両方に含有する請求項１５～２１のいずれかに記載のキット。
          The kit according to any one of claims 15 to 21, further comprising a reagent for measuring hydrogen peroxide in one or both of the first reagent and the second reagent.
PCT/JP2011/060352 2010-04-30 2011-04-28 Method for measuring low-density lipoprotein cholesterol, measurement reagent and measurement kit WO2011136316A1 (en)

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