WO2015076285A1 - Denaturation inhibitor and method for inhibiting denaturation by freeze-drying low-density lipoproteins contained in blood serum or blood plasma - Google Patents

Denaturation inhibitor and method for inhibiting denaturation by freeze-drying low-density lipoproteins contained in blood serum or blood plasma Download PDF

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WO2015076285A1
WO2015076285A1 PCT/JP2014/080604 JP2014080604W WO2015076285A1 WO 2015076285 A1 WO2015076285 A1 WO 2015076285A1 JP 2014080604 W JP2014080604 W JP 2014080604W WO 2015076285 A1 WO2015076285 A1 WO 2015076285A1
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serum
plasma
ldl
component
freeze
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PCT/JP2014/080604
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French (fr)
Japanese (ja)
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健太 金城
瑞季 住田
吾郎 杉澤
有基 片山
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協和メデックス株式会社
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Priority to JP2015549166A priority Critical patent/JP6461005B2/en
Publication of WO2015076285A1 publication Critical patent/WO2015076285A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

  • the present invention relates to a denaturation inhibitor by freeze-drying of low-density lipoprotein in serum or plasma, a method of inhibiting denaturation by freeze-drying of low-density lipoprotein in serum or plasma, and a component in low-density lipoprotein in serum or plasma.
  • Standard for quantification method for producing standard for quantification of components in low-density lipoprotein in serum or plasma, method for quantification of components in low-density lipoprotein in serum or plasma, and serum or plasma
  • the present invention relates to a kit for quantifying components in low-density lipoprotein.
  • Serum and plasma are often used in clinical tests as well as urine. Serum and plasma contain lipoproteins such as high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL). Each lipoprotein contains cholesterol, neutral fat. Ingredients such as phospholipids are included. LDL is a lipoprotein containing Apo B as a constituent component, and is known to be susceptible to denaturation such as oxidation (see Patent Document 1).
  • HDL-C cholesterol in HDL
  • LDL-C cholesterol in LDL
  • LDL-C cholesterol in LDL
  • LDL-C cholesterol in lipoproteins
  • LDL-C cholesterol in lipoproteins
  • a homogeneous method that does not involve separation of lipoproteins from serum or plasma is exclusively used.
  • a standard product in which the concentration of the component is priced is used.
  • the standard product is used to create a calibration curve indicating the relationship between the concentration of the component and the measured value (for example, absorbance) for the component.
  • the concentration of the component in the sample is determined by comparing the calibration curve created using the standard product with the measurement value obtained by the measurement using the actual sample.
  • the component in the sample to be measured is known concentration or content along with other components. Serum type standards contained in are known.
  • liquid standard products There are two types of standard products: liquid standard products and freeze-dried standard products.
  • the components to be measured contained in the standard product need to be stably retained and stored.
  • the production includes a step of freeze-drying serum or plasma as a raw material of the standard product.
  • LDL is a lipoprotein that is susceptible to denaturation, and denaturation proceeds by lyophilization.
  • lyophilization conditions are not uniform, the degree of denaturation of LDL is non-uniform, and this degree of denaturation is not uniform.
  • the calibration curve differs for each product, and there is a problem that accurate measurement cannot be performed.
  • the present inventors have added methionine to serum or plasma, and then freeze-dried serum or plasma, thereby suppressing LDL denaturation due to freeze-drying.
  • the present invention was completed by finding knowledge. That is, the present invention relates to the following [1] to [11].
  • a denaturation inhibitor containing methionine as an active ingredient by lyophilization of LDL in serum or plasma [1] A denaturation inhibitor containing methionine as an active ingredient by lyophilization of LDL in serum or plasma. [2] A method for inhibiting denaturation by lyophilization of LDL in serum or plasma, wherein methionine is added to serum or plasma, and then serum or plasma is freeze-dried.
  • the standard product according to [3] which is produced by a method including the following steps. (1) adding methionine to serum or plasma; (2) freeze-drying the mixture obtained in step (1); and (3) A step of pricing the content of the component in LDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
  • [6] A method for producing a standard product for quantitative determination of components in LDL in serum or plasma, comprising the following steps. (1) adding methionine to serum or plasma; (2) freeze-drying the mixture obtained in step (1); and (3) A step of pricing the content of the component in LDL contained in the freeze-dried product obtained in step (2) using a known amount of the component. [7] The production method of [6], wherein the component in LDL is cholesterol.
  • a method for quantifying components in LDL in serum or plasma comprising the following steps. (1) A step of measuring the component using a reagent for measuring the component in LDL in serum or plasma; (2) Using the standard product described in [3] or [4] and the measurement reagent in step (1), create a calibration curve representing the relationship between the concentration of the component and the measured value for the component Steps; and (3) A step of determining the concentration of the component in serum or plasma from the measurement value obtained in step (1) and the calibration curve prepared in step (2). [9] The quantification method according to [8], wherein the component in LDL is cholesterol.
  • kits for determination of components in LDL in serum or plasma, comprising a reagent for measuring components in LDL in serum or plasma, and a standard product described in [3] or [4] kit.
  • kit according to [10] wherein the component in LDL is cholesterol.
  • the present invention provides a denaturation inhibitor by freeze-drying of LDL in serum or plasma, a method of inhibiting denaturation by freeze-drying of LDL in serum or plasma, and a standard product that enables accurate measurement of components in LDL. Is done.
  • the LDL denaturation inhibitor of the present invention suppresses denaturation of LDL in serum or plasma due to lyophilization, and contains methionine as an active ingredient.
  • the methionine in the present invention is not particularly limited as long as it can suppress denaturation due to lyophilization of LDL in serum or plasma, and any of L-methionine, D-methionine, and DL-methionine may be used.
  • the content of methionine contained in the LDL denaturation inhibitor of the present invention is not particularly limited as long as it is a content capable of inhibiting the denaturation due to lyophilization of LDL in serum or plasma.
  • the content is 0.025 to 5 g, and preferably 0.5 to 1 g.
  • the serum and plasma in the present invention may be any serum and plasma prepared from whole blood collected from humans or animals, and serum prepared from whole blood collected from humans. And plasma are preferred.
  • Serum can be prepared by a method of centrifuging whole blood collected using a blood collection tube not containing an anticoagulant, a method of natural sedimentation, or the like.
  • Plasma can be prepared by a method of centrifuging whole blood collected by an anticoagulant-containing blood collection tube, a method of spontaneous sedimentation, or the like.
  • the anticoagulant include ethylenediaminetetraacetic acid (EDTA) dipotassium salt, heparin, sodium fluoride, sodium citrate and the like.
  • animals include primates such as monkeys, gorillas, orangutans, chimpanzees and baboons.
  • LDL Low density lipoprotein
  • the LDL in the present invention may be either an LDL in a broad sense whose specific gravity is classified as 1.006 to 1.063 or an LDL in a narrow sense whose specific gravity is classified as 1.019 to 1.063.
  • LDL includes intermediate density lipoprotein (IDL) whose specific gravity is classified into 1.006 to 1.019.
  • LDL examples of components in LDL in the present invention include cholesterol and neutral fat, and cholesterol is preferred.
  • lyophilization is used in the usual sense used in the art, and means that a sample is frozen and depressurized in a frozen state to remove moisture from the sample and dry.
  • the lyophilization conditions are not particularly limited, but are usually -80 to 35 ° C., preferably -80 to 30 ° C., 0.667 to 1333 Pa, preferably 13.1 to 133.3 Pa, and 6 to 120 hours, more preferably Perform for 12 to 120 hours.
  • the water content of the freeze-dried product is usually 10% by weight or less, preferably 1% by weight or less.
  • the method for inhibiting denaturation by freeze-drying of LDL in serum or plasma according to the present invention is characterized by freeze-drying serum or plasma after adding methionine to the serum or plasma.
  • the method for inhibiting denaturation of lyophilized LDL in serum or plasma according to the present invention includes the following steps. (1) adding methionine to serum or plasma; and (2) A step of freeze-drying the mixture obtained in step (1).
  • the amount of methionine added to serum or plasma is not particularly limited as long as the amount of denaturation due to freeze-drying of LDL in serum or plasma can be suppressed. It is 0.025-5 g per mL, preferably 0.5-1 g.
  • methionine When adding methionine to serum or plasma, methionine may be added in the state of an aqueous methionine solution.
  • an aqueous medium such as deionized water, distilled water, or a buffer solution can be used.
  • the buffering agent for preparing the buffer solution include a buffering agent described later.
  • the pH of the aqueous methionine solution may be any pH as long as the serum or plasma lipoproteins do not precipitate after the aqueous methionine solution is added to the serum or plasma, and is preferably pH 6-9.
  • the standard for quantifying components in LDL in serum or plasma according to the present invention contains LDL in lyophilized serum or plasma and methionine, and the content of the component is priced It is a product.
  • the standard product of the present invention can be used not only as a standard product for quantifying components in LDL in serum or plasma, but also as a quality control substance in quantifying components in LDL in serum or plasma.
  • the standard product of the present invention is in a lyophilized state, it is dissolved in a known amount of an aqueous medium for use in preparing a calibration curve.
  • the aqueous medium for dissolving the lyophilized standard product is not particularly limited as long as it is an aqueous medium that enables the method of quantifying the components in the LDL of the present invention.
  • deionized water, distilled water, buffer solution Etc examples include a buffering agent described later.
  • the method for producing a standard product of the present invention includes the following steps. (1) adding methionine to serum or plasma; (2) freeze-drying the mixture obtained in step (1); and (3) A step of pricing the content of the LDL component contained in the lyophilized product obtained in step (2) using a known amount of the component.
  • the amount of methionine added to serum or plasma is not particularly limited as long as it is an amount that can suppress denaturation due to lyophilization of LDL in serum or plasma.
  • the amount is 0.025 to 5 g per 100 mL of plasma, preferably 0.5 to 1 g.
  • Serum or plasma is cooled to 2-8 ° C., and an aqueous methionine solution is added thereto.
  • an aqueous methionine solution is added thereto.
  • the pH-adjusted aqueous solution is filtered to about 0.2-0.4 microns. Filter using a membrane. The resulting solution is dispensed into lyophilized vials and half-capped.
  • the solution dispensed in the vial is frozen at a low temperature of -30 ° C or lower, then lyophilized under vacuum conditions of 133.3 Pa, and finally dried at 0 to 20 ° C with gradual warming. Prepare a standard product in a dry state.
  • pricing of a standard product means determining the content of a component in LDL to be measured contained in a manufactured standard product using a standard serum containing a known amount of the component.
  • the standard serum containing a known amount of the component is determined by a method that does not depend on a homogeneous method such as a standard analysis method of the US Center for Disease Control and Prevention (CDC). Serum.
  • CDC US Center for Disease Control and Prevention
  • Serum Using this standard serum and the manufactured standard as a specimen, measurement is performed by a homogeneous method using an actual automatic analyzer, and a measurement value (for example, absorbance) of a standard serum containing a known amount of the component is manufactured.
  • the measured value of the standard product (for example, absorbance) is compared to determine the content of the component in the manufactured standard product.
  • the manufactured standard product is used as a standard serum in the laboratory to determine the measurement value of the specimen.
  • the content of methionine contained in the standard product of the present invention is not particularly limited as long as LDL in the standard product is stably maintained and the components in the LDL in the standard product can be accurately measured.
  • the standard product of the present invention may contain additives such as buffers, metal ions, salts, surfactants, preservatives, sugar compounds and the like as necessary.
  • Buffers include, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur tool Buffering agents, imidazole buffers, malic acid buffers, oxalic acid buffers, glycine buffers, boric acid buffers, carbonate buffers, glycine buffers, Good buffers, and the like can be mentioned.
  • Examples of the good buffer include 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-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 3- [N
  • Examples of metal ions include magnesium ions, calcium ions, manganese ions, and zinc ions.
  • Examples of the salts include sodium chloride and potassium chloride.
  • Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants.
  • Examples of the preservative include antibiotics such as sodium azide, streptomycin, gentamicin, Bioace, Procrine 300 (trademark), Proxel GXL (trademark) and the like.
  • sugar compound examples include monosaccharides and disaccharides, and disaccharides are preferable.
  • monosaccharides include glucose, fructose, and fucose.
  • disaccharide examples include saccharose, trehalose, maltose and the like. Two or more monosaccharides, disaccharides and the like can be used in combination.
  • the amount of the sugar compound added when the sugar compound is mixed with serum or plasma is usually 0.5 to 20 ⁇ g, preferably 1 to 15 ⁇ g, more preferably 1.25 to 12.5 ⁇ g with respect to 100 ⁇ mL of serum or plasma. .
  • the method for quantifying components in LDL in serum or plasma of the present invention comprises the following steps. (1) A step of measuring the component using a reagent for measuring the component in LDL in serum or plasma; (2) using the standard product of the present invention and the measurement reagent of step (1) to create a calibration curve representing the relationship between the concentration of the component and the measured value for the component; and (3) A step of determining the concentration of the component in serum or plasma from the measurement value obtained in step (1) and the calibration curve prepared in step (2).
  • a known method for measuring the component in LDL can be used.
  • the method for measuring components in LDL include a method for measuring LDL-C, a method for measuring neutral fat in LDL (hereinafter referred to as LDL-TG), and the like.
  • LDL-TG a method for measuring neutral fat in LDL
  • LDL-TG a method for measuring neutral fat in LDL
  • a method for measuring the components therein that is, a so-called homogeneous method is preferred.
  • a specimen and an enzyme for measuring cholesterol described in WO2010 / 055916 pamphlet are used: [a] polyoxyethylene / polyoxyalkylene alkylaryl ether; [b] polyoxyethylene One or more surfactants selected from the group consisting of polyoxyalkylene condensates, polyoxyethylene alkenyl ethers, polyoxyethylene branched alkyl ethers and polyoxyethylene polyoxyalkylene branched alkyl ethers; [c] primary One or more surfactants selected from the group consisting of amines, secondary amines, tertiary amines and quaternary ammoniums; and [d] peroxidation produced by the reaction in the presence of a polyanion.
  • the enzyme for measuring cholesterol is a combination of (i) cholesterol ester hydrolase and cholesterol oxidase, or (ii) cholesterol ester hydrolase, oxidized coenzyme, and cholesterol dehydrogenase. It is a combination.
  • the kit for quantifying components in LDL in serum or plasma of the present invention contains the standard product of the present invention and a reagent for measuring components in LDL.
  • a measuring reagent for the component in LDL a known measuring reagent for the component in LDL can be used.
  • the reagent for measuring the components in LDL include an LDL-C measuring reagent and an LDL-TG measuring reagent.
  • LDL is not fractionated by means such as centrifugation, and in addition to LDL, multiple types of lipoproteins such as HDL and VLDL coexist in the same reaction solution.
  • a reagent for measuring the components therein that is, a reagent used in a measuring method by the so-called homogeneous method is preferable.
  • the LDL-C measurement reagent used in the LDL-C measurement method by the homogeneous method is not particularly limited as long as it is a reagent that enables the LDL-C quantification method using the standard product of the present invention.
  • WO2010 / 055916 The enzyme for cholesterol measurement described in the pamphlet; [a] polyoxyethylene / polyoxyalkylene alkyl aryl ether; [b] polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkenyl ether, polyoxyethylene branched alkyl ether And one or more surfactants selected from the group consisting of polyoxyethylene / polyoxyalkylene branched alkyl ethers; [c] a group consisting of primary amines, secondary amines, tertiary amines and quaternary ammoniums One or more surfactants selected from; Beauty include LDL-C measurement reagents containing the [d] a polyanion.
  • the enzyme for measuring cholesterol is a combination of (i) cholesterol ester hydrolase and cholesterol oxidase, or (ii) cholesterol ester hydrolase, oxidized coenzyme, and cholesterol dehydrogenase. It is a combination.
  • a commercially available measuring reagent can also be used as the LDL-C measuring reagent used in the homogeneous method.
  • Examples of commercially available measuring reagents include LDL-C measuring reagents such as Determiner L LDL-C (manufactured by Kyowa Medex), Metabolid LDL-C (manufactured by Kyowa Medex), Colles Test N LDL (manufactured by Sekisui Medical), L Type Wako LDL-C (manufactured by Wako Pure Chemical Industries, Ltd.)
  • LDL-C measuring reagents such as Determiner L LDL-C (manufactured by Kyowa Medex), Metabolid LDL-C (manufactured by Kyowa Medex), Colles Test N LDL (manufactured by Sekisui Medical), L Type Wako LDL-C (manufactured by Wako Pure Chemical Industries, Ltd.)
  • the standard product of the present invention and the kit of the present invention are suitably used in a measurement method using an automatic analyzer in which components in serum or plasma in LDL are automatically and continuously measured.
  • the effect of the LDL denaturation inhibitor of the present invention can be verified, for example, by the following method.
  • Pooled serum or pooled plasma is prepared by mixing serum or plasma collected from a plurality of healthy individuals.
  • the prepared pool serum or pool plasma is divided into two, and the LDL denaturation inhibitor of the present invention is added to one pool serum or pool plasma, and the pool serum or pool to which the LDL denaturation inhibitor of the present invention is added Plasma (group A serum or plasma) and pooled serum or pooled plasma (group B serum or plasma) to which the LDL denaturation inhibitor of the present invention is not added are prepared.
  • a standard A Dispense a certain amount of Group A serum or plasma into multiple containers of the same shape and material, freeze-dry by the above-mentioned method, further price by the above-mentioned method, A standard A) is prepared.
  • a lyophilized standard product (standard product B) is prepared using group B serum or plasma.
  • N prepared standard products A are extracted (standard product A 1 to standard product A N ), and each standard product A (standard product A 1 to standard product A N ) is used as a sample, and LDL-C measurement reagent is used.
  • each standard a (standard a 1 ⁇ standard a N) absorbance to (absorbance a 1 ⁇ absorbance a N) is measured.
  • the absorbance to the standard A (absorbance A 1 to absorbance A N ) is also the absorbance to the standard B (absorbance B 1 to absorbance).
  • B N should also be uniform, but the degree of denaturation of LDL at the time of lyophilization is slightly different in each container, so the absorbance varies. Therefore, if shown that CV A is small compared to the CV B, by denaturation inhibitor of LDL present invention, modified itself by lyophilization of LDL was suppressed, it can be determined.
  • Serum (manufactured by Trina Bioreactives AG; Human TC / TG / HDL / LDL Cholesterol calibrator, LIQUID product code: CL1130), trehalose (manufactured by Hayashibara), bioace (manufactured by KEI Kasei Co., Ltd.), L-methionine (Nacalai Tesque) ), Borosilicate glass vial [manufactured by Yamato Special Glass Co., Ltd. (capacity: 2 mm; diameter: 16 mm; brown)], freeze dryer (manufactured by Tokyo Rika Kikai Co., Ltd .; EYELA DRC-1100, FDU-2100) .
  • Freeze-drying Serum A prepared as described above is dispensed in 0.5 mL portions into glass vials, and freeze-dried by setting primary freeze-drying at -34 ° C for 32 hours and secondary freeze-drying at 20 ° C for 4 hours. Finally, it was closed with a rubber stopper in a vacuum state to produce freeze-dried serum A. The same operation was performed using serum B to E instead of serum A to prepare freeze-dried serum B to E.
  • the prepared freeze-dried serum A was opened, heated at 25 ° C. for 2 hours, then closed again with a rubber stopper, and stored in a refrigerator (4 ° C.) for 1 week to prepare freeze-dried serum A +.
  • the same operation was performed using lyophilized sera B to E instead of lyophilized serum A to prepare lyophilized sera B + to E +.
  • the lyophilized serum prepared with the addition of L-methionine was compared with the lyophilized serum prepared without the addition of L-methionine regardless of the treatment after the opening.
  • the CV was particularly low in lyophilized serum supplemented with 5 to 10 g / L L-methionine.
  • a higher CV means that the absorbance is non-uniform, and this non-uniform absorbance is due to LDL denaturation due to lyophilization. Therefore, when L-methionine is added to serum or plasma, the CV decreases. This indicates that L-methionine suppresses the denaturation of LDL in serum or plasma due to lyophilization. Therefore, it was found that the LDL denaturation inhibitor of the present invention suppresses denaturation of LDL in serum or plasma due to lyophilization, and the components in LDL in serum or plasma can be accurately measured.
  • an LDL denaturation inhibitor in serum or plasma by lyophilization a method for inhibiting LDL denaturation in serum or plasma by lyophilization, and a standard that enables accurate measurement of components in LDL in serum or plasma
  • a method for producing the same a method for quantifying components in LDL in serum or plasma, and a kit for quantifying components in LDL in serum or plasma.
  • LDL denaturation inhibitor in serum or plasma of the present invention method for inhibiting LDL denaturation in serum or plasma, standard product for quantification of components in LDL in serum or plasma and production method thereof, in LDL in serum or plasma
  • the component quantification method and the quantification kit for components in LDL in serum or plasma are useful for diagnosis of metabolic syndrome and the like.

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Abstract

Provided are a denaturation inhibitor obtained by freeze-drying low-density lipoproteins (LDL) contained in blood serum or blood plasma, a method for inhibiting denaturation by freeze-drying LDL contained in blood serum or blood plasma, and a reference standard which makes it possible to accurately measure the components of LDL. The denaturation inhibitor obtained by freeze-drying LDL contained in blood serum or blood plasma contains methionine as an active component. The method for inhibiting denaturation by freeze-drying LDL contained in blood serum or blood plasma is characterized in that the blood serum or blood plasma is freeze-dried after adding methionine thereto. The reference standard for the determination of the components of LDL contained in blood serum or blood plasma is characterized by containing LDL contained in the freeze-dried blood serum, and methionine, wherein a value is given for the content of the components of the LDL.

Description

血清又は血漿中の低密度リポ蛋白の凍結乾燥による変性抑制剤及び変性抑制方法Denaturation inhibitor and method for inhibiting denaturation of lyophilized low density lipoprotein in serum or plasma
 本発明は、血清又は血漿中の低密度リポ蛋白の凍結乾燥による変性抑制剤、血清又は血漿中の低密度リポ蛋白の凍結乾燥による変性抑制方法、血清又は血漿中の低密度リポ蛋白中の成分定量用の標準品、血清又は血漿中の低密度リポ蛋白中の成分定量用の標準品の製造方法、血清又は血漿中の低密度リポ蛋白中の成分の定量方法、及び、血清又は血漿中の低密度リポ蛋白中の成分の定量用キットに関する。 The present invention relates to a denaturation inhibitor by freeze-drying of low-density lipoprotein in serum or plasma, a method of inhibiting denaturation by freeze-drying of low-density lipoprotein in serum or plasma, and a component in low-density lipoprotein in serum or plasma. Standard for quantification, method for producing standard for quantification of components in low-density lipoprotein in serum or plasma, method for quantification of components in low-density lipoprotein in serum or plasma, and serum or plasma The present invention relates to a kit for quantifying components in low-density lipoprotein.
 血清及び血漿は、尿と同様に、臨床検査においてしばしば用いられる。血清及び血漿には、高密度リポ蛋白(HDL)、低密度リポ蛋白(LDL)、超低密度リポ蛋白(VLDL)等のリポ蛋白が含まれており、各リポ蛋白にはコレステロール、中性脂肪、リン脂質等の成分が含まれている。LDLは、アポBを構成成分に含むリポ蛋白であり、酸化等の変性を受け易いことが知られている(特許文献1参照)。 Serum and plasma are often used in clinical tests as well as urine. Serum and plasma contain lipoproteins such as high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL). Each lipoprotein contains cholesterol, neutral fat. Ingredients such as phospholipids are included. LDL is a lipoprotein containing Apo B as a constituent component, and is known to be susceptible to denaturation such as oxidation (see Patent Document 1).
 実際の臨床検査においては、リポ蛋白中の成分であるHDL中のコレステロール(以下、HDL-Cと記す)やLDL中のコレステロール(以下、LDL-Cと記す)等がしばしば測定されている。中でも、LDL-Cは動脈硬化の指標として用いられる。リポ蛋白中の成分の測定においては、血清又は血漿からのリポ蛋白の分離を伴わないホモジニアス法が専ら用いられている。ホモジニアス法によるリポ蛋白中の成分の測定においては、当該成分の濃度が値付けされている標準品が用いられる。標準品は、当該成分の濃度と、当該成分に対する測定値(例えば、吸光度)との間の関係を示す検量線を作成するために使用される。標準品を用いて作成された検量線に、実際の検体を用いた測定で得られた測定値を照らし合わせて、検体中の成分の濃度が決定される。 In actual clinical tests, cholesterol in HDL (hereinafter referred to as HDL-C) and cholesterol in LDL (hereinafter referred to as LDL-C), which are components in lipoproteins, are often measured. Among them, LDL-C is used as an index of arteriosclerosis. For the measurement of components in lipoproteins, a homogeneous method that does not involve separation of lipoproteins from serum or plasma is exclusively used. In the measurement of a component in lipoprotein by the homogeneous method, a standard product in which the concentration of the component is priced is used. The standard product is used to create a calibration curve indicating the relationship between the concentration of the component and the measured value (for example, absorbance) for the component. The concentration of the component in the sample is determined by comparing the calibration curve created using the standard product with the measurement value obtained by the measurement using the actual sample.
 標準品には、測定対象となる成分が単独で一定濃度又は一定含量で含有される、純品タイプの標準品の他、測定対象となる検体中の成分が他の成分と共に既知濃度又は既知含量で含有される、血清タイプの標準品が知られている。 In the standard product, in addition to the pure type standard product that contains the component to be measured alone at a constant concentration or content, the component in the sample to be measured is known concentration or content along with other components. Serum type standards contained in are known.
 標準品の形態としては、液状の標準品と凍結乾燥状態の標準品とがある。いずれの状態の標準品であっても、標準品に含まれる、測定対象となる成分が安定に保持され、保存される必要がある。特に、凍結乾燥状態の標準品の場合、その製造において、標準品の原材料となる血清又は血漿を凍結乾燥する工程を含むことになる。前述の通り、LDLは変性を受け易いリポ蛋白であり、凍結乾燥によって変性が進行するが、凍結乾燥条件が不均一な場合、LDLの変性度合が不均一となり、この変性度合の不均一さに起因して、検量線が製品毎に異なってしまい、正確な測定ができない、という問題があった。これまでにも、冷凍および凍結乾燥時のLDLの変性防止剤としてトレハロースを使用する方法(特許文献2参照)が報告されているが、使用時に水性媒体で溶解した際、粘性が高くなってしまう等の問題があった。また、糖類、高分子物質、又は、親水性有機溶媒を有効成分として含有する、血漿等の生体試料のための凍結保護剤が報告されている(特許文献3参照)。また、抗酸化剤としてメチオニンを含有する脂質測定試薬が報告されている(特許文献4参照)。 * There are two types of standard products: liquid standard products and freeze-dried standard products. In any standard product, the components to be measured contained in the standard product need to be stably retained and stored. In particular, in the case of a standard product in a freeze-dried state, the production includes a step of freeze-drying serum or plasma as a raw material of the standard product. As described above, LDL is a lipoprotein that is susceptible to denaturation, and denaturation proceeds by lyophilization. However, when lyophilization conditions are not uniform, the degree of denaturation of LDL is non-uniform, and this degree of denaturation is not uniform. As a result, the calibration curve differs for each product, and there is a problem that accurate measurement cannot be performed. So far, a method of using trehalose as an anti-denaturing agent for LDL during freezing and freeze-drying (see Patent Document 2) has been reported, but when dissolved in an aqueous medium at the time of use, the viscosity becomes high. There was a problem such as. Moreover, a cryoprotectant for biological samples such as plasma containing saccharides, polymer substances, or hydrophilic organic solvents as active ingredients has been reported (see Patent Document 3). In addition, a lipid measurement reagent containing methionine as an antioxidant has been reported (see Patent Document 4).
WO2003/006989パンフレットWO2003 / 006989 brochure 特開平11-124394号公報Japanese Patent Laid-Open No. 11-124394 特開2008-203269号公報JP 2008-203269 A 特開2004-089191号公報JP 2004-089191 A
 本発明の目的は、血清又は血漿中のLDLの凍結乾燥による変性抑制剤、及び、血清又は血漿中のLDLの凍結乾燥による変性抑制方法を提供することにある。また、本発明の目的は、LDL中の成分の正確な測定を可能とする標準品を提供することにある。 An object of the present invention is to provide a denaturation inhibitor by freeze-drying of LDL in serum or plasma and a method of inhibiting denaturation by freeze-drying of LDL in serum or plasma. Another object of the present invention is to provide a standard product that enables accurate measurement of components in LDL.
 本発明者らは本課題を解決すべく鋭意検討を重ねた結果、血清又は血漿にメチオニンを添加した後、血清又は血漿を凍結乾燥することにより、凍結乾燥によるLDLの変性が抑制される、という知見を見出して本発明を完成させた。すなわち、本発明は、以下の[1]~[11]に関する。 As a result of intensive studies to solve the problem, the present inventors have added methionine to serum or plasma, and then freeze-dried serum or plasma, thereby suppressing LDL denaturation due to freeze-drying. The present invention was completed by finding knowledge. That is, the present invention relates to the following [1] to [11].
[1]メチオニンを有効成分として含有する、血清又は血漿中のLDLの凍結乾燥による変性抑制剤。
[2]血清又は血漿にメチオニンを添加した後、血清又は血漿を凍結乾燥することを特徴とする、血清又は血漿中のLDLの凍結乾燥による変性抑制方法。
[1] A denaturation inhibitor containing methionine as an active ingredient by lyophilization of LDL in serum or plasma.
[2] A method for inhibiting denaturation by lyophilization of LDL in serum or plasma, wherein methionine is added to serum or plasma, and then serum or plasma is freeze-dried.
[3]凍結乾燥された、血清又は血漿中のLDL、及び、メチオニンを含有し、かつ、当該LDL中の成分の含量が値付けされていることを特徴とする、血清又は血漿中のLDL中の成分定量用標準品。
[4]以下の工程を含む方法により製造される、[3]記載の標準品。
(1)血清又は血漿にメチオニンを添加する工程;
(2)工程(1)で得られた混合物を凍結乾燥する工程;及び、
(3)工程(2)で得られた凍結乾燥物中に含まれる、LDL中の成分の含量を、既知量の当該成分を用いて値付けする工程。
[5]LDL中の成分が、コレステロールである、[3]又は[4]記載の標準品。
[3] In LDL in serum or plasma, comprising lyophilized LDL in serum or plasma and methionine, and the content of components in the LDL is priced Standard product for quantitative determination of ingredients.
[4] The standard product according to [3], which is produced by a method including the following steps.
(1) adding methionine to serum or plasma;
(2) freeze-drying the mixture obtained in step (1); and
(3) A step of pricing the content of the component in LDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
[5] The standard product according to [3] or [4], wherein the component in LDL is cholesterol.
[6]以下の工程を含むことを特徴とする、血清又は血漿中のLDL中の成分定量用の標準品の製造方法。
(1)血清又は血漿にメチオニンを添加する工程;
(2)工程(1)で得られた混合物を凍結乾燥する工程;及び、
(3)工程(2)で得られた凍結乾燥物中に含まれる、LDL中の成分の含量を、既知量の当該成分を用いて値付けする工程。
[7]LDL中の成分が、コレステロールである、[6]記載の製造方法。
[6] A method for producing a standard product for quantitative determination of components in LDL in serum or plasma, comprising the following steps.
(1) adding methionine to serum or plasma;
(2) freeze-drying the mixture obtained in step (1); and
(3) A step of pricing the content of the component in LDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
[7] The production method of [6], wherein the component in LDL is cholesterol.
[8]以下の工程を含むことを特徴とする、血清又は血漿中のLDL中の成分の定量方法。
(1)血清又は血漿中のLDL中の成分の測定試薬を用いて、当該成分を測定する工程;
(2)[3]又は[4]記載の標準品と、工程(1)の測定試薬とを用いて、当該成分の濃度と当該成分に対する測定値との間の関係を表す検量線を作成する工程;及び、
(3)工程(1)で得られた測定値と、工程(2)で作成した検量線とから、血清又は血漿中の当該成分の濃度を決定する工程。
[9]LDL中の成分が、コレステロールである、[8]記載の定量方法。
[8] A method for quantifying components in LDL in serum or plasma, comprising the following steps.
(1) A step of measuring the component using a reagent for measuring the component in LDL in serum or plasma;
(2) Using the standard product described in [3] or [4] and the measurement reagent in step (1), create a calibration curve representing the relationship between the concentration of the component and the measured value for the component Steps; and
(3) A step of determining the concentration of the component in serum or plasma from the measurement value obtained in step (1) and the calibration curve prepared in step (2).
[9] The quantification method according to [8], wherein the component in LDL is cholesterol.
[10]血清又は血漿中のLDL中の成分の測定試薬、及び、[3]又は[4]記載の標準品を含有することを特徴とする、血清又は血漿中のLDL中の成分の定量用キット。
[11]LDL中の成分が、コレステロールである、[10]記載のキット。
[10] For determination of components in LDL in serum or plasma, comprising a reagent for measuring components in LDL in serum or plasma, and a standard product described in [3] or [4] kit.
[11] The kit according to [10], wherein the component in LDL is cholesterol.
 本発明により、血清又は血漿中のLDLの凍結乾燥による変性抑制剤、血清又は血漿中のLDLの凍結乾燥による変性抑制方法、及び、LDL中の成分の正確な測定を可能とする標準品が提供される。 The present invention provides a denaturation inhibitor by freeze-drying of LDL in serum or plasma, a method of inhibiting denaturation by freeze-drying of LDL in serum or plasma, and a standard product that enables accurate measurement of components in LDL. Is done.
(LDLの変性抑制剤とメチオニン)
 本発明のLDLの変性抑制剤は、血清又は血漿中のLDLの凍結乾燥による変性を抑制するものであり、メチオニンを有効成分として含有する。本発明におけるメチオニンとしては、血清又は血漿中のLDLの凍結乾燥による変性を抑制し得る限り、特に制限はなく、L-メチオニン、D-メチオニン、DL-メチオニンのいずれでもよい。
(LDL denaturation inhibitor and methionine)
The LDL denaturation inhibitor of the present invention suppresses denaturation of LDL in serum or plasma due to lyophilization, and contains methionine as an active ingredient. The methionine in the present invention is not particularly limited as long as it can suppress denaturation due to lyophilization of LDL in serum or plasma, and any of L-methionine, D-methionine, and DL-methionine may be used.
 本発明のLDLの変性抑制剤に含まれるメチオニンの含量としては、血清又は血漿中のLDLの凍結乾燥による変性を抑制し得る含量であれば特に制限はなく、通常、血清又は血漿100 mLに本発明のLDLの変性抑制剤が添加されたときに0.025~5 gとなる含量であり、0.5~1 gとなる含量が好ましい。 The content of methionine contained in the LDL denaturation inhibitor of the present invention is not particularly limited as long as it is a content capable of inhibiting the denaturation due to lyophilization of LDL in serum or plasma. When the LDL denaturation inhibitor of the invention is added, the content is 0.025 to 5 g, and preferably 0.5 to 1 g.
(血清と血漿)
 本発明における血清と血漿とは、ヒト又は動物より採取して得られた全血から調製される血清及び血漿であればいずれでもよく、ヒトより採取して得られた全血から調製される血清及び血漿が好ましい。血清は、抗凝固剤を含まない採血管を用いて採取された全血を遠心分離する方法、自然沈降させる方法等により調製することができる。血漿は、抗凝固剤入り採血管によって採取された全血を遠心分離する方法、自然沈降させる方法等により調製することができる。抗凝固剤としては、例えばエチレンジアミン四酢酸(EDTA)2カリウム塩、ヘパリン、フッ化ナトリウム、クエン酸ナトリウム等が挙げられる。動物としては、例えばサル、ゴリラ、オランウータン、チンパンジー、ヒヒ等の霊長類等が挙げられる。
(Serum and plasma)
The serum and plasma in the present invention may be any serum and plasma prepared from whole blood collected from humans or animals, and serum prepared from whole blood collected from humans. And plasma are preferred. Serum can be prepared by a method of centrifuging whole blood collected using a blood collection tube not containing an anticoagulant, a method of natural sedimentation, or the like. Plasma can be prepared by a method of centrifuging whole blood collected by an anticoagulant-containing blood collection tube, a method of spontaneous sedimentation, or the like. Examples of the anticoagulant include ethylenediaminetetraacetic acid (EDTA) dipotassium salt, heparin, sodium fluoride, sodium citrate and the like. Examples of animals include primates such as monkeys, gorillas, orangutans, chimpanzees and baboons.
[低密度リポ蛋白(LDL)]
 本発明におけるLDLとしては、比重が1.006~1.063に分類される広義のLDL、比重が1.019~1.063に分類される狭義のLDLのいずれであっても良い。広義のLDLには、比重が1.006~1.019に分類される中間密度リポ蛋白(IDL)も含まれる。
[Low density lipoprotein (LDL)]
The LDL in the present invention may be either an LDL in a broad sense whose specific gravity is classified as 1.006 to 1.063 or an LDL in a narrow sense whose specific gravity is classified as 1.019 to 1.063. In a broad sense, LDL includes intermediate density lipoprotein (IDL) whose specific gravity is classified into 1.006 to 1.019.
(LDL中の成分)
 本発明におけるLDL中の成分としては、例えばコレステロール、中性脂肪等が挙げられ、コレステロールが好ましい。
(Ingredients in LDL)
Examples of components in LDL in the present invention include cholesterol and neutral fat, and cholesterol is preferred.
(凍結乾燥)
 本発明において、凍結乾燥とは、当該分野において使用される通常の意味で使用され、試料を凍結させ、凍結状態のままで減圧して、試料から水分を除き乾燥することを意味する。凍結乾燥の条件は特に制限はないが、通常-80~35℃、好ましくは-80~30℃の温度で、0.667~1333Pa、好ましくは13.1~133.3Paの圧力で6~120時間、より好ましくは12~120時間行う。凍結乾燥品の水分含量は通常、10重量%以下、好ましくは1重量%以下である。
(freeze drying)
In the present invention, lyophilization is used in the usual sense used in the art, and means that a sample is frozen and depressurized in a frozen state to remove moisture from the sample and dry. The lyophilization conditions are not particularly limited, but are usually -80 to 35 ° C., preferably -80 to 30 ° C., 0.667 to 1333 Pa, preferably 13.1 to 133.3 Pa, and 6 to 120 hours, more preferably Perform for 12 to 120 hours. The water content of the freeze-dried product is usually 10% by weight or less, preferably 1% by weight or less.
(LDLの凍結乾燥による変性抑制方法)
 本発明の、血清又は血漿中のLDLの凍結乾燥による変性抑制方法は、血清又は血漿にメチオニンを添加した後に、血清又は血漿を凍結乾燥することを特徴とする。具体的には、本発明の、血清又は血漿中のLDLの凍結乾燥による変性抑制方法は、以下の工程を含む方法である。
(1)血清又は血漿にメチオニンを添加する工程;及び、
(2)工程(1)で得られた混合物を凍結乾燥する工程。
(Method of inhibiting denaturation by freeze-drying of LDL)
The method for inhibiting denaturation by freeze-drying of LDL in serum or plasma according to the present invention is characterized by freeze-drying serum or plasma after adding methionine to the serum or plasma. Specifically, the method for inhibiting denaturation of lyophilized LDL in serum or plasma according to the present invention includes the following steps.
(1) adding methionine to serum or plasma; and
(2) A step of freeze-drying the mixture obtained in step (1).
 本発明の変性抑制方法において、血清又は血漿に添加されるメチオニンの量は、血清又は血漿中のLDLの凍結乾燥による変性が抑制され得る量であれば特に制限はなく、通常、血清又は血漿100 mL当たり、0.025~5 gであり、0.5~1 gが好ましい。 In the method for inhibiting denaturation of the present invention, the amount of methionine added to serum or plasma is not particularly limited as long as the amount of denaturation due to freeze-drying of LDL in serum or plasma can be suppressed. It is 0.025-5 g per mL, preferably 0.5-1 g.
 血清又は血漿にメチオニンを添加する際、メチオニンはメチオニン水溶液の状態で添加してもよい。メチオニン水溶液の調製には、脱イオン水、蒸留水、緩衝液等の水性媒体を使用することができる。緩衝液を調製するための緩衝剤としては、例えば後述の緩衝剤等が挙げられる。メチオニン水溶液のpHは、メチオニン水溶液を血清又は血漿に添加した後に血清又は血漿中のリポ蛋白が沈澱しないpHであればいずれのpHでもよく、pH6~9が好ましい。 When adding methionine to serum or plasma, methionine may be added in the state of an aqueous methionine solution. For the preparation of the aqueous methionine solution, an aqueous medium such as deionized water, distilled water, or a buffer solution can be used. Examples of the buffering agent for preparing the buffer solution include a buffering agent described later. The pH of the aqueous methionine solution may be any pH as long as the serum or plasma lipoproteins do not precipitate after the aqueous methionine solution is added to the serum or plasma, and is preferably pH 6-9.
(標準品)
 本発明の、血清又は血漿中のLDL中の成分定量用標準品は、凍結乾燥された血清又は血漿中のLDL、及び、メチオニンを含有し、かつ、当該成分の含量が値付けされている標準品である。なお、本発明の標準品は、血清又は血漿中のLDL中の成分定量用の標準品としてのみならず、血清又は血漿中のLDL中の成分定量における精度管理物質として用いることもできる。
(Standard goods)
The standard for quantifying components in LDL in serum or plasma according to the present invention contains LDL in lyophilized serum or plasma and methionine, and the content of the component is priced It is a product. The standard product of the present invention can be used not only as a standard product for quantifying components in LDL in serum or plasma, but also as a quality control substance in quantifying components in LDL in serum or plasma.
 なお、本発明の標準品は凍結乾燥状態であるため、検量線作成等においては、既知量の水性媒体に溶解して用いる。凍結乾燥状態の標準品を溶解するための水性媒体としては、本発明のLDL中の成分の定量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る。緩衝液を調製するための緩衝剤としては、例えば後述の緩衝剤等が挙げられる。 Since the standard product of the present invention is in a lyophilized state, it is dissolved in a known amount of an aqueous medium for use in preparing a calibration curve. The aqueous medium for dissolving the lyophilized standard product is not particularly limited as long as it is an aqueous medium that enables the method of quantifying the components in the LDL of the present invention. For example, deionized water, distilled water, buffer solution Etc. Examples of the buffering agent for preparing the buffer solution include a buffering agent described later.
(標準品の製造方法)
 本発明の標準品の製造方法は、以下の工程を含む。
(1)血清又は血漿にメチオニンを添加する工程;
(2)工程(1)で得られた混合物を凍結乾燥する工程;及び、
(3)工程(2)で得られた凍結乾燥物中に含まれる、LDLの成分の含量を、既知量の当該成分を用いて値付けする工程。
(Standard product manufacturing method)
The method for producing a standard product of the present invention includes the following steps.
(1) adding methionine to serum or plasma;
(2) freeze-drying the mixture obtained in step (1); and
(3) A step of pricing the content of the LDL component contained in the lyophilized product obtained in step (2) using a known amount of the component.
 本発明の標準品の製造方法において、血清又は血漿に添加されるメチオニンの量は、血清又は血漿中のLDLの凍結乾燥による変性が抑制され得る量であれば特に制限はなく、通常、血清又は血漿100 mL当たり、0.025~5 gであり、0.5~1 gが好ましい。 In the method for producing a standard product of the present invention, the amount of methionine added to serum or plasma is not particularly limited as long as it is an amount that can suppress denaturation due to lyophilization of LDL in serum or plasma. The amount is 0.025 to 5 g per 100 mL of plasma, preferably 0.5 to 1 g.
 標準品の製造方法の一態様を以下に記す。
 血清又は血漿を2~8℃に冷却し、これにメチオニン水溶液を添加する。この血清又は血漿含有水溶液のpHを約1 mol/Lの水酸化ナトリウム又は約1 mol/Lの塩酸溶液にてpH6~9に調整した後、このpH調整した水溶液を0.2~0.4ミクロン程度の濾過膜を使用して濾過する。得られた溶液を凍結乾燥用のバイアルに分注し、半打栓する。バイアルに分注した溶液を、-30℃以下の低温で凍結させた後、133.3Paの真空条件下で凍結乾燥させ、徐々に加温しながら最終的に0~20℃にて乾燥し、凍結乾燥状態の標準品を作製する。
One embodiment of a standard product manufacturing method is described below.
Serum or plasma is cooled to 2-8 ° C., and an aqueous methionine solution is added thereto. After adjusting the pH of the serum- or plasma-containing aqueous solution to pH 6-9 with about 1 mol / L sodium hydroxide or about 1 mol / L hydrochloric acid solution, the pH-adjusted aqueous solution is filtered to about 0.2-0.4 microns. Filter using a membrane. The resulting solution is dispensed into lyophilized vials and half-capped. The solution dispensed in the vial is frozen at a low temperature of -30 ° C or lower, then lyophilized under vacuum conditions of 133.3 Pa, and finally dried at 0 to 20 ° C with gradual warming. Prepare a standard product in a dry state.
(標準品の値付け)
 本発明において、標準品の値付けとは、製造された標準品中に含まれる、測定すべきLDL中の成分の含量を、既知量の当該成分を含有する標準血清を用いて決定することをいう。ここで、既知量の当該成分を含有する標準血清とは、例えば米国厚生省疾病管理・予防センター(CDC)の基準分析法等のホモジニアス法によらない方法により、当該成分の含量が決定されている血清をいう。この標準血清及び製造された標準品を検体として用いて、実際の自動分析装置を用いるホモジニアス法により測定を行い、既知量の当該成分を含有する標準血清の測定値(例えば、吸光度)と製造された標準品の測定値(例えば、吸光度)とを比較し、製造された標準品中の当該成分の含量を決定する。製造された標準品は検査施設において標準血清として検体の測定値の決定に使用される。
(Standard product pricing)
In the present invention, pricing of a standard product means determining the content of a component in LDL to be measured contained in a manufactured standard product using a standard serum containing a known amount of the component. Say. Here, the standard serum containing a known amount of the component is determined by a method that does not depend on a homogeneous method such as a standard analysis method of the US Center for Disease Control and Prevention (CDC). Serum. Using this standard serum and the manufactured standard as a specimen, measurement is performed by a homogeneous method using an actual automatic analyzer, and a measurement value (for example, absorbance) of a standard serum containing a known amount of the component is manufactured. The measured value of the standard product (for example, absorbance) is compared to determine the content of the component in the manufactured standard product. The manufactured standard product is used as a standard serum in the laboratory to determine the measurement value of the specimen.
(標準品におけるメチオニンの含量)
 本発明の標準品に含まれるメチオニンの含量は、標準品中のLDLが安定に保持され、標準品中のLDL中の成分が正確に測定できる含量であれば特に制限はなく、通常、血清又は血漿100 mLに対し、0.025~5 gであり、0.5~1 gが好ましい。
(Methionine content in standard products)
The content of methionine contained in the standard product of the present invention is not particularly limited as long as LDL in the standard product is stably maintained and the components in the LDL in the standard product can be accurately measured. For 100 mL of plasma, it is 0.025 to 5 g, preferably 0.5 to 1 g.
(添加物)
 本発明の標準品には、必要に応じて、緩衝剤、金属イオン、塩類、界面活性剤、防腐剤、糖化合物等の添加物が含まれていてもよい。緩衝剤としては、例えば乳酸緩衝剤、クエン酸緩衝剤、酢酸緩衝剤、コハク酸緩衝剤、フタル酸緩衝剤、リン酸緩衝剤、トリエタノールアミン緩衝剤、ジエタノールアミン緩衝剤、リジン緩衝剤、バルビツール緩衝剤、イミダゾール緩衝剤、リンゴ酸緩衝剤、シュウ酸緩衝剤、グリシン緩衝剤、ホウ酸緩衝剤、炭酸緩衝剤、グリシン緩衝剤、グッド緩衝剤等が挙げられる。グッド緩衝剤としては、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。
(Additive)
The standard product of the present invention may contain additives such as buffers, metal ions, salts, surfactants, preservatives, sugar compounds and the like as necessary. Buffers include, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur tool Buffering agents, imidazole buffers, malic acid buffers, oxalic acid buffers, glycine buffers, boric acid buffers, carbonate buffers, glycine buffers, Good buffers, and the like can be mentioned. Examples of the good buffer include 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-aminoethanesulfonic acid (TES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), N- [Tris (hydroxymethyl) methyl] -2-hydroxy-3-amino Nopropanesulfonic 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) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3 -Amino-2-hydroxypropanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and the like.
 金属イオンとしては、例えばマグネシウムイオン、カルシウムイオン、マンガンイオン、亜鉛イオン等が挙げられる。塩類としては、例えば塩化ナトリウム、塩化カリウム等が挙げられる。界面活性剤としては、例えば非イオン性界面活性剤、陽イオン性界面活性剤、陰イオン性界面活性剤、両性界面活性剤等が挙げられる。防腐剤としては、例えばアジ化ナトリウム、ストレプトマイシン、ゲンタマイシン等の抗生物質、バイオエース、プロクリン300(商標)、プロキセル(Proxel)GXL(商標)等が挙げられる。 Examples of metal ions include magnesium ions, calcium ions, manganese ions, and zinc ions. Examples of the salts include sodium chloride and potassium chloride. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Examples of the preservative include antibiotics such as sodium azide, streptomycin, gentamicin, Bioace, Procrine 300 (trademark), Proxel GXL (trademark) and the like.
 上記糖化合物としては、例えば単糖類、二糖類等が挙げられるが、二糖類が好ましい。単糖類としては、例えばグルコース、フルクトース、フコース等が挙げられる。二糖類としては、例えばサッカロース、トレハロース、マルトース等が挙げられる。単糖類、二糖類等は2種以上を併用することもできる。また、糖化合物を血清又は血漿と混合する際の糖化合物の添加量は、血清又は血漿100 mLに対し、通常0.5~20 gであり、1~15 gが好ましく、1.25~12.5 gがより好ましい。 Examples of the sugar compound include monosaccharides and disaccharides, and disaccharides are preferable. Examples of monosaccharides include glucose, fructose, and fucose. Examples of the disaccharide include saccharose, trehalose, maltose and the like. Two or more monosaccharides, disaccharides and the like can be used in combination. The amount of the sugar compound added when the sugar compound is mixed with serum or plasma is usually 0.5 to 20 μg, preferably 1 to 15 μg, more preferably 1.25 to 12.5 μg with respect to 100 μmL of serum or plasma. .
(LDL中の成分の定量方法)
 本発明の血清又は血漿中のLDL中の成分の定量方法は、以下の工程を含む。
(1)血清又は血漿中のLDL中の成分の測定試薬を用いて、当該成分を測定する工程;
(2)本発明の標準品と、工程(1)の測定試薬とを用いて、当該成分の濃度と当該成分に対する測定値との間の関係を表す検量線を作成する工程;及び、
(3)工程(1)で得られた測定値と、工程(2)で作成した検量線とから、血清又は血漿中の当該成分の濃度を決定する工程。
(Quantitative method of components in LDL)
The method for quantifying components in LDL in serum or plasma of the present invention comprises the following steps.
(1) A step of measuring the component using a reagent for measuring the component in LDL in serum or plasma;
(2) using the standard product of the present invention and the measurement reagent of step (1) to create a calibration curve representing the relationship between the concentration of the component and the measured value for the component; and
(3) A step of determining the concentration of the component in serum or plasma from the measurement value obtained in step (1) and the calibration curve prepared in step (2).
 上記工程(1)の測定すべきLDL中の成分の測定においては、公知の、LDL中の成分の測定方法を用いることができる。LDL中の成分の測定方法としては、例えばLDL-C測定方法、LDL中の中性脂肪(以下、LDL-TGと記す)の測定方法等が挙げられる。また、LDL中の成分の測定方法としては、LDLを遠心分離等の手段により分画することなく、LDL以外にHDLやVLDL等の複数種のリポ蛋白が同一の反応液に共存した状態でLDL中の成分を測定する方法、所謂ホモジニアス法による測定方法が好ましい。 In the measurement of the component in LDL to be measured in the above step (1), a known method for measuring the component in LDL can be used. Examples of the method for measuring components in LDL include a method for measuring LDL-C, a method for measuring neutral fat in LDL (hereinafter referred to as LDL-TG), and the like. In addition, as a method for measuring the components in LDL, LDL is not fractionated by means such as centrifugation, but LDL and other types of lipoproteins such as HDL and VLDL coexist in the same reaction solution. A method for measuring the components therein, that is, a so-called homogeneous method is preferred.
 ホモジニアス法によるLDL-C測定方法としては、例えばWO2010/055916パンフレットに記載された、検体とコレステロール測定用酵素とを、[a]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;[b]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ケニルエーテル、ポリオキシエチレン分岐アルキ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分岐アルキルエーテルからなる群より選ばれる1つ以上の界面活性剤;[c]第一級アミン、第二級アミン、第三級アミン及び第四級アンモニウムからなる群より選ばれる1つ以上の界面活性剤;及び、[d]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または還元性物質を測定することにより、検体中のLDL-Cを測定する方法等が挙げられる。ここで、コレステロール測定用酵素とは、(i)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の組み合わせ、又は、(ii)コレステロールエステル加水分解酵素、酸化型補酵素、及び、コレステロール脱水素酵素の組み合わせである。 As a method for measuring LDL-C by the homogeneous method, for example, a specimen and an enzyme for measuring cholesterol described in WO2010 / 055916 pamphlet are used: [a] polyoxyethylene / polyoxyalkylene alkylaryl ether; [b] polyoxyethylene One or more surfactants selected from the group consisting of polyoxyalkylene condensates, polyoxyethylene alkenyl ethers, polyoxyethylene branched alkyl ethers and polyoxyethylene polyoxyalkylene branched alkyl ethers; [c] primary One or more surfactants selected from the group consisting of amines, secondary amines, tertiary amines and quaternary ammoniums; and [d] peroxidation produced by the reaction in the presence of a polyanion. By measuring hydrogen or reducing substances And a method for measuring LDL-C in a specimen. Here, the enzyme for measuring cholesterol is a combination of (i) cholesterol ester hydrolase and cholesterol oxidase, or (ii) cholesterol ester hydrolase, oxidized coenzyme, and cholesterol dehydrogenase. It is a combination.
(LDL中の成分の定量用キット)
 本発明の血清又は血漿中のLDL中の成分の定量用キットは、本発明の標準品とLDL中の成分の測定試薬とを含有する。本発明のキットを用いることにより、LDL中の成分の定量を簡便に行うことができる。LDL中の成分の測定試薬としては、公知の、LDL中の成分の測定試薬を用いることができる。LDL中の成分の測定試薬としては、例えばLDL-C測定試薬、LDL-TG測定試薬等が挙げられる。また、LDL中の成分の測定試薬としては、LDLを遠心分離等の手段により分画することなく、LDL以外にHDLやVLDL等の複数種のリポ蛋白が同一の反応液に共存した状態でLDL中の成分を測定する試薬、所謂ホモジニアス法による測定方法に用いられる試薬が好ましい。
(Kit for quantification of components in LDL)
The kit for quantifying components in LDL in serum or plasma of the present invention contains the standard product of the present invention and a reagent for measuring components in LDL. By using the kit of the present invention, the components in LDL can be easily quantified. As a measuring reagent for the component in LDL, a known measuring reagent for the component in LDL can be used. Examples of the reagent for measuring the components in LDL include an LDL-C measuring reagent and an LDL-TG measuring reagent. In addition, as a reagent for measuring the components in LDL, LDL is not fractionated by means such as centrifugation, and in addition to LDL, multiple types of lipoproteins such as HDL and VLDL coexist in the same reaction solution. A reagent for measuring the components therein, that is, a reagent used in a measuring method by the so-called homogeneous method is preferable.
 ホモジニアス法によるLDL-C測定方法に用いられるLDL-C測定試薬としては、本発明の標準品を用いる、LDL-Cの定量方法を可能とする試薬であれば特に制限はなく、例えばWO2010/055916パンフレットに記載された、コレステロール測定用酵素;[a]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;[b]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ケニルエーテル、ポリオキシエチレン分岐アルキ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分岐アルキルエーテルからなる群より選ばれる1つ以上の界面活性剤;[c]第一級アミン、第二級アミン、第三級アミン及び第四級アンモニウムからなる群より選ばれる1つ以上の界面活性剤;及び、[d]ポリアニオンを含有するLDL-C測定試薬等が挙げられる。ここで、コレステロール測定用酵素とは、(i)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の組み合わせ、又は、(ii)コレステロールエステル加水分解酵素、酸化型補酵素、及び、コレステロール脱水素酵素の組み合わせである。また、ホモジニアス法に用いられるLDL-C測定試薬としては、市販の測定試薬も使用することができる。市販の測定試薬としては、例えばLDL-C測定試薬であるデタミナーL LDL-C(協和メデックス社製)、メタボリードLDL-C(協和メデックス社製)、コレステストN LDL(積水メディカル社製)、Lタイプワコー LDL-C(和光純薬工業社製)等が挙げられる。 The LDL-C measurement reagent used in the LDL-C measurement method by the homogeneous method is not particularly limited as long as it is a reagent that enables the LDL-C quantification method using the standard product of the present invention. For example, WO2010 / 055916 The enzyme for cholesterol measurement described in the pamphlet; [a] polyoxyethylene / polyoxyalkylene alkyl aryl ether; [b] polyoxyethylene / polyoxyalkylene condensate, polyoxyethylene alkenyl ether, polyoxyethylene branched alkyl ether And one or more surfactants selected from the group consisting of polyoxyethylene / polyoxyalkylene branched alkyl ethers; [c] a group consisting of primary amines, secondary amines, tertiary amines and quaternary ammoniums One or more surfactants selected from; Beauty include LDL-C measurement reagents containing the [d] a polyanion. Here, the enzyme for measuring cholesterol is a combination of (i) cholesterol ester hydrolase and cholesterol oxidase, or (ii) cholesterol ester hydrolase, oxidized coenzyme, and cholesterol dehydrogenase. It is a combination. A commercially available measuring reagent can also be used as the LDL-C measuring reagent used in the homogeneous method. Examples of commercially available measuring reagents include LDL-C measuring reagents such as Determiner L LDL-C (manufactured by Kyowa Medex), Metabolid LDL-C (manufactured by Kyowa Medex), Colles Test N LDL (manufactured by Sekisui Medical), L Type Wako LDL-C (manufactured by Wako Pure Chemical Industries, Ltd.)
 本発明の標準品や本発明のキットは、血清又は血漿中のLDL中の成分が自動で連続測定される自動分析機を用いた測定方法に好適に用いられる。 The standard product of the present invention and the kit of the present invention are suitably used in a measurement method using an automatic analyzer in which components in serum or plasma in LDL are automatically and continuously measured.
(LDLの変性抑制剤の効果の検証)
 本発明のLDLの変性抑制剤の効果は、例えば次の方法により検証することができる。複数の健常人より採取した血清又は血漿を混合してプール血清又はプール血漿を調製する。調製したプール血清又はプール血漿を2つに分け、片方のプール血清又はプール血漿には本発明のLDLの変性抑制剤を添加し、本発明のLDLの変性抑制剤が添加されたプール血清又はプール血漿(グループAの血清又は血漿)と、本発明のLDLの変性抑制剤が添加されないプール血清又はプール血漿(グループBの血清又は血漿)とを準備する。グループAの血清又は血漿を同じ形状と材質からなる複数の容器に一定量、分注し、前述の方法により凍結乾燥を行い、さらに前述の方法により値付けを行い、凍結乾燥状態の標準品(標準品A)を調製する。同様に、グループBの血清又は血漿を用いて、凍結乾燥状態の標準品(標準品B)を調製する。調製した標準品AをN本抽出し(標準品A~標準品A)、各標準品A(標準品A~標準品A)を検体とし、LDL-C測定試薬を用いて、各標準品A(標準品A~標準品A)に対する吸光度(吸光度A~吸光度A)を測定する。標準品Bに対しても同様の操作を行い、各標準品B(標準品B~標準品B)に対する吸光度(吸光度B~吸光度B)を測定する。次に、測定した吸光度A~吸光度Aに対する変動係数(coefficient of variation:C.V.)C.V.Aを算出する。同様に、測定した吸光度B~吸光度Bに対する変動係数C.V.Bを算出する。算出したC.V.A及びC.V.Bは、各標準品に対する吸光度のばらつきの大きさを示すため、C.V.が小さいことは、吸光度がより均一であることを意味し、C.V.が大きいことは、吸光度がより不均一であることを意味する。本来であれば、同一の血清又は血漿から標準品A及び標準品Bは調製されるため、標準品Aに対する吸光度(吸光度A~吸光度A)も標準品Bに対する吸光度(吸光度B~吸光度B)も、均一になるはずであるが、凍結乾燥時のLDLの変性の度合いが各容器において微妙に異なるために、吸光度にばらつきが出る。従って、C.V.AがC.V.Bに比較して小さいことが示されれば、本発明のLDLの変性抑制剤により、LDLの凍結乾燥による変性自体が抑制された、と判断できる。
(Verification of the effects of LDL denaturation inhibitors)
The effect of the LDL denaturation inhibitor of the present invention can be verified, for example, by the following method. Pooled serum or pooled plasma is prepared by mixing serum or plasma collected from a plurality of healthy individuals. The prepared pool serum or pool plasma is divided into two, and the LDL denaturation inhibitor of the present invention is added to one pool serum or pool plasma, and the pool serum or pool to which the LDL denaturation inhibitor of the present invention is added Plasma (group A serum or plasma) and pooled serum or pooled plasma (group B serum or plasma) to which the LDL denaturation inhibitor of the present invention is not added are prepared. Dispense a certain amount of Group A serum or plasma into multiple containers of the same shape and material, freeze-dry by the above-mentioned method, further price by the above-mentioned method, A standard A) is prepared. Similarly, a lyophilized standard product (standard product B) is prepared using group B serum or plasma. N prepared standard products A are extracted (standard product A 1 to standard product A N ), and each standard product A (standard product A 1 to standard product A N ) is used as a sample, and LDL-C measurement reagent is used. each standard a (standard a 1 ~ standard a N) absorbance to (absorbance a 1 ~ absorbance a N) is measured. Do the same for standard B, and measure each standard B (Standard B 1 ~ Standard B N) absorbance to (absorbance B 1 ~ absorbance B N). Next, a coefficient of variation (CV) CV A with respect to the measured absorbance A 1 to absorbance A N is calculated. Similarly, a coefficient of variation CV B with respect to the measured absorbance B 1 to absorbance B N is calculated. Calculated CV A and CV B, in order to show the magnitude of variation of the absorbance for each standard, it CV is small, it means that absorbance is more uniform, it CV is large, the absorbance is more unsaturated Means uniform. Originally, since the standard A and the standard B are prepared from the same serum or plasma, the absorbance to the standard A (absorbance A 1 to absorbance A N ) is also the absorbance to the standard B (absorbance B 1 to absorbance). B N ) should also be uniform, but the degree of denaturation of LDL at the time of lyophilization is slightly different in each container, so the absorbance varies. Therefore, if shown that CV A is small compared to the CV B, by denaturation inhibitor of LDL present invention, modified itself by lyophilization of LDL was suppressed, it can be determined.
 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、本実施例においては、下記メーカーの試薬、及び、機器類を使用した。 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 this example, reagents and instruments from the following manufacturers were used.
 血清(Trina Bioreactives AG社製;Human TC/TG/HDL/LDL Cholesterol calibrator, LIQUID 製品コード:CL1130)、トレハロース(林原社製)、バイオエース(ケイ・アイ化成社製)、L-メチオニン(ナカライテスク社製)、ホウ珪酸ガラスバイアル[大和特殊硝子社製(容量:2 mL;直径:16 mm;茶褐色)]、凍結乾燥機(東京理化器械社製;EYELA DRC-1100型、FDU-2100型)。 Serum (manufactured by Trina Bioreactives AG; Human TC / TG / HDL / LDL Cholesterol calibrator, LIQUID product code: CL1130), trehalose (manufactured by Hayashibara), bioace (manufactured by KEI Kasei Co., Ltd.), L-methionine (Nacalai Tesque) ), Borosilicate glass vial [manufactured by Yamato Special Glass Co., Ltd. (capacity: 2 mm; diameter: 16 mm; brown)], freeze dryer (manufactured by Tokyo Rika Kikai Co., Ltd .; EYELA DRC-1100, FDU-2100) .
 以下の方法により、メチオニンの凍結乾燥によるLDLの変性抑制効果を検討した。
(1)凍結乾燥用血清の調製
 以下の組成からなる血清A~Eを調製した。
<血清A~E>
 血清        1 L
 トレハロース    40 g
 バイオエース    0.05 g
 L-メチオニン(第1表参照)
By the following method, the effect of inhibiting the denaturation of LDL by lyophilization of methionine was examined.
(1) Preparation of lyophilized serum Serums A to E having the following compositions were prepared.
<Serum A to E>
Serum 1 L
Trehalose 40 g
Bioace 0.05 g
L-methionine (see Table 1)
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
(2)凍結乾燥
 上記で調製した血清Aをガラスバイアルに0.5 mLずつ分注し、一次凍結乾燥を-34℃で32時間、二次凍結乾燥を20℃で4時間に設定して凍結乾燥を行い、最後に、真空状態のままゴム栓にて閉栓し、凍結乾燥血清Aを製造した。
 血清Aの代わりに、血清B~Eを用いて同様の操作を行い、凍結乾燥血清B~Eを調製した。
(2) Freeze-drying Serum A prepared as described above is dispensed in 0.5 mL portions into glass vials, and freeze-dried by setting primary freeze-drying at -34 ° C for 32 hours and secondary freeze-drying at 20 ° C for 4 hours. Finally, it was closed with a rubber stopper in a vacuum state to produce freeze-dried serum A.
The same operation was performed using serum B to E instead of serum A to prepare freeze-dried serum B to E.
 調製した凍結乾燥血清Aを開栓し、25℃で2時間加温したのち、再度ゴム栓にて閉栓し、冷蔵(4℃)で1週間保存することにより、凍結乾燥血清A+を調製した。凍結乾燥血清Aの代わりに凍結乾燥血清B~Eを用いて同様の操作を行い、凍結乾燥血清B+~E+を調製した。 The prepared freeze-dried serum A was opened, heated at 25 ° C. for 2 hours, then closed again with a rubber stopper, and stored in a refrigerator (4 ° C.) for 1 week to prepare freeze-dried serum A +. The same operation was performed using lyophilized sera B to E instead of lyophilized serum A to prepare lyophilized sera B + to E +.
(3)バイアル間差の検討
 上記(2)で調製した凍結乾燥血清Aのバイアルを20本準備し、各バイアルに精製水0.5 mLの精製水を加えて再構成した血清を検体(20検体)として用いて、LDL-C測定試薬として「デタミナーL LDL-C」を用いて、「デタミナーL LDL-C」に付随の添付文書に記載の操作手順に従い、各検体について全3回測定を行い、各検体に対する吸光度を測定した。測定した吸光度から吸光度のC.V.を算出した。その結果を第2表に示す。
(3) Examination of difference between vials Prepare 20 vials of freeze-dried serum A prepared in (2) above, and add reconstituted serum by adding 0.5 mL of purified water to each vial (20 samples) Using “Determiner L LDL-C” as an LDL-C measurement reagent, according to the operation procedure described in the attached document for “Determiner L LDL-C”, measurement is performed three times for each sample. Absorbance for each specimen was measured. The absorbance CV was calculated from the measured absorbance. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
 第2表から明らかな様に、L-メチオニンを添加して調製した凍結乾燥血清においては、開栓後の処理の有無にかかわらず、L-メチオニンを添加しないで調製した凍結乾燥血清に比較してC.V.が低く、特に5~10 g/LのL-メチオニンを添加した凍結乾燥血清において、C.V.は顕著に低かった。C.V.が高い程、吸光度が不均一であることを意味し、この吸光度の不均一は、凍結乾燥によるLDLの変性に起因することから、L-メチオニンを血清又は血漿に添加した場合にC.V.が低くなることは、L-メチオニンが、血清又は血漿中のLDLの凍結乾燥による変性を抑制することを示している。従って、本発明のLDLの変性抑制剤により、血清又は血漿中のLDLの凍結乾燥による変性が抑制され、血清又は血漿中のLDL中の成分を正確に測定することができることが分かった。 As is apparent from Table 2, the lyophilized serum prepared with the addition of L-methionine was compared with the lyophilized serum prepared without the addition of L-methionine regardless of the treatment after the opening. The CV was particularly low in lyophilized serum supplemented with 5 to 10 g / L L-methionine. A higher CV means that the absorbance is non-uniform, and this non-uniform absorbance is due to LDL denaturation due to lyophilization. Therefore, when L-methionine is added to serum or plasma, the CV decreases. This indicates that L-methionine suppresses the denaturation of LDL in serum or plasma due to lyophilization. Therefore, it was found that the LDL denaturation inhibitor of the present invention suppresses denaturation of LDL in serum or plasma due to lyophilization, and the components in LDL in serum or plasma can be accurately measured.
 本発明により、凍結乾燥による血清又は血漿中のLDLの変性抑制剤、凍結乾燥による血清又は血漿中のLDLの変性抑制方法、血清又は血漿中のLDL中の成分の正確な測定を可能とする標準品とその製造方法、血清又は血漿中のLDL中の成分の定量方法、及び、血清又は血漿中のLDL中の成分の定量キットが提供される。 According to the present invention, an LDL denaturation inhibitor in serum or plasma by lyophilization, a method for inhibiting LDL denaturation in serum or plasma by lyophilization, and a standard that enables accurate measurement of components in LDL in serum or plasma And a method for producing the same, a method for quantifying components in LDL in serum or plasma, and a kit for quantifying components in LDL in serum or plasma.
 本発明の血清又は血漿中のLDLの変性抑制剤、血清又は血漿中のLDLの変性抑制方法、血清又は血漿中のLDL中の成分定量用標準品とその製造方法、血清又は血漿中のLDL中の成分の定量方法、及び、血清又は血漿中のLDL中の成分の定量キットは、メタボリックシンドローム等の診断に有用である。
 
LDL denaturation inhibitor in serum or plasma of the present invention, method for inhibiting LDL denaturation in serum or plasma, standard product for quantification of components in LDL in serum or plasma and production method thereof, in LDL in serum or plasma The component quantification method and the quantification kit for components in LDL in serum or plasma are useful for diagnosis of metabolic syndrome and the like.

Claims (11)

  1. メチオニンを有効成分として含有する、血清又は血漿中の低密度リポ蛋白の凍結乾燥による変性抑制剤。 A denaturation inhibitor by freeze-drying of low-density lipoprotein in serum or plasma, containing methionine as an active ingredient.
  2. 血清又は血漿にメチオニンを添加した後、血清又は血漿を凍結乾燥することを特徴とする、血清又は血漿中の低密度リポ蛋白の凍結乾燥による変性抑制方法。 A method for inhibiting denaturation by freeze-drying of low-density lipoprotein in serum or plasma, wherein methionine is added to serum or plasma, followed by freeze-drying of serum or plasma.
  3. 凍結乾燥された、血清又は血漿中の低密度リポ蛋白、及び、メチオニンを含有し、かつ、当該低密度リポ蛋白中の成分の含量が値付けされていることを特徴とする、血清又は血漿中の低密度リポ蛋白中の成分定量用標準品。 In serum or plasma, comprising lyophilized low-density lipoprotein and methionine in serum or plasma, and the content of components in the low-density lipoprotein is priced Standard for quantitative determination of components in low-density lipoprotein.
  4. 以下の工程を含む方法により製造される、請求項3記載の標準品。
    (1)血清又は血漿にメチオニンを添加する工程;
    (2)工程(1)で得られた混合物を凍結乾燥する工程;及び、
    (3)工程(2)で得られた凍結乾燥物中に含まれる、低密度リポ蛋白中の成分の含量を、既知量の当該成分を用いて値付けする工程。
    The standard product according to claim 3, which is produced by a method comprising the following steps.
    (1) adding methionine to serum or plasma;
    (2) freeze-drying the mixture obtained in step (1); and
    (3) A step of pricing the content of the component in the low-density lipoprotein contained in the lyophilized product obtained in step (2) using a known amount of the component.
  5. 低密度リポ蛋白中の成分が、コレステロールである、請求項3又は4記載の標準品。 The standard product according to claim 3 or 4, wherein the component in the low density lipoprotein is cholesterol.
  6. 以下の工程を含むことを特徴とする、血清又は血漿中の低密度リポ蛋白中の成分定量用の標準品の製造方法。
    (1)血清又は血漿にメチオニンを添加する工程;
    (2)工程(1)で得られた混合物を凍結乾燥する工程;及び、
    (3)工程(2)で得られた凍結乾燥物中に含まれる、低密度リポ蛋白中の成分の含量を、既知量の当該成分を用いて値付けする工程。
    A method for producing a standard product for quantitative determination of components in low-density lipoprotein in serum or plasma, which comprises the following steps.
    (1) adding methionine to serum or plasma;
    (2) freeze-drying the mixture obtained in step (1); and
    (3) A step of pricing the content of the component in the low-density lipoprotein contained in the lyophilized product obtained in step (2) using a known amount of the component.
  7. 低密度リポ蛋白中の成分が、コレステロールである、請求項6記載の製造方法。 The production method according to claim 6, wherein the component in the low density lipoprotein is cholesterol.
  8. 以下の工程を含むことを特徴とする、血清又は血漿中の低密度リポ蛋白中の成分の定量方法。
    (1)血清又は血漿中の低密度リポ蛋白中の成分の測定試薬を用いて、当該成分を測定する工程;
    (2)請求項3又は4記載の標準品と、工程(1)の測定試薬とを用いて、当該成分の濃度と当該成分に対する測定値との間の関係を表す検量線を作成する工程;及び、
    (3)工程(1)で得られた測定値と、工程(2)で作成した検量線とから、血清又は血漿中の当該成分の濃度を決定する工程。
    A method for quantifying a component in low-density lipoprotein in serum or plasma, comprising the following steps.
    (1) a step of measuring the component using a reagent for measuring the component in low-density lipoprotein in serum or plasma;
    (2) A step of creating a calibration curve representing the relationship between the concentration of the component and the measured value for the component using the standard product according to claim 3 or 4 and the measurement reagent of step (1); as well as,
    (3) A step of determining the concentration of the component in serum or plasma from the measurement value obtained in step (1) and the calibration curve prepared in step (2).
  9. 低密度リポ蛋白中の成分が、コレステロールである、請求項8記載の定量方法。 The quantification method according to claim 8, wherein the component in the low density lipoprotein is cholesterol.
  10. 血清又は血漿中の低密度リポ蛋白中の成分の測定試薬、及び、請求項3又は4記載の標準品を含有することを特徴とする、血清又は血漿中の低密度リポ蛋白中の成分の定量用キット。 A reagent for measuring a component in low-density lipoprotein in serum or plasma, and a standard product according to claim 3 or 4, comprising a reagent for determination of a component in low-density lipoprotein in serum or plasma For kit.
  11. 低密度リポ蛋白中の成分が、コレステロールである、請求項10記載のキット。
     
    The kit according to claim 10, wherein the component in the low density lipoprotein is cholesterol.
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