WO2015076284A1 - Stabilizer and stabilization method for high-density lipoproteins in blood serum or blood plasma - Google Patents

Stabilizer and stabilization method for high-density lipoproteins in blood serum or blood plasma Download PDF

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Publication number
WO2015076284A1
WO2015076284A1 PCT/JP2014/080603 JP2014080603W WO2015076284A1 WO 2015076284 A1 WO2015076284 A1 WO 2015076284A1 JP 2014080603 W JP2014080603 W JP 2014080603W WO 2015076284 A1 WO2015076284 A1 WO 2015076284A1
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serum
plasma
hdl
density lipoprotein
component
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PCT/JP2014/080603
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French (fr)
Japanese (ja)
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健太 金城
拓郎 奥田
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協和メデックス株式会社
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Priority to JP2015549165A priority Critical patent/JP6795887B2/en
Publication of WO2015076284A1 publication Critical patent/WO2015076284A1/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
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a stabilizer for high-density lipoprotein in serum or plasma, a method for stabilizing high-density lipoprotein in serum or plasma, a method for preserving high-density lipoprotein in serum or plasma, Standards for quantifying components in high density lipoprotein, methods for producing standard products for quantifying components in high density lipoproteins in serum or plasma, methods for quantifying components in high density lipoproteins in serum or plasma, The present invention also relates to a kit for quantifying components in high-density lipoprotein in serum or plasma.
  • Serum and plasma are one of the specimens often used in clinical tests. 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. These lipoproteins are separated by agarose gel electrophoresis based on the difference in charge and by polyacrylamide gel electrophoresis based on the difference in particle diameter. It is known that lipoproteins in serum and plasma are denatured during storage. Since the denatured lipoprotein changes in charge and particle size from the original lipoprotein, the denatured lipoprotein can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.
  • HDL high density lipoprotein
  • LDL low density lipoprotein
  • VLDL very low density lipoprotein
  • Patent Document 1 As a method for stabilizing lipoprotein for suppressing lipoprotein denaturation, a method using 2-methyl-4-isothiazolin-3-one (see Patent Document 1), a method using guanidine sulfate (Patent Document 2). And a method using a buffer having a sulfo group (see Patent Document 3) and the like are known.
  • HDL-C cholesterol in HDL
  • LDL-C cholesterol in LDL
  • Patent Document 4 Method for measuring HDL-C using ionic surfactant, polyanion and albumin
  • Patent Document 5 Method for measuring HDL-C using quaternary ammonium salt and polyanion
  • Patent Document 6 Method for measuring LDL-C using an ethylene polycyclic surfactant
  • Patent Document 8 Method for measuring LDL-C using an arylsulfonic acid derivative
  • Serum or plasma lipoproteins are easily denatured during storage or by lyophilization. This denaturation of lipoproteins often affects the measurement of components in lipoproteins and often precludes accurate measurement of components in lipoproteins.
  • the object of the present invention is to stabilize HDL in serum or plasma, to stabilize HDL in serum or plasma, to accurately measure components in HDL in serum or plasma, in serum or plasma, Provided are a method for storing HDL, a standard for quantifying components in HDL and a method for producing the same, a method for quantifying components in HDL in serum or plasma, and a kit for quantifying components in HDL in serum or plasma. There is.
  • the present inventors have stabilized HDL in serum or plasma by adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  • the present invention was completed by finding the knowledge that components in HDL such as HDL-C can be accurately quantified. That is, the present invention relates to the following [1] to [18].
  • a stabilizer for HDL in serum or plasma containing a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative as an active ingredient.
  • a method for stabilizing HDL in serum or plasma comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  • a method for stabilizing HDL in serum or plasma comprising the following steps.
  • a method for preserving HDL in serum or plasma comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  • a method for preserving HDL in serum or plasma comprising the following steps. (1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and (2) A step of storing the mixture obtained in step (1).
  • a method for preserving HDL in serum or plasma comprising the following steps.
  • a method for producing a standard product for quantitative determination of components in HDL in serum or plasma comprising the following steps. (1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and (2) A step of pricing the concentration of the component in HDL contained in the mixture obtained in step (1) using a known amount of the component. [13] A method for producing a standard product for quantitative determination of components in HDL in serum or plasma, comprising the following steps.
  • a method for quantifying components in HDL in serum or plasma comprising the following steps. (1) a step of measuring the component using a reagent for measuring the component in HDL in serum or plasma; (2) Calibration using the standard product according to any one of [7] to [10] and the measurement reagent of step (1) to express the relationship between the concentration of the component and the measured value for the component Creating a line; 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).
  • the quantification method according to [15] wherein the component in HDL is cholesterol.
  • a stabilizer for HDL in serum or plasma a method for stabilizing HDL in serum or plasma, serum or plasma for accurately measuring components in HDL such as HDL-C in serum or plasma, Method for storing HDL in plasma, standard product for quantifying components in HDL and method for producing the same, method for quantifying components in HDL in serum or plasma, and kit for quantifying components in HDL in serum or plasma Is provided.
  • 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.
  • HDL High density lipoprotein in the present invention is a lipoprotein having a specific gravity of 1.063 to 1.21.
  • Examples of components in HDL in the present invention include cholesterol and neutral fat, and cholesterol is preferred.
  • the stabilizer of the present invention stabilizes HDL in serum or plasma.
  • the stabilizer of the present invention contains a polyoxyethylene polycyclic phenyl ether derivative (hereinafter referred to as POE polycyclic phenyl ether derivative) or a naphthalenesulfonic acid derivative as an active ingredient.
  • Examples of the POE polycyclic phenyl ether derivative in the present invention include polyoxyethylene polycyclic phenyl ether (hereinafter referred to as POE polycyclic phenyl ether), polyoxyethylene polycyclic phenyl ether sulfate (hereinafter referred to as POE polycyclic phenyl ether sulfate).
  • polyoxyethylene / polyoxypropylene polycyclic phenyl ether hereinafter referred to as POE / POP polycyclic phenyl ether).
  • POE polycyclic phenyl ether sulfates include, for example, New Coal 707-SF, New Coal 707-SFC, New Coal 707-SN, New Coal 714-SF, New Coal 714-SN, New Coal 723-SF, New Coal 740-SF, New Coal 780-SF, New Coal 2607-SF, New Coal 2614-SF (above, manufactured by Nippon Emulsifier Co., Ltd.), Hightenol NF-08, Hightenol NF-0825, High Tenol NF-13, Haitenol NF-17 (Daiichi Kogyo Seiyaku Co., Ltd.) and the like.
  • POE / POP polycyclic phenyl ether examples include, for example, New Coal 707-F, New Coal 710-F, New Coal 714-F, New Coal 2608-F, New Coal 2600-FB, New Coal 2616-F, New Coal 3612-FA (manufactured by Nippon Emulsifier Co., Ltd.) and the like.
  • naphthalene sulfonic acid derivatives examples include naphthalene sulfonic acid formaldehyde condensates.
  • Specific examples (commercial products) of naphthalene sulfonic acid formaldehyde condensates include, for example, Disrol SH, Escort (30) (manufactured by Nippon Emulsifier Co., Ltd.), Demol N, Demol NL, Demol RN, Demol RN-L, Demol T , Demol T-45, Demol MS, Demol SN-B (above, manufactured by Kao Corporation) and the like.
  • the content of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative contained in the stabilizer of the present invention is not particularly limited as long as it is a content capable of stabilizing HDL in serum or plasma.
  • the stabilizer of the present invention is added to mL, the content is 0.01-5 g, and the content is preferably 0.05-1 g.
  • 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 13.3 to 133.3 Pa, and 6 to 120 hours, more preferably 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 stabilizing HDL of the present invention includes a step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  • the amount of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative added to serum or plasma is not particularly limited as long as HDL is stabilized in serum or plasma, and is usually per 100 mL of serum or plasma. 0.01 to 5 g, preferably 0.05 to 1 g.
  • the POE polycyclic phenyl ether derivative When a POE polycyclic phenyl ether derivative is added to serum or plasma, the POE polycyclic phenyl ether derivative may be added as the POE polycyclic phenyl ether derivative itself, or as a POE polycyclic phenyl ether derivative aqueous solution. Good.
  • the naphthalenesulfonic acid derivative when the naphthalenesulfonic acid derivative is added to serum or plasma, the naphthalenesulfonic acid derivative may be added as the naphthalenesulfonic acid derivative itself, or may be added as a naphthalenesulfonic acid derivative aqueous solution.
  • an aqueous medium such as deionized water, distilled water, or a buffer solution
  • a buffering agent for preparing the buffer solution include a buffering agent described later.
  • the pH of these aqueous solutions is not particularly limited as long as the lipoprotein in serum or plasma does not precipitate after the aqueous solution is added to serum or plasma, and is preferably pH 6-9.
  • Another aspect of the HDL stabilization method of the present invention includes the following steps. (1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; (2) freeze-drying the mixture obtained in step (1); and (3) A step of dissolving the lyophilized product obtained in step (2) with an aqueous medium.
  • the aqueous medium in step (3) is not particularly limited as long as it dissolves HDL in serum or plasma and can stably maintain HDL in an aqueous solution of HDL, and examples thereof include the above-mentioned aqueous medium. It is done.
  • the method for storing HDL of the present invention includes a step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  • Another aspect of the HDL storage method of the present invention includes the following steps. (1) adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and (2) A step of storing the mixture obtained in step (1).
  • another aspect of the HDL storage method of the present invention includes the following steps. (1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; (2) a step of freeze-drying the mixture obtained in step (1); (3) dissolving the lyophilized product obtained in step (2) with an aqueous medium; and (4) A step of storing the aqueous solution obtained in step (3).
  • Storing conditions in the HDL storage method of the present invention are not particularly limited as long as HDL is stored stably.
  • the storage temperature in the HDL storage method is not particularly limited as long as the HDL is stored stably, and is usually -80 to 45 ° C, preferably -5 to 30 ° C, and particularly preferably 2 to 10 ° C. .
  • the storage period in the HDL storage method is not particularly limited as long as HDL is stably stored, and is usually 1 day to 2 years.
  • the standard for quantifying components in HDL in serum or plasma of the present invention may be in solution or lyophilized.
  • the standard product of the present invention contains HDL in serum or plasma and a POE polycyclic phenyl ether derivative or naphthalene sulfonic acid derivative, and the content of the component in the HDL is a value. It is attached.
  • the standard product is in a lyophilized state, contains HDL in freeze-dried serum or plasma, and POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative, and the HDL The content of the components in it is priced.
  • the standard product of the present invention can be used not only as a standard product for quantifying components in HDL in serum or plasma, but also as a quality control substance in quantifying components in HDL in serum or plasma.
  • the method for producing the standard product of the present invention includes the following steps. (1) adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and (2) A step of pricing the concentration of the component in HDL contained in the mixture obtained in step (1) using a known amount of the component.
  • the method for producing the standard product of the present invention includes the following steps. (1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 HDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
  • the amount of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative added to serum or plasma is not particularly limited as long as HDL is stabilized in serum or plasma. In general, it is 0.01 to 5 g, preferably 0.05 to 1 g per 100 mL of serum or plasma.
  • Serum or plasma is cooled to 2-8 ° C., and an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma.
  • an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma.
  • the pH-adjusted aqueous solution is filtered to about 0.2-0.4 microns. Filter using a membrane to obtain a standard product.
  • Serum or plasma is cooled to 2-8 ° C., and an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma.
  • an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma.
  • 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.
  • the standard product in a lyophilized state is used after dissolving in a known amount of an aqueous medium in preparing a calibration curve.
  • the aqueous medium for dissolving the lyophilized standard is not particularly limited as long as it is an aqueous medium that enables the method of quantifying the components in HDL of the present invention.
  • deionized water, distilled water, buffer solution Etc examples of the buffering agent for preparing the buffer solution include a buffering agent described later.
  • pricing of a standard product means determining the content of a component in HDL 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 the POE polycyclic phenyl ether derivative and naphthalene sulfonic acid derivative contained in the standard product of the present invention should be such that the HDL in the standard product is stably maintained and the components in the HDL 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 HDL 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 HDL 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 HDL can be used.
  • the method for measuring components in HDL include a method for measuring HDL-C, a method for measuring neutral fat in HDL (hereinafter referred to as HDL-TG), and the like.
  • HDL-TG a method for measuring neutral fat in HDL
  • HDL-TG a method for measuring neutral fat in HDL
  • 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 WO 2004/035816 are reacted in an aqueous medium containing a nonionic surfactant, a polyanion, and albumin.
  • 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 HDL in serum or plasma of the present invention contains the standard product of the present invention and a reagent for measuring components in HDL. By using the kit of the present invention, the components in HDL can be easily quantified.
  • a reagent for measuring a component in HDL a known reagent for measuring a component in HDL can be used. Examples of the reagent for measuring components in HDL include HDL-C measuring reagent and HDL-TG measuring reagent.
  • HDL is not fractionated by means of centrifugation or the like, but in a state where a plurality of lipoproteins such as LDL and VLDL coexist in the same reaction solution in addition to HDL.
  • a reagent for measuring the components therein that is, a reagent used in a measuring method by the so-called homogeneous method is preferable.
  • Examples of the HDL-C measuring reagent used in the HDL-C measuring method by the homogeneous method include HDL-C containing an enzyme for measuring cholesterol, a nonionic surfactant, a polyanion and albumin described in, for example, WO 2004/035816 pamphlet.
  • Examples include a measurement reagent, an enzyme for measuring cholesterol, a quaternary ammonium salt having a specific structure, and a HDL-C measurement reagent containing a polyanion described in WO 2006/118199 pamphlet.
  • 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.
  • a commercially available measuring reagent can also be used as the HDL-C measuring reagent used in the homogeneous method.
  • Examples of commercially available measuring reagents include Determiner L HDL-C (manufactured by Kyowa Medex), Metabolid HDL-C (manufactured by Kyowa Medex), and “Seiken” HDL-EX N (manufactured by Denka Seiken). ) And the like.
  • 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 HDL are automatically and continuously measured.
  • the effects of the HDL stabilizer, the stabilization method, and the storage method 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 HDL stabilizer of the present invention is added to one pool serum or pool plasma, and the pool sera or pool to which the HDL stabilizer 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 HDL stabilizer of the present invention is not added are prepared.
  • a certain amount of group A serum or plasma is dispensed into a plurality of containers of the same shape and material, and if necessary, freeze-dried by the method described above.
  • pricing is performed by the above-described method to prepare a standard product (standard product A).
  • a standard product (standard product B) is prepared using serum or plasma of group B.
  • the sample is stored for 7 days at 5 ° C. before storage of standard solution A, and after storage of standard solution A ( 1 after storage of standard product A to N after storage of standard product A).
  • Serum (manufactured by Discovery Life Science), trehalose (manufactured by Hayashibara), New Coal 740 (60) (POE polycyclic phenyl ether derivative; manufactured by Nippon Emulsifier Co., Ltd.), New Coal 740-SF (POE polycyclic phenyl ether derivative; Japan) Emulsifier Co., Ltd.), Demol N (Naphthalenesulfonic acid derivative; Kao Co., Ltd.), Borosilicate glass vial [manufactured by Yamato Special Glass Co., Ltd. (capacity: 2 mL; diameter: 16 mm; brown)], freeze dryer (Tokyo Rika Instruments) Manufactured by EYELA DRC-1100, FDU-2100).
  • the serum 1 before storage (the serum 1 before storage 1 serum 1 before storage 3) a 5 ° C. for 7 days Save and sera obtained 1 after storage (serum 1 after storage 1 serum 1 after storage 3)
  • “Metabolide HDL-C” as an HDL-C measurement reagent, and measuring each sample according to the operation procedure described in the package insert accompanying “Metabolide HDL-C” after the absorbance 1 store (absorbance 1 after storage 1 to the absorbance 1 after storage 3) were measured for each sample, the mean value was calculated absorbance 1 after storage 1 to the absorbance 1 after storage 3 as the mean absorbance 1 after storage.
  • Measure by the same method except using freeze-dried serum 2-4 and freeze-dried serum 0 instead of freeze-dried serum 1, and calculate average absorbance 2 after storage -average absorbance 4 storage and after average absorbance 0 storage did.
  • the residual rate (%) was calculated according to the following formula (II) using the calculated average absorbance before 1 storage and after the average absorbance after 1 storage .
  • the freeze-dried serum 1 was measured three times in accordance with the above procedure, and the residual ratio in each measurement of all three measurements was calculated, and the average value (average residual ratio) of the calculated residual ratio was determined.
  • the combination of the average absorbance 1 before storage and the average absorbance 1 after storage instead of the combination of the average absorbance 2 before storage and the average absorbance 2 storage, the combination of the average absorbance 3 before storage and the average absorbance 3 storage , and the average absorbance 4 before storage mean absorbance 4 combination after storage, and, in the same procedure as above except for using the mean absorbance 0 after storage and the average absorbance 0 combination after storage, respectively, each of the freeze-lyophilized serum 2-4 and lyophilized serum 0
  • measurement was performed three times in total, and the residual rate in each measurement of all three measurements was calculated for each freeze-dried serum, and the average value (average residual rate) of the calculated residual rate was calculated. Determined for each lyophilized serum. Further, the calculated residual ratio was subjected to a Dunnett test (Dunnett test) with a significance level of 0.05. The results are shown in Table 2.
  • the lyophilized serum prepared by adding the stabilizer of the present invention has a higher residual rate than the lyophilized serum prepared without adding the stabilizer of the present invention. It was. In the standard product for HDL-C determination, even when the standard product is stored in solution, a very high residual rate of 100% is required for accurate HDL-C measurement. Is done. Therefore, the difference in the residual ratio between the case where the stabilizer of the present invention is added and the case where it is not added in Table 2 is important. In addition, when Dunnett's test was performed with a significance level of 0.05, all the lyophilized sera prepared by adding the stabilizer of the present invention remained in the lyophilized serum prepared by adding the stabilizer of the present invention.
  • a stabilizer for HDL in serum or plasma a method for stabilizing HDL in serum or plasma, a method for storing HDL in serum or plasma, and an accurate measurement of components in HDL in serum or plasma.
  • a standard product and a production method thereof a method for quantifying components in HDL in serum or plasma, and a kit for quantifying components in HDL in serum or plasma.
  • Stabilizer for HDL in serum or plasma of the present invention method for stabilizing HDL in serum or plasma, method for storing HDL in serum or plasma, standard for quantification of components in HDL in serum or plasma, and The production method, the method for quantifying components in HDL in serum or plasma, and the kit for quantifying components in HDL in serum or plasma are useful for diagnosis of metabolic syndrome and the like.

Abstract

In order to accurately measure the components of high-density lipoproteins (HDL) in blood serum or blood plasma, the present invention provides a stabilizer for HDL in blood serum or blood plasma, a stabilization method for HDL in blood serum or blood plasma, a storage method for HDL in blood serum or blood plasma, and a reference standard for quantifying the components of HDL. A stabilizer for HDL in blood serum or blood plasma having a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonate derivative as an active component. A stabilization method and a storage method for HDL in blood serum or blood plasma, characterized in that a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonate derivative is added to the blood serum or blood plasma. A reference standard for quantifying the components of HDL in blood serum or blood plasma, characterized by containing HDL in blood serum or blood plasma, and a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonate derivative, wherein a value is assigned for the content of the components of the HDL.

Description

血清又は血漿中の高密度リポ蛋白の安定化剤及び安定化方法Stabilizer and method for stabilizing high-density lipoprotein in serum or plasma
　本発明は、血清又は血漿中の高密度リポ蛋白の安定化剤、血清又は血漿中の高密度リポ蛋白の安定化方法、血清又は血漿中の高密度リポ蛋白の保存方法、血清又は血漿中の高密度リポ蛋白中の成分定量用の標準品、血清又は血漿中の高密度リポ蛋白中の成分定量用の標準品の製造方法、血清又は血漿中の高密度リポ蛋白中の成分の定量方法、及び、血清又は血漿中の高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 stabilizer for high-density lipoprotein in serum or plasma, a method for stabilizing high-density lipoprotein in serum or plasma, a method for preserving high-density lipoprotein in serum or plasma, Standards for quantifying components in high density lipoprotein, methods for producing standard products for quantifying components in high density lipoproteins in serum or plasma, methods for quantifying components in high density lipoproteins in serum or plasma, The present invention also relates to a kit for quantifying components in high-density lipoprotein in serum or plasma.
　血清及び血漿は、臨床検査においてしばしば用いられる検体の１つである。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。これらのリポ蛋白は荷電の違いに基づき、アガロースゲル電気泳動により、また、粒子径の違いに基づき、ポリアクリルアミドゲル電気泳動により分離される。血清及び血漿中のリポ蛋白は保存中に変性することが知られている。変性したリポ蛋白は元のリポ蛋白と荷電や粒子径が変化するため、アガロースゲル電気泳動やポリアクリルアミドゲル電気泳動によりリポ蛋白の変性を検出することができる。 Serum and plasma are one of the specimens often used in clinical tests. 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. These lipoproteins are separated by agarose gel electrophoresis based on the difference in charge and by polyacrylamide gel electrophoresis based on the difference in particle diameter. It is known that lipoproteins in serum and plasma are denatured during storage. Since the denatured lipoprotein changes in charge and particle size from the original lipoprotein, the denatured lipoprotein can be detected by agarose gel electrophoresis or polyacrylamide gel electrophoresis.
　これまでにリポ蛋白の変性を抑制するためのリポ蛋白の安定化方法として、２－メチル－４－イソチアゾリン－３－オンを用いる方法（特許文献１参照）、硫酸グアニジンを用いる方法（特許文献２参照）、スルホ基を有する緩衝剤を用いる方法（特許文献３参照）等が知られている。 So far, as a method for stabilizing lipoprotein for suppressing lipoprotein denaturation, a method using 2-methyl-4-isothiazolin-3-one (see Patent Document 1), a method using guanidine sulfate (Patent Document 2). And a method using a buffer having a sulfo group (see Patent Document 3) and the like are known.
　血清及び血漿中の特定のリポ蛋白中の成分を測定する方法として、当該特定のリポ蛋白を分離することなく測定する方法、所謂、ホモジニアス法が報告されている。特に、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）の測定においては、界面活性剤を用いるホモジニアス法が報告されており、例えば、非イオン性界面活性剤、ポリアニオン、及び、アルブミンを用いるＨＤＬ－Ｃの測定方法（特許文献４）、第四級アンモニウム塩、及び、ポリアニオンを用いるＨＤＬ－Ｃの測定方法（特許文献５）、ポリオキシエチレン多環系界面活性剤を用いるＬＤＬ－Ｃの測定方法（特許文献６～８参照）やアリールスルホン酸誘導体を用いるＬＤＬ－Ｃの測定方法（特許文献８参照）が報告されている。 As a method for measuring components in specific lipoproteins in serum and plasma, a method for measuring the specific lipoproteins without separating them, a so-called homogeneous method has been reported. In particular, in the measurement of cholesterol in HDL (hereinafter referred to as HDL-C) and cholesterol in LDL (hereinafter referred to as LDL-C), a homogeneous method using a surfactant has been reported. Method for measuring HDL-C using ionic surfactant, polyanion and albumin (Patent Document 4), Method for measuring HDL-C using quaternary ammonium salt and polyanion (Patent Document 5), polyoxy A method for measuring LDL-C using an ethylene polycyclic surfactant (see Patent Documents 6 to 8) and a method for measuring LDL-C using an arylsulfonic acid derivative (see Patent Document 8) have been reported.
特開平１０－０８７６９３号公報Japanese Patent Laid-Open No. 10-087793 特開平９－２１６８３５号公報Japanese Patent Laid-Open No. 9-216835 ＷＯ２００６／０５４５１９パンフレットWO 2006/0554519 pamphlet ＷＯ２００４／０３５８１６パンフレットWO2004 / 035816 pamphlet ＷＯ２００６／１１８１９９パンフレットWO2006 / 118199 pamphlet ＷＯ２００４／０４８６０５パンフレットWO2004 / 048605 pamphlet 特開２００９－２８２０４４号公報JP 2009-282044 A ＷＯ２０１１／１３６３１６パンフレットWO2011 / 136316 Pamphlet
　血清又は血漿中のリポ蛋白は保存中に、又は、凍結乾燥により変性し易い。このリポ蛋白の変性は、リポ蛋白中の成分の測定に影響を与え、リポ蛋白中の成分の正確な測定を妨げてしまうことがしばしば問題となる。 Serum or plasma lipoproteins are easily denatured during storage or by lyophilization. This denaturation of lipoproteins often affects the measurement of components in lipoproteins and often precludes accurate measurement of components in lipoproteins.
　本発明の目的は、血清又は血漿中のＨＤＬ中の成分を正確に測定するための、血清又は血漿中のＨＤＬの安定化剤、血清又は血漿中のＨＤＬの安定化方法、血清又は血漿中のＨＤＬの保存方法、ＨＤＬ中の成分定量用の標準品とその製造方法、血清又は血漿中のＨＤＬ中の成分の定量方法、及び、血清又は血漿中のＨＤＬ中の成分の定量用キットを提供することにある。 The object of the present invention is to stabilize HDL in serum or plasma, to stabilize HDL in serum or plasma, to accurately measure components in HDL in serum or plasma, in serum or plasma, Provided are a method for storing HDL, a standard for quantifying components in HDL and a method for producing the same, a method for quantifying components in HDL in serum or plasma, and a kit for quantifying components in HDL in serum or plasma. There is.
　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、血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加することにより、血清又は血漿中でＨＤＬが安定化され、ＨＤＬ－Ｃ等のＨＤＬ中の成分を正確に定量できる、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］～［１８］に関する。 As a result of intensive studies to solve this problem, the present inventors have stabilized HDL in serum or plasma by adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma. The present invention was completed by finding the knowledge that components in HDL such as HDL-C can be accurately quantified. That is, the present invention relates to the following [1] to [18].
［１］ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を有効成分として含有する、血清又は血漿中のＨＤＬの安定化剤。
［２］血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加することを特徴とする、血清又は血漿中のＨＤＬの安定化方法。
［３］以下の工程を含むことを特徴とする、血清又は血漿中のＨＤＬの安定化方法。
（１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（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を水性媒体で溶解する工程。 [1] A stabilizer for HDL in serum or plasma containing a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative as an active ingredient.
[2] A method for stabilizing HDL in serum or plasma, comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
[3] A method for stabilizing HDL in serum or plasma, comprising the following steps.
(1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
(2) freeze-drying the mixture obtained in step (1); and
(3) A step of dissolving the lyophilized product obtained in step (2) with an aqueous medium.
［４］血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加することを特徴とする、血清又は血漿中のＨＤＬの保存方法。
［５］以下の工程を含むことを特徴とする、血清又は血漿中のＨＤＬの保存方法。
（１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
（２）工程（１）で得られた混合物を保存する工程。
［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ＨＤＬの保存方法。
（１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程； 
（２）工程（１）で得られた混合物を凍結乾燥するする工程；
（３）工程（２）で得られた凍結乾燥物を水性媒体で溶解する工程；及び、
（４）工程（３）で得られた水溶液を保存する工程。 [4] A method for preserving HDL in serum or plasma, comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
[5] A method for preserving HDL in serum or plasma, comprising the following steps.
(1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
(2) A step of storing the mixture obtained in step (1).
[6] A method for preserving HDL in serum or plasma, comprising the following steps.
(1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
(2) A step of freeze-drying the mixture obtained in step (1);
(3) dissolving the lyophilized product obtained in step (2) with an aqueous medium; and
(4) A step of storing the aqueous solution obtained in step (3).
［７］血清又は血漿中のＨＤＬ、及び、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該ＨＤＬ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ＨＤＬ中の成分定量用標準品。
［８］以下の工程を含む方法により製造される、［７］記載の標準品。
（１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
（２）工程（１）で得られた混合物中に含まれる、ＨＤＬ中の成分の濃度を、既知量の当該成分を用いて値付けする工程。
［９］凍結乾燥された血清又は血漿中のＨＤＬ、及び、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該ＨＤＬ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ＨＤＬ中の成分定量用標準品。
［１０］以下の工程を含む方法により製造される、［９］記載の標準品。
（１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（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、ＨＤＬ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。
［１１］ＨＤＬ中の成分が、コレステロールである、［７］～［１０］のいずれかに記載の標準品。 [7] It contains HDL in serum or plasma and a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative, and the content of the component in the HDL is priced. Standard for quantification of components in HDL in serum or plasma.
[8] The standard product according to [7], which is produced by a method including the following steps.
(1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
(2) A step of pricing the concentration of the component in HDL contained in the mixture obtained in step (1) using a known amount of the component.
[9] It contains HDL in lyophilized serum or plasma and a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative, and the content of the component in the HDL is priced A standard product for quantitative determination of components in HDL in serum or plasma.
[10] The standard product according to [9], which is produced by a method including the following steps.
(1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 HDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
[11] The standard product according to any one of [7] to [10], wherein the component in HDL is cholesterol.
［１２］以下の工程を含むことを特徴とする、血清又は血漿中のＨＤＬ中の成分定量用の標準品の製造方法。
（１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
（２）工程（１）で得られた混合物中に含まれる、ＨＤＬ中の成分の濃度を、既知量の当該成分を用いて値付けする工程。
［１３］以下の工程を含むことを特徴とする、血清又は血漿中のＨＤＬ中の成分定量用の標準品の製造方法。
（１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（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、ＨＤＬ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。
［１４］ＨＤＬ中の成分が、コレステロールである、［１２］又は［１３］記載の製造方法。 [12] A method for producing a standard product for quantitative determination of components in HDL in serum or plasma, comprising the following steps.
(1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
(2) A step of pricing the concentration of the component in HDL contained in the mixture obtained in step (1) using a known amount of the component.
[13] A method for producing a standard product for quantitative determination of components in HDL in serum or plasma, comprising the following steps.
(1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 HDL contained in the freeze-dried product obtained in step (2) using a known amount of the component.
[14] The production method of [12] or [13], wherein the component in HDL is cholesterol.
［１５］以下の工程を含むことを特徴とする、血清又は血漿中のＨＤＬ中の成分の定量方法。
（１）血清又は血漿中のＨＤＬ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）［７］～［１０］のいずれかに記載の標準品と、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。
［１６］ＨＤＬ中の成分が、コレステロールである、［１５］記載の定量方法。 [15] A method for quantifying components in HDL in serum or plasma, comprising the following steps.
(1) a step of measuring the component using a reagent for measuring the component in HDL in serum or plasma;
(2) Calibration using the standard product according to any one of [7] to [10] and the measurement reagent of step (1) to express the relationship between the concentration of the component and the measured value for the component Creating a line; 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).
[16] The quantification method according to [15], wherein the component in HDL is cholesterol.
［１７］血清又は血漿中のＨＤＬ中の成分の測定試薬、及び、［７］～［１０］のいずれかに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ＨＤＬ中の成分の定量用キット。
［１８］ＨＤＬ中の成分が、コレステロールである、［１７］記載のキット。 [17] A reagent for measuring a component in HDL in serum or plasma, and a standard product according to any one of [7] to [10], Component quantification kit.
[18] The kit according to [17], wherein the component in HDL is cholesterol.
　本発明により、血清又は血漿中のＨＤＬ－Ｃ等のＨＤＬ中の成分を正確に測定するための、血清又は血漿中のＨＤＬの安定化剤、血清又は血漿中のＨＤＬの安定化方法、血清又は血漿中のＨＤＬの保存方法、ＨＤＬ中の成分定量用の標準品とその製造方法、血清又は血漿中のＨＤＬ中の成分の定量方法、及び、血清又は血漿中のＨＤＬ中の成分の定量用キットが提供される。 According to the present invention, a stabilizer for HDL in serum or plasma, a method for stabilizing HDL in serum or plasma, serum or plasma for accurately measuring components in HDL such as HDL-C in serum or plasma, Method for storing HDL in plasma, standard product for quantifying components in HDL and method for producing the same, method for quantifying components in HDL in serum or plasma, and kit for quantifying components in HDL in serum or plasma Is provided.
（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.
（高密度リポ蛋白：ＨＤＬ）
　本発明におけるＨＤＬは、比重が1.063～1.21であるリポ蛋白である。 (High density lipoprotein: HDL)
HDL in the present invention is a lipoprotein having a specific gravity of 1.063 to 1.21.
（ＨＤＬ中の成分）
　本発明におけるＨＤＬ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 HDL)
Examples of components in HDL in the present invention include cholesterol and neutral fat, and cholesterol is preferred.
（安定化剤）
　本発明の安定化剤は、血清又は血漿中のＨＤＬを安定化するものである。本発明の安定化剤は、ポリオキシエチレン多環フェニルエーテル誘導体（以下、ＰＯＥ多環フェニルエーテル誘導体と記す）又はナフタレンスルホン酸誘導体を有効成分として含有する。本発明におけるＰＯＥ多環フェニルエーテル誘導体としては、例えばポリオキシエチレン多環フェニルエーテル（以下、ＰＯＥ多環フェニルエーテルと記す）、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（以下、ＰＯＥ多環フェニルエーテル硫酸エステルと記す）、ポリオキシエチレン・ポリオキシプロピレン多環フェニルエーテル（以下、ＰＯＥ・ＰＯＰ多環フェニルエーテルと記す）等が挙げられる。 (Stabilizer)
The stabilizer of the present invention stabilizes HDL in serum or plasma. The stabilizer of the present invention contains a polyoxyethylene polycyclic phenyl ether derivative (hereinafter referred to as POE polycyclic phenyl ether derivative) or a naphthalenesulfonic acid derivative as an active ingredient. Examples of the POE polycyclic phenyl ether derivative in the present invention include polyoxyethylene polycyclic phenyl ether (hereinafter referred to as POE polycyclic phenyl ether), polyoxyethylene polycyclic phenyl ether sulfate (hereinafter referred to as POE polycyclic phenyl ether sulfate). And polyoxyethylene / polyoxypropylene polycyclic phenyl ether (hereinafter referred to as POE / POP polycyclic phenyl ether).
　ＰＯＥ多環フェニルエーテルの具体例（市販品）としては、例えばニューコール７０３、ニューコール７０４、ニューコール７０６、ニューコール７０７、ニューコール７０８、ニューコール７０９、ニューコール７１０、ニューコール７１１、ニューコール７１２、ニューコール７１４、ニューコール７１４（８０）、ニューコール７１９、ニューコール７２３、ニューコール７２３（６０）、ニューコール７２９、ニューコール７３３、ニューコール７４０、ニューコール７４０（６０）、ニューコール７４７、ニューコール７８０（６０）、ニューコール６１０、ニューコール６１０（８０）、ニューコール２６０４、ニューコール２６０７、ニューコール２６０９、ニューコール２６１４（以上、日本乳化剤社製）、ノイゲンＥＡ－８７、ノイゲンＥＡ－１３７、ノイゲンＥＡ－１５７、ノイゲンＥＡ－１６７、ノイゲンＥＡ－１７７、ノイゲンＥＡ－１９７Ｄ、ノイゲンＥＡ－２０７Ｄ（以上、第一工業製薬社製）等が挙げられる。 As specific examples (commercially available products) of POE polycyclic phenyl ether, for example, New Coal 703, New Coal 704, New Coal 706, New Coal 707, New Coal 708, New Coal 709, New Coal 710, New Coal 711, New Coal 712, New Call 714, New Call 714 (80), New Call 719, New Call 723, New Call 723 (60), New Call 729, New Call 733, New Call 740, New Call 740 (60), New Call 747 , New Coal 780 (60), New Coal 610, New Coal 610 (80), New Coal 2604, New Coal 2607, New Coal 2609, New Coal 2614 (above, manufactured by Nippon Emulsifier Co., Ltd.), Neugen EA 87, Noigen EA-137, Noigen EA-157, Noigen EA-167, Noigen EA-177, Noigen EA-197d, Noigen EA-207D (above, Dai-ichi Kogyo Seiyaku Co., Ltd.).
　ＰＯＥ多環フェニルエーテル硫酸エステルの具体例（市販品）としては、例えばニューコール７０７－ＳＦ、ニューコール７０７－ＳＦＣ、ニューコール７０７－ＳＮ、ニューコール７１４－ＳＦ、ニューコール７１４－ＳＮ、ニューコール７２３－ＳＦ、ニューコール７４０－ＳＦ、ニューコール７８０－ＳＦ、ニューコール２６０７－ＳＦ、ニューコール２６１４－ＳＦ（以上、日本乳化剤社製）、ハイテノールＮＦ－０８、ハイテノールＮＦ－０８２５、ハイテノールＮＦ－１３、ハイテノールＮＦ－１７（以上、第一工業製薬社製）等が挙げられる。 Specific examples (commercially available) of POE polycyclic phenyl ether sulfates include, for example, New Coal 707-SF, New Coal 707-SFC, New Coal 707-SN, New Coal 714-SF, New Coal 714-SN, New Coal 723-SF, New Coal 740-SF, New Coal 780-SF, New Coal 2607-SF, New Coal 2614-SF (above, manufactured by Nippon Emulsifier Co., Ltd.), Hightenol NF-08, Hightenol NF-0825, High Tenol NF-13, Haitenol NF-17 (Daiichi Kogyo Seiyaku Co., Ltd.) and the like.
　ＰＯＥ・ＰＯＰ多環フェニルエーテルの具体例（市販品）としては、例えばニューコール７０７－Ｆ、ニューコール７１０－Ｆ、ニューコール７１４－Ｆ、ニューコール２６０８－Ｆ、ニューコール２６００－ＦＢ、ニューコール２６１６－Ｆ、ニューコール３６１２－ＦＡ（以上、日本乳化剤社製）等が挙げられる。 Specific examples (commercial products) of POE / POP polycyclic phenyl ether include, for example, New Coal 707-F, New Coal 710-F, New Coal 714-F, New Coal 2608-F, New Coal 2600-FB, New Coal 2616-F, New Coal 3612-FA (manufactured by Nippon Emulsifier Co., Ltd.) and the like.
　ナフタレン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ムアルデヒド縮合物等が挙げられる。ナフタレンスルホン酸ホルムアルデヒド縮合物の具体例（市販品）としては、例えばディスロールＳＨ、エスコール（３０）（以上、日本乳化剤社製）、デモールＮ、デモールＮＬ、デモールＲＮ、デモールＲＮ－Ｌ、デモールＴ、デモールＴ－４５、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 naphthalene sulfonic acid derivatives include naphthalene sulfonic acid formaldehyde condensates. Specific examples (commercial products) of naphthalene sulfonic acid formaldehyde condensates include, for example, Disrol SH, Escort (30) (manufactured by Nippon Emulsifier Co., Ltd.), Demol N, Demol NL, Demol RN, Demol RN-L, Demol T , Demol T-45, Demol MS, Demol SN-B (above, manufactured by Kao Corporation) and the like.
　本発明の安定化剤に含まれるＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体の含量としては、血清又は血漿中のＨＤＬを安定化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に本発明の安定化剤が添加されたときに0.01～5 gとなる含量であり、0.05～1 gとなる含量が好ましい。 The content of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative contained in the stabilizer of the present invention is not particularly limited as long as it is a content capable of stabilizing HDL in serum or plasma. When the stabilizer of the present invention is added to mL, the content is 0.01-5 g, and the content is preferably 0.05-1 g.
（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13.3～133.3Pa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 13.3 to 133.3 Pa, and 6 to 120 hours, more preferably 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.
（ＨＤＬの安定化方法）
　本発明のＨＤＬの安定化方法は、血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程を含む。血清又は血漿に添加されるＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体の量は、血清又は血漿中でＨＤＬが安定化され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、0.01～5 gであり、0.05～1 gが好ましい。 (HDL stabilization method)
The method for stabilizing HDL of the present invention includes a step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma. The amount of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative added to serum or plasma is not particularly limited as long as HDL is stabilized in serum or plasma, and is usually per 100 mL of serum or plasma. 0.01 to 5 g, preferably 0.05 to 1 g.
　血清又は血漿にＰＯＥ多環フェニルエーテル誘導体を添加する際、ＰＯＥ多環フェニルエーテル誘導体は、ＰＯＥ多環フェニルエーテル誘導体そのものを添加してもよいが、ＰＯＥ多環フェニルエーテル誘導体水溶液として添加してもよい。また、血清又は血漿にナフタレンスルホン酸誘導体を添加する際、ナフタレンスルホン酸誘導体は、ナフタレンスルホン酸誘導体そのものを添加してもよいが、ナフタレンスルホン酸誘導体水溶液として添加してもよい。これらの水溶液の調製には、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。これらの水溶液のpHは、当該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であれば特に制限はなく、pH6～9が好ましい。 When a POE polycyclic phenyl ether derivative is added to serum or plasma, the POE polycyclic phenyl ether derivative may be added as the POE polycyclic phenyl ether derivative itself, or as a POE polycyclic phenyl ether derivative aqueous solution. Good. In addition, when the naphthalenesulfonic acid derivative is added to serum or plasma, the naphthalenesulfonic acid derivative may be added as the naphthalenesulfonic acid derivative itself, or may be added as a naphthalenesulfonic acid derivative aqueous solution. For the preparation of these aqueous solutions, 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 these aqueous solutions is not particularly limited as long as the lipoprotein in serum or plasma does not precipitate after the aqueous solution is added to serum or plasma, and is preferably pH 6-9.
　また、本発明のＨＤＬの安定化方法の別の態様は、以下の工程を含む。
（１）血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（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を水性媒体で溶解する工程。 Another aspect of the HDL stabilization method of the present invention includes the following steps.
(1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
(2) freeze-drying the mixture obtained in step (1); and
(3) A step of dissolving the lyophilized product obtained in step (2) with an aqueous medium.
　工程（３）における水性媒体としては、血清又は血漿中のＨＤＬを溶解し、ＨＤＬの水溶液中でＨＤＬが安定に保持され得るものであれば特に制限はなく、例えば、前述の水性媒体等が挙げられる。 The aqueous medium in step (3) is not particularly limited as long as it dissolves HDL in serum or plasma and can stably maintain HDL in an aqueous solution of HDL, and examples thereof include the above-mentioned aqueous medium. It is done.
（ＨＤＬの保存方法）
　本発明のＨＤＬの保存方法は、血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程を含む。また、本発明のＨＤＬの保存方法の別の態様は、以下の工程を含む。
（１）血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
（２）工程（１）で得られた混合物を保存する工程。 (How to save HDL)
The method for storing HDL of the present invention includes a step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma. Another aspect of the HDL storage method of the present invention includes the following steps.
(1) adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
(2) A step of storing the mixture obtained in step (1).
　さらに、本発明のＨＤＬの保存方法の別の態様は、以下の工程を含む。
（１）血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（２）工程（１）で得られた混合物を凍結乾燥する工程；
（３）工程（２）で得られた凍結乾燥物を水性媒体で溶解する工程；及び、
（４）工程（３）で得られた水溶液を保存する工程。 Furthermore, another aspect of the HDL storage method of the present invention includes the following steps.
(1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
(2) a step of freeze-drying the mixture obtained in step (1);
(3) dissolving the lyophilized product obtained in step (2) with an aqueous medium; and
(4) A step of storing the aqueous solution obtained in step (3).
　本発明のＨＤＬの保存方法における保存条件としては、ＨＤＬが安定に保存される条件であれば特に制限はない。ＨＤＬの保存方法における保存温度は、ＨＤＬが安定に保存される温度であれば特に制限はなく、通常、-80～45℃であり、-5～30℃が好ましく、2～10℃が特に好ましい。ＨＤＬの保存方法における保存期間は、ＨＤＬが安定に保存される期間であれば特に制限はなく、通常、1日～2年間である。 Storing conditions in the HDL storage method of the present invention are not particularly limited as long as HDL is stored stably. The storage temperature in the HDL storage method is not particularly limited as long as the HDL is stored stably, and is usually -80 to 45 ° C, preferably -5 to 30 ° C, and particularly preferably 2 to 10 ° C. . The storage period in the HDL storage method is not particularly limited as long as HDL is stably stored, and is usually 1 day to 2 years.
（標準品）
　本発明の血清又は血漿中のＨＤＬ中の成分定量用標準品は、溶液状態でも凍結乾燥状態でもよい。標準品が溶液状態の場合、本発明の標準品は、血清又は血漿中のＨＤＬ、及び、ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該ＨＤＬ中の成分の含量が値付けされている。また、標準品が凍結乾燥状態の場合、本発明の標準品は、凍結乾燥された血清又は血漿中のＨＤＬ、及び、ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該ＨＤＬ中の成分の含量が値付けされている。なお、本発明の標準品は、血清又は血漿中のＨＤＬ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ＨＤＬ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 HDL in serum or plasma of the present invention may be in solution or lyophilized. When the standard product is in a solution state, the standard product of the present invention contains HDL in serum or plasma and a POE polycyclic phenyl ether derivative or naphthalene sulfonic acid derivative, and the content of the component in the HDL is a value. It is attached. When the standard product is in a lyophilized state, the standard product of the present invention contains HDL in freeze-dried serum or plasma, and POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative, and the HDL The content of the components in it is priced. In addition, the standard product of the present invention can be used not only as a standard product for quantifying components in HDL in serum or plasma, but also as a quality control substance in quantifying components in HDL in serum or plasma.
（標準品の製造方法）
　本発明の標準品が溶液状態の場合、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、ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
（２）工程（１）で得られた混合物中に含まれる、ＨＤＬ中の成分の濃度を、既知量の当該成分を用いて値付けする工程。 (Standard product manufacturing method)
When the standard product of the present invention is in a solution state, the method for producing the standard product of the present invention includes the following steps.
(1) adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
(2) A step of pricing the concentration of the component in HDL contained in the mixture obtained in step (1) using a known amount of the component.
　また、本発明の標準品が凍結乾燥状態の場合、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、ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
（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、ＨＤＬ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。 When the standard product of the present invention is in a freeze-dried state, the method for producing the standard product of the present invention includes the following steps.
(1) A step of adding a POE polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 HDL contained in the freeze-dried 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ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体の量は、血清又は血漿中でＨＤＬが安定化され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、0.01～5 gであり、0.05～1 gが好ましい。 In the method for producing a standard product of the present invention, the amount of the POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative added to serum or plasma is not particularly limited as long as HDL is stabilized in serum or plasma. In general, it is 0.01 to 5 g, preferably 0.05 to 1 g per 100 mL of serum or plasma.
　本発明の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、冷却した血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体水溶液を添加する。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して標準品とする。 One embodiment of the method for producing a standard product of the present invention will be described below.
Serum or plasma is cooled to 2-8 ° C., and an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma. 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 to obtain a standard product.
　また、本発明の標準品の製造方法の別の態様を以下に記す。
　血清又は血漿を2～8℃に冷却し、冷却した血清又は血漿にＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体水溶液を添加する。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製造する。 Moreover, another aspect of the manufacturing method of the standard goods of this invention is described below.
Serum or plasma is cooled to 2-8 ° C., and an aqueous solution of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative is added to the cooled serum or plasma. 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. Manufacture standard products in a dry state.
　なお、凍結乾燥状態の標準品は、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ＨＤＬ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。 Note that the standard product in a lyophilized state is used after dissolving in a known amount of an aqueous medium in preparing a calibration curve. The aqueous medium for dissolving the lyophilized standard is not particularly limited as long as it is an aqueous medium that enables the method of quantifying the components in HDL 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ＨＤＬ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 HDL 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ＰＯＥ多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体の含量）
　本発明の標準品に含まれるＰＯＥ多環フェニルエーテル誘導体及びナフタレンスルホン酸誘導体の含量は、標準品中のＨＤＬが安定に保持され、標準品中のＨＤＬ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、0.01～5 gであり、0.05～1 gが好ましい。 (Content of POE polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative in the standard product)
The content of the POE polycyclic phenyl ether derivative and naphthalene sulfonic acid derivative contained in the standard product of the present invention should be such that the HDL in the standard product is stably maintained and the components in the HDL in the standard product can be accurately measured. There is no particular limitation, and it is usually 0.01 to 5 g and preferably 0.05 to 1 g with respect to 100 mL of serum or plasma.
（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. .
（ＨＤＬ中の成分の定量方法）
　本発明の血清又は血漿中のＨＤＬ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ＨＤＬ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 determination method of components in HDL)
The method for quantifying components in HDL 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 HDL 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ＨＤＬ中の成分の測定においては、公知の、ＨＤＬ中の成分の測定方法を用いることができる。ＨＤＬ中の成分の測定方法としては、例えばＨＤＬ－Ｃ測定方法、ＨＤＬ中の中性脂肪（以下、ＨＤＬ－ＴＧと記す）の測定方法等が挙げられる。また、ＨＤＬ中の成分の測定方法としては、ＨＤＬを遠心分離等の手段により分画することなく、ＨＤＬ以外にＬＤＬ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ＨＤＬ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 HDL to be measured in the above step (1), a known method for measuring the component in HDL can be used. Examples of the method for measuring components in HDL include a method for measuring HDL-C, a method for measuring neutral fat in HDL (hereinafter referred to as HDL-TG), and the like. In addition, as a method of measuring components in HDL, HDL is not fractionated by means such as centrifugation, but in a state where a plurality of lipoproteins such as LDL and VLDL coexist in the same reaction solution in addition to HDL. 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ＨＤＬ－Ｃ測定方法としては、例えばＷＯ２００４／０３５８１６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、非イオン性界面活性剤、ポリアニオン及びアルブミンを含有する水性媒体中で反応させ、生成した過酸化水素または還元型補酵素を測定することにより、検体中のＨＤＬ－Ｃを測定する方法や、ＷＯ２００６／１１８１９９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、特定の構造の第四級アンモニウム塩、及び、ポリアニオンを含有する水性媒体中で反応させ、生成した過酸化水素または還元型補酵素を測定することにより、検体中のＨＤＬ－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 HDL-C by a homogeneous method, for example, a specimen and an enzyme for measuring cholesterol described in WO 2004/035816 are reacted in an aqueous medium containing a nonionic surfactant, a polyanion, and albumin. A method of measuring HDL-C in a specimen by measuring the generated hydrogen peroxide or reduced coenzyme, or a specimen and an enzyme for measuring cholesterol described in WO2006 / 118199 pamphlet having a specific structure. Examples include a method of measuring HDL-C in a specimen by reacting in an aqueous medium containing a quaternary ammonium salt and a polyanion and measuring the produced hydrogen peroxide or reduced coenzyme. 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.
（ＨＤＬ中の成分の定量用キット）
　本発明の血清又は血漿中のＨＤＬ中の成分の定量用キットは、本発明の標準品とＨＤＬ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、ＨＤＬ中の成分の定量を簡便に行うことができる。ＨＤＬ中の成分の測定試薬としては、公知の、ＨＤＬ中の成分の測定試薬を用いることができる。ＨＤＬ中の成分の測定試薬としては、例えばＨＤＬ－Ｃ測定試薬、ＨＤＬ－ＴＧ測定試薬等が挙げられる。また、ＨＤＬ中の成分の測定試薬としては、ＨＤＬを遠心分離等の手段により分画することなく、ＨＤＬ以外にＬＤＬ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ＨＤＬ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 quantitative determination of components in HDL)
The kit for quantifying components in HDL in serum or plasma of the present invention contains the standard product of the present invention and a reagent for measuring components in HDL. By using the kit of the present invention, the components in HDL can be easily quantified. As a reagent for measuring a component in HDL, a known reagent for measuring a component in HDL can be used. Examples of the reagent for measuring components in HDL include HDL-C measuring reagent and HDL-TG measuring reagent. In addition, as a reagent for measuring components in HDL, HDL is not fractionated by means of centrifugation or the like, but in a state where a plurality of lipoproteins such as LDL and VLDL coexist in the same reaction solution in addition to HDL. 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ＨＤＬ－Ｃ測定方法に用いられるＨＤＬ－Ｃ測定試薬としては、例えばＷＯ２００４／０３５８１６パンフレットに記載された、コレステロール測定用酵素、非イオン性界面活性剤、ポリアニオン及びアルブミンを含有するＨＤＬ－Ｃ測定試薬や、ＷＯ２００６／１１８１９９パンフレットに記載された、コレステロール測定用酵素、特定の構造の第四級アンモニウム塩、及び、ポリアニオンを含有するＨＤＬ－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ＨＤＬ－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ＨＤＬ－Ｃ測定試薬であるデタミナーＬ　ＨＤＬ－Ｃ（協和メデックス社製）、メタボリードＨＤＬ－Ｃ（協和メデックス社製）、「生研」ＨＤＬ－ＥＸ　Ｎ（デンカ生研社製）等が挙げられる。 Examples of the HDL-C measuring reagent used in the HDL-C measuring method by the homogeneous method include HDL-C containing an enzyme for measuring cholesterol, a nonionic surfactant, a polyanion and albumin described in, for example, WO 2004/035816 pamphlet. Examples include a measurement reagent, an enzyme for measuring cholesterol, a quaternary ammonium salt having a specific structure, and a HDL-C measurement reagent containing a polyanion described in WO 2006/118199 pamphlet. 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 HDL-C measuring reagent used in the homogeneous method. Examples of commercially available measuring reagents include Determiner L HDL-C (manufactured by Kyowa Medex), Metabolid HDL-C (manufactured by Kyowa Medex), and “Seiken” HDL-EX N (manufactured by Denka Seiken). ) And the like.
　本発明の標準品や本発明のキットは、血清又は血漿中のＨＤＬ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 HDL are automatically and continuously measured.
（ＨＤＬの安定化剤、安定化方法、及び、保存方法の効果の検証）
　本発明のＨＤＬ安定化剤、安定化方法、及び、保存方法の効果は、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ＨＤＬの安定化剤を添加し、本発明のＨＤＬの安定化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ＨＤＬの安定化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、必要に応じて、前述の方法により凍結乾燥を行う。次に、前述の方法により値付けを行い、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、標準品（標準品Ｂ）を調製する。 (Verification of effects of HDL stabilizer, stabilization method, and storage method)
The effects of the HDL stabilizer, the stabilization method, and the storage method 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 HDL stabilizer of the present invention is added to one pool serum or pool plasma, and the pool sera or pool to which the HDL stabilizer 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 HDL stabilizer of the present invention is not added are prepared. A certain amount of group A serum or plasma is dispensed into a plurality of containers of the same shape and material, and if necessary, freeze-dried by the method described above. Next, pricing is performed by the above-described method to prepare a standard product (standard product A). Similarly, a standard product (standard product B) is prepared using serum or plasma of group B.
　本発明の安定化剤を添加した直後の標準品ＡをＮ本（標準品Ａ１～標準品ＡＮ）用意し、調製直後の標準品溶液Ａ保存前（Ａ保存前１～Ａ保存前Ｎ）とする。凍結乾燥状態の標準品の場合は、各標準品Ａ（標準品Ａ１～標準品ＡＮ）について一定量の精製水で溶解し、調製直後の標準品溶液Ａ保存前（Ａ保存前１～Ａ保存前Ｎ）とする。 Prepare N standard products A (standard product A 1 to standard product A N ) immediately after adding the stabilizer of the present invention, and store the standard product solution A immediately after preparation ( 1 before storage 1 to N before storage A). ). In the case of lyophilized standard products, each standard product A (standard product A 1 to standard product A N ) is dissolved in a fixed amount of purified water, and immediately after preparation, the standard product solution A is stored before storage (A 1 to 1 before storage ). A) N before storage .
　さらに、溶解直後の標準品溶液Ａ保存前を５℃で７日間保存し、標準品溶液Ａ保存後（標準品Ａ保存後１～標準品Ａ保存後Ｎ）とする。 Further, immediately after dissolution, the sample is stored for 7 days at 5 ° C. before storage of standard solution A, and after storage of standard solution A ( 1 after storage of standard product A to N after storage of standard product A).
　それぞれの標準品溶液Ａ保存前（標準品Ａ保存前１～標準品Ａ保存前Ｎ）を検体とし、ＨＤＬ－Ｃ測定試薬を用いて、吸光度Ａ保存前（吸光度Ａ保存前１～吸光度Ａ保存前Ｎ）を測定する。同様に、それぞれの標準品溶液Ａ保存後（標準品Ａ保存後１～標準品Ａ保存後Ｎ）を検体として用いて、ＨＤＬ－Ｃ測定試薬と反応させ、吸光度Ａ保存後（吸光度Ａ保存後１～吸光度Ａ保存後Ｎ）を測定する。得られた吸光度Ａ保存前と吸光度Ａ保存後それぞれについて平均値（平均吸光度Ａ保存前と平均吸光度Ａ保存後）を算出し、以下の式（Ｉ）に従い、残存率（％）を算出する。 Samples before storage of standard solution A ( 1 before storage of standard product A to N before storage of standard product A) were used as specimens and stored with HDL-C measurement reagent before storage of absorbance A ( 1 storage before storage of absorbance A to storage of absorbance A). Measure the previous N ). Similarly, each using standard solution A after storage (standard A storage after 1 to Standard A after storage N) as a sample, is reacted with HDL-C measurement reagent, absorbance A after storage (absorbance A after storage 1 to Measure N ) after storage of absorbance A. An average value ( before storage of average absorbance A and after storage of average absorbance A) is calculated for each of the obtained absorbance A before storage and after storage of absorbance A, and the residual rate (%) is calculated according to the following formula (I).
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　算出した残存率（％）が100に近い程、血清又は血漿中でＨＤＬが安定化され、ＨＤＬ中の成分が正確に測定されることを意味する。すなわち、残存率（％）が100に近い程、血清又は血漿中でＨＤＬが安定化されたことを意味する。 The closer the calculated residual rate (%) is to 100, the more stable HDL is in serum or plasma, and the more accurate the components in HDL are measured. That is, the closer the residual rate (%) is to 100, the more stable HDL is in serum or plasma.
　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（ディスカバリーライフサイエンス社製）、トレハロース（林原社製）、ニューコール７４０（６０）（ＰＯＥ多環フェニルエーテル誘導体；日本乳化剤社製）、ニューコール７４０－ＳＦ（ＰＯＥ多環フェニルエーテル誘導体；日本乳化剤社製）、デモールＮ（ナフタレンスルホン酸誘導体；花王社製）、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 Discovery Life Science), trehalose (manufactured by Hayashibara), New Coal 740 (60) (POE polycyclic phenyl ether derivative; manufactured by Nippon Emulsifier Co., Ltd.), New Coal 740-SF (POE polycyclic phenyl ether derivative; Japan) Emulsifier Co., Ltd.), Demol N (Naphthalenesulfonic acid derivative; Kao Co., Ltd.), Borosilicate glass vial [manufactured by Yamato Special Glass Co., Ltd. (capacity: 2 mL; diameter: 16 mm; brown)], freeze dryer (Tokyo Rika Instruments) Manufactured by EYELA DRC-1100, FDU-2100).
　以下の方法により、ＰＯＥ多環フェニルエーテル誘導体及びナフタレンスルホン酸誘導体によるＨＤＬの安定化効果を検討した。
（１）凍結乾燥用血清の調製
　以下の組成からなる血清１～４及び血清０を調製した。
＜血清１～４及び血清０＞
　血清　　　　　　　　1 L
　トレハロース　　　　40 g
　ＨＤＬの安定化剤（第１表参照） By the following method, the stabilization effect of HDL by a POE polycyclic phenyl ether derivative and a naphthalenesulfonic acid derivative was examined.
(1) Preparation of lyophilized serum Serum 1 to 4 and serum 0 having the following composition were prepared.
<Serum 1 to 4 and Serum 0>
Serum 1 L
Trehalose 40 g
HDL stabilizer (see Table 1)
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（２）凍結乾燥血清の製造
　上記で調製した血清１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１を製造した。
　血清１の代わりに、血清２～４及び血清０を用いて同様の操作を行い、凍結乾燥血清２～４及び凍結乾燥血清０を調製した。 (2) Manufacture of lyophilized serum Serum 1 prepared above was dispensed in 0.5 mL portions into glass vials, with primary lyophilization set at -34 ° C for 32 hours and secondary lyophilization set at 20 ° C for 4 hours. Freeze-drying was performed, and finally it was closed with a rubber stopper in a vacuum state to produce freeze-dried serum 1.
Similar procedures were performed using serum 2-4 and serum 0 instead of serum 1, and freeze-dried serum 2-4 and freeze-dried serum 0 were prepared.
（３）残存率の算出
　上記（２）で調製した凍結乾燥血清１のバイアルを３本準備し、各バイアルに精製水0.5 mLを加えて再構成した血清１保存前（血清１保存前１～血清１保存前３）を検体（３検体）として用いて、ＨＤＬ－Ｃ測定試薬として「メタボリードＨＤＬ－Ｃ」を用いて、「メタボリードＨＤＬ－Ｃ」に付随の添付文書に記載の操作手順に従い、各検体について測定を行い、各検体に対する吸光度１保存前（吸光度１保存前１～吸光度１保存前３）を測定し、吸光度１保存前１～吸光度１保存前３の平均値を平均吸光度１保存前として算出した。凍結乾燥血清１の代わりに、凍結乾燥血清２～４及び凍結乾燥血清０を用いる以外は同様の方法により測定を行い、平均吸光度２保存前～平均吸光度４保存前及び平均吸光度０保存前を算出した。 (3) Calculation of residual ratio Prepare three vials of freeze-dried serum 1 prepared in (2) above, and reconstitute each vial with 0.5 mL of purified water ( before serum 1 storage 1 ~ Serum 1 before storage 3 ) is used as a sample (3 samples), “Metabolido HDL-C” is used as a reagent for HDL-C measurement, and “Metabolide HDL-C” is followed according to the operation procedure described in the package insert. was measured for each sample, before the absorbance 1 stored for each specimen (absorbance 1 before storage 1 to the absorbance 1 before storage 3) was measured, the average absorbance 1 save the average absorbance 1 before storage 1 to the absorbance 1 before storage 3 Calculated as before . Measure by the same method except using freeze-dried serum 2-4 and freeze-dried serum 0 instead of freeze-dried serum 1, and calculate average absorbance 2 before storage -average absorbance 4 before storage and average absorbance 0 before storage did.
　次いで、上記血清１保存前（血清１保存前１～血清１保存前３）を５℃で７日間保存して得られた血清１保存後（血清１保存後１～血清１保存後３）を検体（３検体）として用いて、ＨＤＬ－Ｃ測定試薬として「メタボリードＨＤＬ－Ｃ」を用いて、「メタボリードＨＤＬ－Ｃ」に付随の添付文書に記載の操作手順に従い、各検体について測定を行い、各検体に対する吸光度１保存後（吸光度１保存後１～吸光度１保存後３）を測定し、吸光度１保存後１～吸光度１保存後３の平均値を平均吸光度１保存後として算出した。凍結乾燥血清１の代わりに、凍結乾燥血清２～４及び凍結乾燥血清０を用いる以外は同様の方法により測定を行い、平均吸光度２保存後～平均吸光度４保存後及び平均吸光度０保存後を算出した。 Then, the serum 1 before storage (the serum 1 before storage 1 serum 1 before storage 3) a 5 ° C. for 7 days Save and sera obtained 1 after storage (serum 1 after storage 1 serum 1 after storage 3) Using each sample as a sample (3 samples), using “Metabolide HDL-C” as an HDL-C measurement reagent, and measuring each sample according to the operation procedure described in the package insert accompanying “Metabolide HDL-C” after the absorbance 1 store (absorbance 1 after storage 1 to the absorbance 1 after storage 3) were measured for each sample, the mean value was calculated absorbance 1 after storage 1 to the absorbance 1 after storage 3 as the mean absorbance 1 after storage. Measure by the same method except using freeze-dried serum 2-4 and freeze-dried serum 0 instead of freeze-dried serum 1, and calculate average absorbance 2 after storage -average absorbance 4 storage and after average absorbance 0 storage did.
　凍結乾燥血清１について、算出した平均吸光度１保存前と平均吸光度１保存後とを用いて、以下の式（ＩＩ）に従い、残存率（％）を算出した。 For lyophilized serum 1, the residual rate (%) was calculated according to the following formula (II) using the calculated average absorbance before 1 storage and after the average absorbance after 1 storage .
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000003
　凍結乾燥血清１について、全３回、上記手順による測定を行い、全３回の測定の各測定における残存率を算出すると共に、算出された残存率の平均値（平均残存率）を決定した。 The freeze-dried serum 1 was measured three times in accordance with the above procedure, and the residual ratio in each measurement of all three measurements was calculated, and the average value (average residual ratio) of the calculated residual ratio was determined.
　平均吸光度１保存前と平均吸光度１保存後の組み合わせの代わりに、平均吸光度２保存前と平均吸光度２保存後の組み合わせ、平均吸光度３保存前と平均吸光度３保存後の組み合わせ、平均吸光度４保存前と平均吸光度４保存後の組み合わせ、及び、平均吸光度０保存後と平均吸光度０保存後の組み合わせをそれぞれ用いる以外は上記と同様の手順で、凍結乾燥血清２～４及び凍結乾燥血清０の各凍結乾燥血清に対して、測定を全３回行い、全３回の測定の各測定における残存率を各凍結乾燥血清に対して算出すると共に、算出された残存率の平均値（平均残存率）を各凍結乾燥血清に対して決定した。さらに、算出した残存率について、有意水準を0.05としてDunnett法による検定（Dunnett検定）を行った。その結果を第２表に示す。 Instead of the combination of the average absorbance 1 before storage and the average absorbance 1 after storage, the combination of the average absorbance 2 before storage and the average absorbance 2 storage, the combination of the average absorbance 3 before storage and the average absorbance 3 storage , and the average absorbance 4 before storage mean absorbance 4 combination after storage, and, in the same procedure as above except for using the mean absorbance 0 after storage and the average absorbance 0 combination after storage, respectively, each of the freeze-lyophilized serum 2-4 and lyophilized serum 0 For dry serum, measurement was performed three times in total, and the residual rate in each measurement of all three measurements was calculated for each freeze-dried serum, and the average value (average residual rate) of the calculated residual rate was calculated. Determined for each lyophilized serum. Further, the calculated residual ratio was subjected to a Dunnett test (Dunnett test) with a significance level of 0.05. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　第２表から明らかな様に、本発明の安定化剤を添加して調製した凍結乾燥血清においては、本発明の安定化剤を添加しないで調製した凍結乾燥血清に比較して残存率が高かった。ＨＤＬ－Ｃ定量用の標準品においては、標準品を溶液状態で保存した場合でも、正確なＨＤＬ－Ｃの測定のためには、できる限り100%に近い残存率、という極めて高い残存率が要求される。従って、第２表における、本発明の安定化剤が添加された場合と、添加されていない場合の残存率の差は、重要な意義を有するのである。しかも、有意水準を0.05としてDunnett検定を行ったところ、本発明の安定化剤を添加して調製した全ての凍結乾燥血清において、本発明の安定化剤を添加して調製した凍結乾燥血清に対する残存率と、本発明の安定化剤を添加しないで調製した凍結乾燥血清に対する残存率との間で有意に差があることが分かった。従って、本発明の安定化剤により血清又は血漿中のＨＤＬが安定化され、これにより、血清又は血漿中のＨＤＬ中の成分を正確に測定できることが分かった。 As is apparent from Table 2, the lyophilized serum prepared by adding the stabilizer of the present invention has a higher residual rate than the lyophilized serum prepared without adding the stabilizer of the present invention. It was. In the standard product for HDL-C determination, even when the standard product is stored in solution, a very high residual rate of 100% is required for accurate HDL-C measurement. Is done. Therefore, the difference in the residual ratio between the case where the stabilizer of the present invention is added and the case where it is not added in Table 2 is important. In addition, when Dunnett's test was performed with a significance level of 0.05, all the lyophilized sera prepared by adding the stabilizer of the present invention remained in the lyophilized serum prepared by adding the stabilizer of the present invention. It was found that there was a significant difference between the rate and the survival rate for lyophilized serum prepared without the addition of the stabilizer of the present invention. Therefore, it has been found that the HDL in serum or plasma is stabilized by the stabilizer of the present invention, whereby the components in HDL in serum or plasma can be accurately measured.
　本発明により、血清又は血漿中のＨＤＬの安定化剤、血清又は血漿中のＨＤＬの安定化方法、血清又は血漿中のＨＤＬの保存方法、血清又は血漿中のＨＤＬ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ＨＤＬ中の成分の定量方法、及び、血清又は血漿中のＨＤＬ中の成分の定量用キットが提供される。 According to the present invention, a stabilizer for HDL in serum or plasma, a method for stabilizing HDL in serum or plasma, a method for storing HDL in serum or plasma, and an accurate measurement of components in HDL in serum or plasma. Provided are a standard product and a production method thereof, a method for quantifying components in HDL in serum or plasma, and a kit for quantifying components in HDL in serum or plasma.
　本発明の血清又は血漿中のＨＤＬの安定化剤、血清又は血漿中のＨＤＬの安定化方法、血清又は血漿中のＨＤＬの保存方法、血清又は血漿中のＨＤＬ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ＨＤＬ中の成分の定量方法、及び、血清又は血漿中のＨＤＬ中の成分の定量用キットは、メタボリックシンドローム等の診断に有用である。
  Stabilizer for HDL in serum or plasma of the present invention, method for stabilizing HDL in serum or plasma, method for storing HDL in serum or plasma, standard for quantification of components in HDL in serum or plasma, and The production method, the method for quantifying components in HDL in serum or plasma, and the kit for quantifying components in HDL in serum or plasma are useful for diagnosis of metabolic syndrome and the like.

Claims (18)

  1. ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を有効成分として含有する、血清又は血漿中の高密度リポ蛋白の安定化剤。 A stabilizer for high-density lipoprotein in serum or plasma, comprising a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative as an active ingredient.
  2. 血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加することを特徴とする、血清又は血漿中の高密度リポ蛋白の安定化方法。 A method for stabilizing high-density lipoprotein in serum or plasma, comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  3. 以下の工程を含むことを特徴とする、血清又は血漿中の高密度リポ蛋白の安定化方法。
    （１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
    （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を水性媒体で溶解する工程。     A method for stabilizing high-density lipoprotein in serum or plasma, comprising the following steps.
    (1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
    (2) freeze-drying the mixture obtained in step (1); and
    (3) A step of dissolving the lyophilized product obtained in step (2) with an aqueous medium.
  4. 血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加することを特徴とする、血清又は血漿中の高密度リポ蛋白の保存方法。 A method for preserving high-density lipoprotein in serum or plasma, comprising adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma.
  5. 以下の工程を含むことを特徴とする、血清又は血漿中の高密度リポ蛋白の保存方法。
    （１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
    （２）工程（１）で得られた混合物を保存する工程。     A method for preserving high-density lipoprotein in serum or plasma, comprising the following steps.
    (1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
    (2) A step of storing the mixture obtained in step (1).
  6. 以下の工程を含むことを特徴とする、血清又は血漿中の高密度リポ蛋白の保存方法。
    （１）血清又は血漿にポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
    （２）工程（１）で得られた混合物を凍結乾燥する工程；
    （３）工程（２）で得られた凍結乾燥物を水性媒体で溶解する工程；及び、
    （４）工程（３）で得られた水溶液を保存する工程。     A method for preserving high-density lipoprotein in serum or plasma, comprising the following steps.
    (1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma;
    (2) a step of freeze-drying the mixture obtained in step (1);
    (3) dissolving the lyophilized product obtained in step (2) with an aqueous medium; and
    (4) A step of storing the aqueous solution obtained in step (3).
  7. 血清又は血漿中の高密度リポ蛋白、及び、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該高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、血清又は血漿中の高密度リポ蛋白中の成分定量用標準品。 It contains high-density lipoprotein in serum or plasma and polyoxyethylene polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative, and the content of the component in the high-density lipoprotein is priced A standard product for quantitative determination of components in high-density lipoprotein in serum or plasma.
  8. 以下の工程を含む方法により製造される、請求項７記載の標準品。
    （１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
    （２）工程（１）で得られた混合物中に含まれる、高密度リポ蛋白中の成分の濃度を、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 7, which is produced by a method including the following steps.
    (1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
    (2) A step of pricing the concentration of the component in the high-density lipoprotein contained in the mixture obtained in step (1) using a known amount of the component.
  9. 凍結乾燥された血清又は血漿中の高密度リポ蛋白、及び、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を含有し、かつ、当該高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、血清又は血漿中の高密度リポ蛋白中の成分定量用標準品。 Contains high-density lipoprotein in lyophilized serum or plasma, and polyoxyethylene polycyclic phenyl ether derivative or naphthalenesulfonic acid derivative, and the content of the component in the high-density lipoprotein is priced A standard product for quantitative determination of components in high-density lipoprotein in serum or plasma.
  10. 以下の工程を含む方法により製造される、請求項９記載の標準品。
    （１）血清又は血漿に、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
    （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、高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 9, which is produced by a method including the following steps.
    (1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 high-density lipoprotein contained in the lyophilized product obtained in step (2) using a known amount of the component.
  11. 高密度リポ蛋白中の成分が、コレステロールである、請求項７～１０のいずれかに記載の標準品。 The standard product according to any one of claims 7 to 10, wherein the component in the high-density lipoprotein is cholesterol.
  12. 以下の工程を含むことを特徴とする、血清又は血漿中の高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、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；及び、
    （２）工程（１）で得られた混合物中に含まれる、高密度リポ蛋白中の成分の濃度を、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 high-density lipoprotein in serum or plasma, which comprises the following steps.
    (1) adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative to serum or plasma; and
    (2) A step of pricing the concentration of the component in the high-density lipoprotein contained in the mixture obtained in step (1) using a known amount of the component.
  13. 以下の工程を含むことを特徴とする、血清又は血漿中の高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、ポリオキシエチレン多環フェニルエーテル誘導体又はナフタレンスルホン酸誘導体を添加する工程；
    （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、高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 high-density lipoprotein in serum or plasma, which comprises the following steps.
    (1) A step of adding a polyoxyethylene polycyclic phenyl ether derivative or a naphthalenesulfonic acid derivative 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 high-density lipoprotein contained in the lyophilized product obtained in step (2) using a known amount of the component.
  14. 高密度リポ蛋白中の成分が、コレステロールである、請求項１２又は１３記載の製造方法。 The production method according to claim 12 or 13, wherein the component in the high-density lipoprotein is cholesterol.
  15. 以下の工程を含むことを特徴とする、血清又は血漿中の高密度リポ蛋白中の成分の定量方法。
    （１）血清又は血漿中の高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７～１０のいずれかに記載の標準品と、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 components in high-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 high-density lipoprotein in serum or plasma;
    (2) Using the standard product according to any one of claims 7 to 10 and the measurement reagent in step (1), a calibration curve representing the relationship between the concentration of the component and the measured value for the component Creating step; 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).
  16. 高密度リポ蛋白中の成分が、コレステロールである、請求項１５記載の定量方法。 The quantification method according to claim 15, wherein the component in the high-density lipoprotein is cholesterol.
  17. 血清又は血漿中の高密度リポ蛋白中の成分の測定試薬、及び、請求項７～１０のいずれかに記載の標準品を含有することを特徴とする、血清又は血漿中の高密度リポ蛋白中の成分の定量用キット。 In a high-density lipoprotein in serum or plasma, comprising a reagent for measuring a component in high-density lipoprotein in serum or plasma and the standard product according to any one of claims 7 to 10 Kit for quantitative determination of ingredients.
  18. 高密度リポ蛋白中の成分が、コレステロールである、請求項１７記載のキット。
          The kit according to claim 17, wherein the component in the high-density lipoprotein is cholesterol.
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