Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • 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
• • 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

Definitions

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

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• 2014
  • 2014-11-19 WO PCT/JP2014/080604 patent/WO2015076285A1/ja active Application Filing
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