[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2023085323A1 - 溶血ヘモグロビンによる測定誤差を低減する方法 - Google Patents

溶血ヘモグロビンによる測定誤差を低減する方法 Download PDF

Info

Publication number
WO2023085323A1
WO2023085323A1 PCT/JP2022/041727 JP2022041727W WO2023085323A1 WO 2023085323 A1 WO2023085323 A1 WO 2023085323A1 JP 2022041727 W JP2022041727 W JP 2022041727W WO 2023085323 A1 WO2023085323 A1 WO 2023085323A1
Authority
WO
WIPO (PCT)
Prior art keywords
polyoxyethylene
ether
nonionic surfactant
sample
oxidase
Prior art date
Application number
PCT/JP2022/041727
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
美月 木内
Original Assignee
東洋紡株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 東洋紡株式会社 filed Critical 東洋紡株式会社
Priority to JP2023559867A priority Critical patent/JPWO2023085323A1/ja
Priority to CN202280082188.1A priority patent/CN118382704A/zh
Publication of WO2023085323A1 publication Critical patent/WO2023085323A1/ja

Links

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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase

Definitions

  • the present invention relates to a reagent kit, a measuring method, and the like that can reduce measurement errors by suppressing the influence of hemolyzed hemoglobin that may be contained in a sample in the measurement of a biological component or the like by an enzymatic method.
  • enzymatic measurement methods using enzymes having high substrate specificity for each component have been widely used as methods for measuring biological components and the like.
  • a method in which an oxidase is caused to act on a biological component or a substance derived from a biological component to be measured, and the resulting hydrogen peroxide is measured is often used.
  • hydrogen peroxide generated after decomposing substances other than the components to be measured by the first reagent is decomposed into water by catalase, and then hydrogen peroxide is generated from the components to be measured by the second reagent using oxidase.
  • This hydrogen peroxide is reacted with a coloring agent (also referred to as a chromogen) in the presence of peroxidase to convert the coloring agent into a dye (e.g., quinoneimine dye), and the dye is colorimetrically determined by measuring the absorbance.
  • a coloring agent also referred to as a chromogen
  • hemolysis in the specimen may interfere with the measurement system when measuring biological components in clinical diagnosis using such an enzymatic method.
  • hemoglobin does not normally exist in serum/plasma specimens, but when hemolysis occurs, hemoglobin in red blood cells will be present in the specimen. Since hemoglobin is a reducing substance, hydrogen peroxide is reduced, often leading to lower measured values.
  • the present invention is a method for measuring biological components by an enzymatic method in which an oxidase is acted on, and the generated hydrogen peroxide is reacted with a coloring agent in the presence of peroxidase for colorimetric determination.
  • a coloring agent in the presence of peroxidase for colorimetric determination.
  • hemoglobin interference is suppressed.
  • amphoteric surfactants and other components which were thought to be possible, makes the measurement more susceptible to hemolytic hemoglobin, resulting in a deterioration in measured values.
  • the present invention has been made in order to solve the above-mentioned newly discovered problems, and the hydrogen peroxide produced by the action of an oxidase is reacted with a coloring agent in the presence of peroxidase to obtain a colorimetric product.
  • a method of suppressing interference of hemoglobin which may be mixed in a sample due to hemolysis, and suppressing a change in absorbance independent of a biological component to be measured, in a method of measuring a biological component or the like by a quantitative enzymatic method. is.
  • the present inventors have discovered an enzymatic method in which the above-described oxidase is acted on, and the generated hydrogen peroxide is reacted with a coloring agent in the presence of peroxidase for colorimetric determination.
  • the inventors have found that the measurement error due to the interference of hemolyzed hemoglobin that can occur in the method of measuring biological components etc. can be reduced by allowing a specific nonionic surfactant to act in the reaction solution, and have completed the present invention. .
  • the typical present invention has the following configurations.
  • [Claim 1] In the method of causing an oxidase to act on a biological component or a substance derived from the biological component in a sample and reacting the generated hydrogen peroxide with a coloring agent in the presence of peroxidase for colorimetric determination, hemolyzed hemoglobin by the action of at least one nonionic surfactant selected from the group consisting of nonionic surfactants and polyoxyethylene-based nonionic surfactants having an HLB value of 12.7 to 13.6
  • the nonionic surfactant comprises at least one selected from the group consisting of N-acyl-N-methylglucamides, polyoxyethylene alkyl ethers, and polyoxyalkylene derivatives.
  • n-octanoyl-N-methyl-glucamine As the nonionic surfactant, n-octanoyl-N-methyl-glucamine, n-nonanoyl-N-methyl-glucamine, n-decanoyl-N-methyl-glucamine, polyoxyethylene lauryl ether, poly Oxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene behenyl ether, polyoxyalkylene alkyl ether, polyoxyethylene polyoxypropylene decyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene di Item 3.
  • the method according to Item 1 or 2 comprising at least one selected from the group consisting of styrenated phenyl ether, polyoxyethylene nonylphenyl ether, and polyoxyethylene polyoxypropylene glycol.
  • the nonionic surfactant is selected from the group consisting of n-nonanoyl-N-methyl-D-glucamine, polyoxyethylene oleyl ether, polyoxyalkylene alkyl ether, and polyoxyethylene tribenzylphenyl ether.
  • the method according to any one of [Item 8] The method according to any one of items 1 to 7, wherein the biological component or the substance derived from the biological component is creatinine.
  • [Item 9] The method according to any one of Items 1 to 8, wherein a negative measurement error caused by hemolyzed hemoglobin is reduced in the sample.
  • [Item 10] Selected from the group consisting of an oxidase, a peroxidase, a coloring agent, and a glucamine-based nonionic surfactant and a polyoxyethylene-based nonionic surfactant having an HLB value of 12.7 to 13.6
  • nonionic surfactant comprises at least one selected from the group consisting of N-acyl-N-methylglucamides, polyoxyethylene alkyl ethers, and polyoxyalkylene derivatives. Reagent kit as described.
  • the influence of measurement error caused by hemolyzed hemoglobin which may be contained in a biological sample, can be greatly suppressed when measuring a biological component or the like by an enzymatic method using an oxidase, and more accurate measurement results can be obtained. becomes possible.
  • the method of the present invention requires only the addition of a specific nonionic surfactant to the reaction solution and allows it to act, eliminating the need for pretreatment such as removing hemoglobin in advance from the sample to be measured. , it is possible to easily obtain highly accurate measurement results.
  • the present invention is an enzymatic method (herein referred to as simply referred to as "an enzyme method using an oxidase-peroxidase-coloring agent system", etc.), suppressing the influence of hemolyzed hemoglobin that may be contained in the sample on the measurement value, and reducing the measurement error (e.g., negative measurement error). It is a method to reduce.
  • an oxidase is allowed to act on a biological component or a substance derived from the biological component in a sample, and the generated hydrogen peroxide is reacted with a coloring agent in the presence of peroxidase.
  • At least one nonionic surface selected from the group consisting of a glucamine-based nonionic surfactant and a polyoxyethylene-based nonionic surfactant having an HLB value of 12.7 to 13.6 in the colorimetric determination method.
  • the biological component measuring method to which the present invention is applied is a biological component measuring method based on an enzymatic method.
  • hydrogen peroxide is generated by using peroxidase, which is reacted with a coloring agent in the presence of peroxidase, and colorimetrically determined.
  • a biological component measuring method using this principle has already been established in the technical field. Therefore, by applying the knowledge to the present invention, the amount or concentration of any biological component to be measured or any substance derived from the biological component can be measured. There are no particular restrictions on the order and timing of addition of the coloring agents.
  • the present invention can be implemented in a method for measuring creatinine, a method for measuring uric acid, a method for measuring glycated hemoglobin, a method for measuring triglyceride, and more preferably a method for measuring creatinine. As shown in the results of Examples described later, it is clear that the present invention provides highly accurate results in creatinine measurement.
  • enzymes such as oxidases used in the biological component measurement method using an oxidase-peroxidase-coloring agent system are not particularly limited, and those skilled in the art can appropriately select and use them according to the biological component to be measured. can be done.
  • the reaction of creatinine amidinohydrolase with creatinine as a substrate does not directly produce hydrogen peroxide, so the reaction of creatinine amidinohydrolase Sarcosine is produced by reacting the creatine produced in step with creatine amidohydrolase previously added to the reagent, and sarcosine is reacted with sarcosine oxidase (oxidase) previously added to the reagent to produce hydrogen peroxide.
  • oxidase sarcosine oxidase
  • uric acid When measuring uric acid (UA), it can be quantified by an oxidase-peroxidase-coloring agent system by generating hydrogen peroxide generated by the reaction of uricase (oxidase) with uric acid as a substrate.
  • oxidase uricase
  • uric acid as a substrate
  • glycated hemoglobin oxidase for example, fructosyl amino acid oxidase
  • oxidase oxidase
  • triglyceride When measuring triglyceride (TG), lipoprotein lipase using triglyceride (TG) as a substrate and glycerol kinase and glycerol 3-phosphate oxidase (oxidase) as coupled enzymes are used to generate hydrogen peroxide. , quantification of triglyceride concentration by an oxidase-peroxidase-color former system is possible. Even when measuring other biological components, hydrogen peroxide can be generated by an enzymatic reaction based on a method well known in the art in the same manner as described above.
  • the present invention can be practiced in a method of measuring creatinine with a peroxidase-chromogen system using creatinine amidinohydrolase, creatine amidohydrolase, sarcosine oxidase (an oxidase) to produce hydrogen peroxide. .
  • the measurement is performed on the substrate of an oxidase that can generate hydrogen oxide.
  • an enzyme that catalyzes a reaction that can change a target which may involve several steps of enzymatic reactions
  • the enzyme used in the enzymatic method using an oxidase-peroxidase-coloring agent system can be selected according to the biological component to be measured or a component derived from the biological component.
  • enzymes for generating substrates for oxidases include creatinine amide hydrolase, creatinine amidinohydrolase, cholesterol esterase, lipoprotein lipase, glycerol kinase, phospholipase, purine phosphorylase, acyl-CoA synthetase, urea amide lyase, purine nucleosides. phosphorylase and the like.
  • oxidizing enzymes that can be used in the present invention include sarcosine oxidase, cholesterol oxidase, glucose oxidase, glycerol oxidase, glycerol-3-phosphate oxidase, choline oxidase, xanthine oxidase, acyl-CoA oxidase, pyruvate oxidase, Examples include, but are not limited to, uricase, fructosyl amino acid oxidase, xanthine oxidase, and the like.
  • a method using a liquid reagent (or kit) configured to be applied to a general-purpose automatic analyzer (e.g., Hitachi 7170 automatic analyzer), a method such as freeze-drying, etc.
  • a method using a reagent (or kit) composed of a combination of a dry formulation and a solution manufactured by , and a method using a kit or sensor called a so-called dry system in which an enzyme or the like is supported on an appropriate carrier. can be exemplified.
  • it is a method of measuring with an automatic analyzer using one or more liquid reagents, and more preferably, a liquid reagent in which the reagent is divided into two (hereinafter also referred to as a two-reagent liquid reagent) is used. It is a method of analyzing with an automatic analyzer.
  • the first type of reagent (hereinafter also referred to as the first reagent or R1 reagent) is first added to the sample and reacted for a certain period of time (this is referred to as the first reaction), and then the second type of reagent (hereinafter , Second reagent or R2 reagent) is further added and reacted for a certain period of time (this is referred to as the second reaction), and the change in absorbance between the end of the first reaction and the end of the second reaction is measured.
  • the target component can be colorimetrically determined.
  • a similar component to be measured that may be contained in the sample may cause a measurement error, or may cause several steps of conjugation reaction.
  • reaction intermediates may cause measurement errors, and in such cases, it is preferable to first perform an elimination reaction that eliminates components that may cause measurement errors in the first reaction.
  • an enzyme other than the enzyme that directly acts on the substance to be measured is allowed to act on the sample to generate hydrogen peroxide, and after eliminating the reaction intermediates with catalase, in the second reaction, an enzyme that directly acts on the substance to be measured is added to the reaction system to generate hydrogen peroxide, which is reacted with a coloring agent in the presence of peroxidase, and at the same time, the action of catalase is substantially stopped for colorimetric determination.
  • creatinine amidohydrolase enzymes other than creatinine amidohydrolase (creatine amidinohydrolase and sarcosine oxidase) are allowed to act on the sample in the first reaction, and hydrogen peroxide generated due to creatine etc. is eliminated by catalase,
  • creatinine amidohydrolase is added to the reaction system, and hydrogen peroxide generated due to creatinine to be measured is reacted with a coloring agent in the presence of peroxidase for colorimetric determination (at this time and, at the same time, substantially stop the action of catalase).
  • the first reagent containing an enzyme other than the enzyme that directly acts on the target substance is used to perform the first reaction of the elimination system. followed by a second reaction with a second reagent containing an enzyme that directly acts on the substance to be measured.
  • Nonionic surfactant uses at least one nonionic surfactant selected from the group consisting of glucamine-based nonionic surfactants and polyoxyethylene-based nonionic surfactants having an HLB value of 12.7 to 13.6.
  • One of the characteristics is that the sample is acted on in the reaction liquid. This makes it possible to highly suppress the influence caused by hemolyzed hemoglobin that may be contained in the specimen when measuring biological components in an oxidase-peroxidase-coloring agent system enzymatic method, thereby reducing measurement errors. It is possible to reduce the amount of radiation and measure biological components with high accuracy.
  • the manner in which these specific nonionic surfactants act in the reaction solution is not particularly limited.
  • the nonionic surfactant may be directly added to the sample to act, or
  • the reagent to be reacted may contain the nonionic surfactant.
  • the first reagent to be reacted with the sample contains the nonionic surfactant.
  • the first reagent contains the nonionic surfactant.
  • the glucamine-based nonionic surfactant that can be used in the present invention is not particularly limited as long as it is a nonionic surfactant having an N-methyl-glucamine group.
  • glucamine-based nonionic surfactants include N-acyl-N-methylglucamide, preferably n-octanoyl-N-methyl-glucamine and n-nonanoyl-N-methyl-glucamine. , n-decanoyl-N-methyl-glucamine, preferably n-nonanoyl-N-methyl-D-glucamine.
  • Commercially available glucamine-based nonionic surfactants can also be suitably used, for example, MEGA-8, MEGA-9, MEGA-10 manufactured by Dojindo Laboratories, etc. can be used.
  • the polyoxyethylene-based nonionic surfactant that can be used in the present invention is a nonionic surfactant having a polyoxyethylene (ethylene oxide, EO) chain and an HLB value of 12.7 to 13.6. If so, it is not particularly limited.
  • polyoxyethylene-based nonionic surfactants include polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene behenyl ether, etc.), polyoxyalkylene derivatives (e.g., polyoxyalkylene alkyl ether, polyoxyethylene polyoxypropylene decyl ether, polyoxyethylene tribenzylphenyl ether, polyoxyethylene distyrenated phenyl ether, polyoxyethylene nonylphenyl ether , polyoxyethylene polyoxypropylene glycol, etc.), but not limited thereto.
  • polyoxyethylene alkyl ethers e.g., polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene behenyl ether, etc.
  • polyoxyalkylene derivatives
  • polyoxyethylene oleyl ether Preferred are polyoxyethylene oleyl ether, polyoxyethylene alkylene alkyl ether and polyoxyethylene tribenzylphenyl ether having an HLB value of 12.7 to 13.6.
  • Commercially available polyoxyethylene-based nonionic surfactants having an HLB value of 12.7 to 13.6 can also be suitably used.
  • Emulgen 109P, Emulgen 709, Emulgen 2020G-HA, Emulgen B-66, Emulgen MS-110, Emulgen LS-110, Emulgen A-60, NOF's Nonion P-210, Nonion E-212, etc. can do.
  • the amounts of the glucamine-based nonionic surfactant and the polyoxyethylene-based nonionic surfactant having an HLB value of 12.7 to 13.6 are not particularly limited as long as the effects of the present invention are exhibited.
  • a reaction solution containing a sample and the nonionic surfactant for example, when the first reagent for the first reaction contains the nonionic surfactant, the sample and the first reagent are The content of the nonionic surfactant in the reaction solution after mixing) is 0.1 to 1 w/v%, preferably 0.1 to 0.7 w/v%, more preferably 0 .1 to 0.5 w/v%.
  • Coexistence of a sample that may contain hemolyzed hemoglobin and the nonionic surfactant at such a concentration makes it possible to more effectively reduce measurement errors caused by hemolyzed hemoglobin.
  • peroxidase As the peroxidase used in the present invention, any kind of enzyme may be used as long as it catalyzes the reaction between hydrogen peroxide and a coloring agent (also referred to as a redox-based coloring reagent). , peroxidases derived from basidiomycetes. Among these, horseradish, rice, and soybean-derived peroxidases are preferred, and horseradish-derived peroxidases are more preferred, in terms of purity, availability, price, and the like.
  • PEO-131 manufactured by Toyobo
  • PEO-301 manufactured by Toyobo
  • PEO-302 manufactured by Toyobo
  • Peroxidase activity is defined in the following manner. 14 mL of distilled water, 2 mL of 5% (W/V) pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were sequentially mixed, and then pre-heated at 20° C. for 5 minutes. and add 1 mL of the sample solution to initiate the enzymatic reaction. After reacting for 20 seconds, 1 mL of 2N aqueous sulfuric acid solution is added to terminate the reaction, and the produced purpurogallin is extracted 5 times with 15 mL of ether.
  • the total volume is adjusted to 100 mL, and the absorbance at a wavelength of 420 nm is measured ( ⁇ ODtest).
  • ⁇ ODtest the absorbance at a wavelength of 420 nm is measured.
  • 14 mL of distilled water, 2 mL of 5% pyrogallol aqueous solution, 1 mL of 0.147 M hydrogen peroxide solution and 2 mL of 100 mM phosphate buffer (pH 6.0) were sequentially mixed, and then 1 mL of 2N sulfuric acid aqueous solution was added and mixed. , then add 1 mL of sample solution to prepare. This liquid is subjected to ether extraction in the same manner as above, and the absorbance is measured ( ⁇ OD blank).
  • the amount of purpurogallin produced is calculated from the difference in absorbance between ⁇ ODtest and ⁇ ODblank, and the peroxidase activity is calculated.
  • the amount of enzyme that produces 1.0 mg of purpurogallin in 20 seconds under the above conditions is defined as 1 purpurogallin unit (U).
  • the calculation formula is as shown below.
  • U/mg ⁇ U/mL ⁇ x 1/C 0.117: absorbance at 420 nm of 1 mg% purpurogallin ether solution
  • C Enzyme concentration at dissolution (c mg/mL) (One propurgarine unit corresponds to 13.5 international units (using o-dianisidine as a substrate under reaction conditions of 25°C).)
  • the sample solution was dissolved in ice-cooled 0.1 M phosphate buffer pH 6.0 in advance, and diluted with the same buffer to 3.0 to 6.0 purpurogallin units (U)/mL. It is preferable to use it for measurement.
  • coloring agent used in the present invention any type of coloring agent may be used as long as it reacts with hydrogen peroxide to lead to a dye and develop a color.
  • a combination of a hydrogen donor and a coupler, leuco bodies, tetrazolium salts, and the like from the viewpoint that the effect of the present invention can be obtained more effectively, it is preferable to use a combination of a hydrogen donor and a coupler as the coloring agent.
  • the amount of the coloring agent used or the form of addition All of these can be obtained as commercially available products.
  • a representative example using a combination of a hydrogen donor and a coupler is the Trinder method in which the hydrogen donor and coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye.
  • Phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives and the like are known as hydrogen donors used in the Trinder method and the like, and these can be preferably used in the present invention.
  • Aniline derivatives are preferred.
  • couplers include 4-aminoantipyrine (4-AA), aminoantipyrine derivatives, vanillindiaminesulfonic acid, methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthiazolinone hydrazone (SMBTH), and the like. and these can be preferably used in the present invention.
  • 4-aminoantipyrine 4-aminoantipyrine.
  • Leuco derivatives include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives and the like. Specifically, 4,4′-benzylidenebis(N,N-dimethylaniline), 4,4′-bis[N-ethyl-N-(3-sulfopropylamino)-2,6-dimethylphenyl]methane , 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole, 4,4′-bis(dimethylamino)diphenylamine, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine salt (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA67), etc.
  • Tetrazolium salts include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium salt, 3,3′-[3,3′-dimethoxy-(1 ,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt, 3,3′-[3,3′-dimethoxy-( 1,1′-biphenyl)-4,4′-diyl]-bis(2,5-diphenyl-2H-tetrazolium) salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium salt, 3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2,5-diphenyl
  • the reaction reagent used in the present invention is a liquid reagent, it preferably contains a buffer component.
  • Buffers include Tris buffers, phosphate buffers, borate buffers, carbonate buffers, GOOD buffers and the like. There are no particular restrictions on the amount used, the set pH, the mode of addition, and the like. All of these can be obtained as commercially available products.
  • GOOD buffers include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl- 2-aminoethanesulfonic acid (CHES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2- Hydroxy-3-aminopropanesulfonic acid (CAPSO), 3-[N,N-bis(2-hydroxyethyl)amino]-2
  • reaction reagent of the present invention may contain antiseptics, chelating agents, antibiotics, antibacterial agents, salts, enzyme stabilizers, color former stabilizers, etc., as long as they do not affect the reaction.
  • Preservatives include azides, chelating agents, antibiotics, antimicrobial agents, and the like.
  • Chelating agents include ethylenediaminetetraacetic acid and salts thereof.
  • Antibiotics include gentamicin, kanamycin, chloramphenicol and the like.
  • Antibacterial agents include methylisothiazolinone, imidazolidinyl urea, ProClin and the like.
  • Salts include sodium chloride, potassium chloride, aluminum chloride and the like.
  • Enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like.
  • Color former stabilizers include chelating agents such as ethylenediaminetetraacetic acid and salts thereof, cyclodextrins and the like.
  • the reaction reagent of the present invention preferably contains a component capable of reducing the effects of such substances reduced in the body, more preferably ascorbate oxidase and bilirubin oxidase, and particularly preferably ascorbate oxidase. .
  • the reaction reagents used in the present invention do not contain surfactants other than nonionic surfactants.
  • surfactants other than nonionic surfactants.
  • a component such as an amphoteric surfactant, which was conventionally believed to be able to suppress interference with hemoglobin, is used, It has been confirmed that the measurement value may deteriorate due to the fact that it is easily affected by hemolyzed hemoglobin. Therefore, in certain embodiments, it is desirable not to coexist with components that can exacerbate such measurement errors, preferably no anionic, cationic, or amphoteric surfactants.
  • the reaction reagent used in the present invention is preferably a reagent that does not substantially contain an anionic surfactant, a cationic surfactant, or an amphoteric surfactant.
  • Surfactants other than polyoxyethylene-based nonionic surfactants with an HLB value of 12.7 to 13.6 e.g., sucrose fatty acid esters, polyoxyethylene-based nonionic surfactants with an HLB value higher than 13.6
  • a reagent that does not substantially contain active agents, anionic surfactants, cationic surfactants, amphoteric surfactants can also be used.
  • the term "substantially not contained" means that the content of Say no.
  • the present invention may be practiced in the measurement of biological components or substances derived from said biological components in any sample that may contain hemolyzed hemoglobin.
  • a sample is not particularly limited as long as it is a sample collected from a living body, and examples thereof include blood (particularly serum, plasma, etc.), urine, ascites, and body fluids of a living body such as cerebrospinal fluid.
  • blood-derived samples such as serum and plasma, and samples collected from humans such as urine-derived samples are preferable, and serum and plasma are more preferable.
  • these blood-derived samples contain hemolyzed hemoglobin and can cause measurement errors, the present invention has the advantage of highly reducing such measurement errors.
  • At least one nonionic surfactant selected from the group consisting of glucamine-based nonionic surfactants and polyoxyethylene-based nonionic surfactants having an HLB value of 12.7 to 13.6 is allowed to coexist in the reaction solution and act on the sample, it is possible to suppress measurement errors caused by hemolyzed hemoglobin in the sample in an enzymatic method using an oxidase-peroxidase-color former system.
  • the present invention provides an oxidase, a peroxidase, a coloring agent, and a glucamine-based nonionic surfactant and a polyoxyethylene-based nonionic surfactant having an HLB value of 12.7 to 13.6.
  • a reagent kit used for measuring a biological component or a substance derived from the biological component, containing at least one nonionic surfactant selected from the group consisting of: The types and amounts of oxidases, peroxidases, coloring agents, and nonionic surfactants that can be used in this kit, as well as other components that may coexist or components that should not coexist, etc., are described above. It is the same as a thing.
  • the reagent kit of the present invention contains at least one selected from the group consisting of N-acyl-N-methylglucamides, polyoxyethylene alkyl ethers, and polyoxyalkylene derivatives as nonionic surfactants.
  • the reagent kit of the present invention may preferably be a reagent kit for creatinine measurement.
  • the oxidase, peroxidase, coloring agent, nonionic surfactant, etc. may be filled in the same container as one reagent, or divided into a plurality of containers. , may be configured to be used by mixing at the time of use.
  • the reagent kit is configured such that the first reagent to be mixed with the sample first contains the nonionic surfactant.
  • the reagent kit of the present invention contains information on how to use the kit (for example, the nonionic surfactant in an amount such that the final concentration when mixed with the reaction solution is 0.1 to 1 w / v%. instructions to add a reagent containing a nonionic surfactant, etc.).
  • Example 1 Confirmation of effect of addition of polyoxyethylene-based nonionic surfactant to creatinine measurement reagent> 5 g/L of Emulgen 420 (polyoxyethylene oleyl ether, HLB value 13.6) was added to the first reagent of the reagents for measuring creatinine described below (the final concentration in the reaction solution was 0.49 w/v%).
  • Emulgen 420 polyoxyethylene oleyl ether, HLB value 13.6
  • a first reagent containing no surfactant was also prepared and measured as a control.
  • a first reagent and a second reagent having the following compositions were prepared as reagents for measuring creatinine.
  • First reagent Ascorbic acid oxidase (Toyobo ASO-311) 3 KU/mL Sarcosine oxidase (SAO-351 manufactured by Toyobo Co., Ltd.) 10 KU / mL Creatinine amidohydrolase (CRH-221 manufactured by Toyobo) 40.0 KU/mL N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline 0.14g/L
  • Hitachi 7180 automatic analyzer was used for the measurement. 120 ⁇ L of the first reagent was added to 2.7 ⁇ L of the sample and incubated for 5 minutes to form the first reaction. After that, 40 ⁇ L of the second reagent was added and incubated for 5 minutes to obtain the second reaction. Absorbance at 546 nm (main wavelength) and absorbance at 800 nm (sub-wavelength) were measured by a two-point end method in which the absorbances of the first reaction and the second reaction were corrected for liquid volume and the difference was taken. It was obtained by calculating the absorbance obtained by subtracting the sub-wavelength from the main wavelength.
  • samples 1 to 5 of human serum were calculated by comparing with a calibration curve prepared from the measured absorbance of purified water and 5 mg/dL creatinine aqueous solution. Further, the degree of divergence from hemolyzed hemoglobin-free was calculated by the following formula.
  • Deviation from hemolytic Hb-free ⁇ (measured creatinine value when hemolyzed hemoglobin (hemolyzed Hb) was added ⁇ creatinine measured value when hemolyzed hemoglobin was not added)/measured creatinine value when hemolyzed hemoglobin was not added ⁇ 100 (%)
  • Table 1 The results with no surfactant added are shown in Table 1 below, and the results with Emulgen 420 added are shown in Table 2 below.
  • the creatinine measurement reagent in which 5 g/L of polyoxyethylene oleyl ether (Emulgen 420, manufactured by Kao Corporation) with an HLB value of 13.6 was added to the first reagent did not lower the measured value. do not have. From the results of this example, it was found that the present invention can greatly reduce the negative effects caused by hemolyzed hemoglobin, which is an interfering substance, and can improve the divergence of measured values.
  • polyoxyethylene oleyl ether Emulgen 420, manufactured by Kao Corporation
  • Example 2 Confirmation of effect by adding various nonionic surfactants 1>
  • surfactants capable of ameliorating the negative effects of hemolyzed hemoglobin in creatinine measurement reagents were further evaluated.
  • Emulgen 420 polyoxyethylene oleyl ether, HLB value 13.6
  • MEGA9 n-nonanoyl- N-methyl-D-glucamine
  • the concentrations of the nonionic surfactants added were 2 g/L and 5 g/L (the final concentrations in the reaction solution were 0.20 w/v % and 0.49 w/v %, respectively). Then, the measured values of the same samples 1 to 5 as in Example 1 were confirmed. Table 3 shows the degree of divergence between the measured value of creatinine (measured value of CRE) and hemolyzed hemoglobin-free calculated in the same manner as in Example 1.
  • Emulgen 420 polyoxyethylene oleyl ether, HLB value: 13.6
  • MEGA9 n-nonanoyl-N-methyl-D-glucamine
  • Example 3 Confirmation 2 of effect by adding various nonionic surfactants>
  • further surfactants capable of ameliorating the negative effects of hemolyzed hemoglobin in creatinine measurement reagents were searched.
  • Emulgen B-66 polyoxyethylene tribenzylphenyl ether, HLB value 13.2
  • Emulgen MS-110 polyoxyethylene alkylene alkyl ether, HLB value 12.7 were used and evaluated. gone.
  • a reagent was prepared by adding 2 g/L of each nonionic surfactant to the first reagent in the same manner as in Example 1 (the final concentration in the reaction solution was 0.20 w/v %).
  • Samples 1 to 3 prepared in Example 1 were measured using the first reagent to which Emulgen B-66 was added, and the measured values were confirmed. Separately, using the first reagent to which Emulgen MS-110 was added, samples 1 and 5 prepared in Example 1 were measured, and the measured values were confirmed. Table 4 below shows the results of the degree of discrepancy between the measured creatinine value when Emulgen B-66 was added and the hemolyzed hemoglobin-free case, and Table 5 below shows the results when Emulgen MS-110 was added.
  • Emulgen B-66 polyoxyethylene tribenzylphenyl ether, HLB value 13.2
  • Emulgen MS-110 polyoxyethylene alkylene alkyl ether, HLB value 12.2
  • amphoteric surfactant (CHAPS), anionic surfactant (Na taurodeoxycholate), nonionic surfactant with HLB value higher than 13.6 (sucrose fatty acid ester (HLB value 16.0) and polyoxyethylene lauryl ether (HLB value 18.1) were found to exacerbate measurement errors caused by hemolyzed hemoglobin.From these results, oxidase-peroxidase-color development was observed. In order to suppress the negative measurement error caused by hemolyzed hemoglobin in the agent-based enzymatic method, it became clear that it is necessary to select and use the specific nonionic surfactant of the present invention.
  • the reagent kit for measuring biological components and the measuring method of the present invention can easily obtain highly accurate measurement results. be able to.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
PCT/JP2022/041727 2021-11-12 2022-11-09 溶血ヘモグロビンによる測定誤差を低減する方法 WO2023085323A1 (ja)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2023559867A JPWO2023085323A1 (zh) 2021-11-12 2022-11-09
CN202280082188.1A CN118382704A (zh) 2021-11-12 2022-11-09 降低溶血血红蛋白所引起的测定误差的方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021184801 2021-11-12
JP2021-184801 2021-11-12

Publications (1)

Publication Number Publication Date
WO2023085323A1 true WO2023085323A1 (ja) 2023-05-19

Family

ID=86335812

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/041727 WO2023085323A1 (ja) 2021-11-12 2022-11-09 溶血ヘモグロビンによる測定誤差を低減する方法

Country Status (3)

Country Link
JP (1) JPWO2023085323A1 (zh)
CN (1) CN118382704A (zh)
WO (1) WO2023085323A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6427499A (en) * 1987-04-20 1989-01-30 Tokuyama Soda Kk Quantitative determination of polyamine
JPH09224697A (ja) * 1996-02-22 1997-09-02 Kainosu:Kk 生体成分の測定方法および測定用組成物
JPH11103888A (ja) * 1997-10-01 1999-04-20 Toyobo Co Ltd 生体成分測定用試薬および測定方法
WO2013147309A1 (ja) * 2012-03-30 2013-10-03 積水メディカル株式会社 血液試料中の物質の測定法
CN109706218A (zh) * 2019-03-08 2019-05-03 浙江达美生物技术有限公司 一种测定脂蛋白残粒胆固醇的试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6427499A (en) * 1987-04-20 1989-01-30 Tokuyama Soda Kk Quantitative determination of polyamine
JPH09224697A (ja) * 1996-02-22 1997-09-02 Kainosu:Kk 生体成分の測定方法および測定用組成物
JPH11103888A (ja) * 1997-10-01 1999-04-20 Toyobo Co Ltd 生体成分測定用試薬および測定方法
WO2013147309A1 (ja) * 2012-03-30 2013-10-03 積水メディカル株式会社 血液試料中の物質の測定法
CN109706218A (zh) * 2019-03-08 2019-05-03 浙江达美生物技术有限公司 一种测定脂蛋白残粒胆固醇的试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WIDMANN, C ET AL.: "In vitro activity of MEKK2 and MEKK3 in detergents is a function of a valine to serine difference in the catalytic domain", BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ELSEVIER, AMSTERDAM; NL, vol. 1547, no. 1, 5 May 2001 (2001-05-05), AMSTERDAM; NL , pages 167 - 173, XP004239384, ISSN: 0167-4838, DOI: 10.1016/S0167-4838(01)00183-2 *

Also Published As

Publication number Publication date
JPWO2023085323A1 (zh) 2023-05-19
CN118382704A (zh) 2024-07-23

Similar Documents

Publication Publication Date Title
US8119360B2 (en) Method, reagent and kit for determination of cholesterol in remnant-like particles (RLP)
JP6569193B2 (ja) 生体成分の測定方法および測定用組成物
JP6446875B2 (ja) 生体成分の測定方法および測定用組成物
EP0878716B1 (en) Measurement of LDL-cholesterol
JP6435605B2 (ja) 生体成分の測定方法および測定用組成物
US9671348B2 (en) Method for measuring component by treating aqueous sample with alpha-keto acid and then converting component to hydrogen peroxide
JP5332611B2 (ja) 高密度リポ蛋白中コレステロールの測定方法およびその試薬
JP6459268B2 (ja) 生体成分の測定方法および測定用組成物
WO2010055916A1 (ja) 低密度リポ蛋白中のコレステロールの測定方法、測定用試薬及び測定用キット
JP7579919B2 (ja) リポ蛋白コレステロールの定量方法及び定量キット
JP7484998B2 (ja) 測定感度を改善した生体成分測定キット及び生体成分測定方法
EP2857503B1 (en) Method for stabilizing ascorbic acid oxidase
WO2023085323A1 (ja) 溶血ヘモグロビンによる測定誤差を低減する方法
JP7206631B2 (ja) ４－ヒドロキシアンチピリンの定量方法、並びに生体成分測定キットの製造方法及び生体成分測定キット
JPH11103888A (ja) 生体成分測定用試薬および測定方法
WO2023190087A1 (ja) クレアチニン高値検体測定時の正確性向上
JPH1084997A (ja) Ｌｄｌ−コレステロールの測定法
JP7314932B2 (ja) 生体成分測定試薬キットの感度低下抑制方法
JPH10311833A (ja) Ｌｄｌ−コレステロールの測定方法
JP7314931B2 (ja) 生体成分測定試薬キットの感度低下抑制方法
JP7276328B2 (ja) 生体成分測定試薬キットの感度低下抑制方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22892821

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023559867

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 202280082188.1

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22892821

Country of ref document: EP

Kind code of ref document: A1