CN111020003A - Liquid stable β -hydroxybutyric acid determination reagent - Google Patents

Liquid stable β -hydroxybutyric acid determination reagent Download PDF

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CN111020003A
CN111020003A CN201911376198.2A CN201911376198A CN111020003A CN 111020003 A CN111020003 A CN 111020003A CN 201911376198 A CN201911376198 A CN 201911376198A CN 111020003 A CN111020003 A CN 111020003A
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reagent
acid
buffer solution
hydroxybutyrate
liquid stable
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叶志杰
黄艳妮
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Guilin Anglo American Institute Of Biotechnology
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    • 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
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
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Abstract

The invention discloses a liquid stable β -hydroxybutyric acid determination reagent, which comprises a first reagent and a second reagent, wherein the first reagent and the second reagent have a reaction ratio of 4:1, the first reagent comprises 20-200 mmol/L of a buffer solution with a pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyric dehydrogenase and 1-5 KU/L of diaphorase, and the second reagent comprises 20-200 mmol/L of an acidic buffer solution with a pH value of 4.0-5.0, 10-100 mmol/L of monovalent metal halide, 30-80 g/L of water-soluble saturated monohydric alcohol, 0.1-1 g/L of iodonitrotetrazolium blue and 5-10 g/L of coenzyme I.

Description

Liquid stable β -hydroxybutyric acid determination reagent
Technical Field
The invention relates to the technical field of medical detection, in particular to a liquid stable β -hydroxybutyric acid determination reagent.
Background
The β -hydroxybutyric acid accounts for about 70% of the total amount of ketone bodies and is a main component of the ketone bodies, so that the concentration of β -hydroxybutyric acid can reflect the level of the ketone bodies in the human body and can be used as an important index for measuring the curative effect of diabetic ketoacidosis, and meanwhile, the detection of β -hydroxybutyric acid is beneficial to the early diagnosis of type 1 diabetes mellitus complication acidosis and has important significance for the early diagnosis of diabetes mellitus.
At present, β -hydroxybutyric acid detection methods in blood are gas chromatography, spectrophotometry and enzyme methods, wherein the enzyme methods are gradually paid attention to clinical importance due to the characteristics of accuracy, simplicity, sensitivity, high specificity and the like.
Disclosure of Invention
The invention aims to provide a liquid stable β -hydroxybutyric acid determination reagent, which solves the problem of stable coexistence of aqueous solutions of INT and coenzyme I under acidic conditions through a solution system combining water-soluble saturated monohydric alcohol and monovalent metal halide, and simultaneously realizes stable preservation of biological enzymes and non-interference reaction by adopting GOOD' S biological buffer solutions of two acid-base systems.
In order to achieve the purpose, the invention provides a liquid-stable β -hydroxybutyric acid determination reagent which comprises a first reagent and a second reagent, wherein the reaction ratio of the first reagent to the second reagent is 4:1, the first reagent comprises 20-200 mmol/L of a buffer solution with a pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyric acid dehydrogenase and 1-5 KU/L of diaphorase, and the second reagent comprises 20-200 mmol/L of an acidic buffer solution with a pH value of 4.0-5.0, 10-100 mmol/L of monovalent metal halide, 30-80 g/L of water-soluble saturated monohydric alcohol, 0.1-1 g/L of iodonitrotetrazolium blue and 5-10 g/L of coenzyme I.
The first reagent further comprises 0.5-5 g/L of a nonionic surfactant, and the nonionic surfactant comprises one of triton X-100 or lauryl ether 35.
The first reagent comprises 2-5 g/L of metal complexing agent, and the metal complexing agent comprises one of ethylene diamine tetraacetic acid sodium salt, divinyl triamino pentaacetic dianhydride or sodium oxalate.
The first reagent comprises 20-50 g/L of polyhydric alcohol, and the polyhydric alcohol comprises one of sucrose, mannitol or trehalose.
The first reagent comprises 0.1-1 g/L preservative, and the preservative comprises one of sodium azide, dichloroacetamide or ethyl acetate sodium.
Wherein the buffer solution in the reagent I comprises one of N-trimethyl-3-aminopropane sulfonic acid, trihydroxymethyl glycine or N, N-dihydroxyethyl glycine buffer solution.
And the reagent II further comprises 2-5 g/L of a metal complexing agent, and the metal complexing agent comprises one of ethylene diamine tetraacetic acid sodium salt, divinyl triamino pentaacetic dianhydride or sodium oxalate.
Wherein the acidic buffer solution in the second reagent comprises one of 2-morpholine ethanesulfonic acid-hydrochloric acid, N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid-hydrochloric acid and piperazine-1, 4-diethylsulfonic acid-hydrochloric acid buffer solution.
Wherein the monovalent metal halide salt comprises one of sodium chloride, potassium chloride, sodium bromide, or potassium bromide.
Wherein the water-soluble saturated monohydric alcohol comprises one of methanol, ethanol or isopropanol.
According to the β -hydroxybutyrate determination reagent with stable liquid, the reaction ratio of the reagent I and the reagent II is 4:1, the reagent I comprises 20-200 mmol/L of buffer solution with the pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyrate dehydrogenase and 1-5 KU/L of diaphorase, the reagent II comprises 20-200 mmol/L of acidic buffer solution with the pH value of 4.0-5.0, 10-100 mmol/L of monovalent metal halide, 30-80 g/L of water-soluble saturated monohydric alcohol, 0.1-1 g/L of iodonitrotetrazolium blue and 5-10 g/L of coenzyme I, the problem of stable coexistence of aqueous solutions of INT and coenzyme I under an acidic condition is solved through a solution system combining the water-soluble saturated monohydric alcohol and the monovalent metal halide, the problems of stable storage and non-interference reaction of biological enzyme are solved through GOOD' S biological buffer solutions of two acid-base systems, and the stability of the reagent with the current enzyme method is realized.
Detailed Description
The embodiment of the invention is described in detail below, wherein the liquid-stable β -hydroxybutyrate assay reagent comprises a first reagent and a second reagent, the reaction ratio of the first reagent to the second reagent is 4:1, the first reagent comprises 20-200 mmol/L of a buffer solution with a pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyrate dehydrogenase and 1-5 KU/L of diaphorase, and the second reagent comprises 20-200 mmol/L of an acidic buffer solution with a pH value of 4.0-5.0, 10-100 mmol/L of a monovalent metal halide, 30-80 g/L of a water-soluble saturated monohydric alcohol, 0.1-1 g/L of nitrotetrazolium iodide and 5-10 g/L of coenzyme I.
The method comprises the steps of adding a reagent I which is an active biological enzyme solution, adjusting the pH value in the reaction process, adding a reagent I which is a stabilized acid INT and a coenzyme I solution, forming a complete detection reagent when the two solutions are detected, wherein the pH value of a reaction system is 7.0-8.0, the reaction ratio of the reagent I to the reagent I is 4:1, adding 0.5-5 g/L of a nonionic surfactant in the reagent I, wherein the nonionic surfactant comprises one of Triton X-100 or lauryl ether 35, the dissolubility of sample serum in the reagent and the permeability of the reaction system can be improved, adding 2-5 g/L of a metal complexing agent in the reagent I or/and the reagent II, wherein the metal complexing agent comprises one of ethylene diamine tetraacetic acid sodium salt, divinyl triamino pentaacetic dianhydride or sodium oxalate, the influence of heavy metal ion on the enzyme activity can be prevented, adding 20-50 g/L of polyhydric alcohol in the reagent I, wherein the polyhydric alcohol comprises one of sucrose, mannitol or trehalose, the buffering agent I, the buffering agent, the.
The principle is explained in that under the catalytic action of β -hydroxybutyrate dehydrogenase, β -hydroxybutyrate is oxidized to generate acetoacetate, meanwhile oxidized coenzyme I is reduced to reduced coenzyme I, reduced coenzyme I and INT (iodonitrotetrazolium chloride) generate formazane (red substance) under the catalysis of diaphorase, and the absorbance value of the formazane (red substance) is in positive correlation with the concentration of β -hydroxybutyrate at a specific wavelength.
Figure RE-GDA0002405410710000041
Figure RE-GDA0002405410710000042
The β -hydroxybutyric acid content of the samples was determined by measuring the change in the absorbance at a wavelength of 505 nm.
In a stability load experiment in which the first reagent and the second reagent are placed at 37 ℃ for 7 days and a storage stability experiment in which the first reagent and the second reagent are stored at 4-8 ℃ for 12 months, the reagent has no obvious change in appearance, sensitivity, accuracy and linear range, and completely meets the requirements of clinical detection.
The reagent can adopt a manual testing method and can also detect a sample through an analysis instrument such as a full-automatic biochemical analyzer, and the like, and the following method is specifically adopted:
Figure RE-GDA0002405410710000043
the calculation method comprises the following steps:
Figure RE-GDA0002405410710000051
in order to ensure the reliability of the test result, the reagent detection needs to be calibrated by β -hydroxybutyric acid standard or standard serum before the sample detection.
The invention embodiment 1 provides a liquid stable β -hydroxybutyric acid determination reagent, which comprises a reagent I and a reagent II, wherein the reagent I comprises 25mmol/L of tris (hydroxymethyl) methylglycine (Tricine) buffer solution with the pH value of 8.5, 50g/L of sucrose, 5KU/L of β -hydroxybutyric acid dehydrogenase, 2KU/L of diaphorase, X-1002 g/L of triton, 2g/L of disodium ethylenediamine tetraacetate and 0.5g/L of sodium azide, and the reagent II comprises 20mmol/L of 2-morpholinoethanesulfonic acid-hydrochloric acid (MES-HCL) buffer solution with the pH value of 4.5, 50mmol/L of sodium chloride, 30g/L of ethanol, 0.75g/L of iodonitrotetrazolium blue, 6.5g/L of coenzyme I and 2g/L of disodium ethylenediamine tetraacetate.
Example 2 of the present invention provides a liquid-stable β -hydroxybutyrate assay reagent, which comprises a reagent I and a reagent II, wherein the reagent I comprises 50mol/L of N-tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) buffer solution with a pH value of 8.0, 30g/L of trehalose, 6KU/L of β -hydroxybutyrate dehydrogenase, 1.5KU/L of diaphorase, 355g/L of lauryl ether, 3g/L of sodium oxalate and 1g/L of dichloroacetamido, and the reagent II comprises 30mmol/L of N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid-hydrochloric acid (HEPES-HCL) buffer solution with a pH value of 4.5, 5mmol/L of potassium chloride, 30mmol/L of sodium bromide, 45g/L of isopropanol, 0.5g/L of iodonitrotetrazolium blue, 8g/L of coenzyme I, 2g/L of disodium ethylenediamine tetraacetate, 2g/L of disodium edetate,
Embodiment 3 of the present invention provides a liquid-stable β -hydroxybutyrate assay reagent, which comprises a reagent I and a reagent II, wherein the reagent I comprises 30mol/L of N, N-dihydroxyethylglycine (Bicine) buffer solution with a pH value of 9.0, 50g/L of mannitol, 4KU/L of β -hydroxybutyrate dehydrogenase, 1.5KU/L of diaphorase, 1.154 g/L of fatty alcohol polyoxyethylene ether AEO-154g/L of fatty alcohol polyoxyethylene ether, 3g/L of divinyltriamino-pentaacetic dianhydride, and 2g/L of ethyl acetate sodium, and the reagent II comprises 20mmol/L of piperazine-1, 4-diethylsulfonic acid-hydrochloric acid (PIPES-HC) buffer solution with a pH value of 4.0, 5mmol/L of potassium bromide, 25mmol/L of sodium bromide, 40g/L of methanol, 1.5g/L of iodonitrotetrazolium blue, 9g/L of coenzyme I, and 2g/L of divinyltriamino-pentaacetic dianhydride.
In a stability load test of placing the reagent I and the reagent II in the examples 1, 2 and 3 at 37 ℃ for 7 days and a storage stability test of storing the reagent I and the reagent II at the storage temperature of 4-8 ℃ for 12 months, the iodonitrotetrazolium blue in the reagent has no precipitation phenomenon and no obvious change of the appearance of the reagent, and when the detection is carried out according to the detection method disclosed by the invention, the upper limit of the concentration of β -hydroxybutyric acid in the detection of the reagent can reach 4.5 mmol/L.
According to the β -hydroxybutyrate determination reagent with stable liquid, the reaction ratio of the reagent I and the reagent II is 4:1, the reagent I comprises 20-200 mmol/L of buffer solution with the pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyrate dehydrogenase and 1-5 KU/L of diaphorase, the reagent II comprises 20-200 mmol/L of acidic buffer solution with the pH value of 4.0-5.0, 10-100 mmol/L of monovalent metal halide, 30-80 g/L of water-soluble saturated monohydric alcohol, 0.1-1 g/L of iodonitrotetrazolium blue and 5-10 g/L of coenzyme I, the problem of stable coexistence of aqueous solutions of INT and coenzyme I under an acidic condition is solved through a solution system combining the water-soluble saturated monohydric alcohol and the monovalent metal halide, the problems of stable storage and non-interference reaction of biological enzyme are solved through GOOD' S biological buffer solutions of two acid-base systems, and the stability of the reagent with the current enzyme method is realized.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A liquid-stable β -hydroxybutyric acid assay reagent, characterized in that,
the reagent I and the reagent II react according to a ratio of 4:1, the reagent I comprises 20-200 mmol/L of buffer solution with a pH value of 8.0-9.0, 4-10 KU/L of β -hydroxybutyrate dehydrogenase and 1-5 KU/L of diaphorase, and the reagent II comprises 20-200 mmol/L of acidic buffer solution with a pH value of 4.0-5.0, 10-100 mmol/L of monovalent metal halide, 30-80 g/L of water-soluble saturated monohydric alcohol, 0.1-1 g/L of iodonitrotetrazolium blue and 5-10 g/L of coenzyme I.
2. The liquid stable β -hydroxybutyrate assay reagent of claim 1,
the first reagent further comprises 0.5-5 g/L of a nonionic surfactant, and the nonionic surfactant comprises one of triton X-100 or lauryl ether 35.
3. The liquid stable β -hydroxybutyrate assay reagent of claim 2,
the first reagent comprises 2-5 g/L of metal complexing agent, and the metal complexing agent comprises one of ethylene diamine tetraacetic acid sodium salt, divinyl triamino pentaacetic dianhydride or sodium oxalate.
4. The liquid stable β -hydroxybutyrate assay reagent of claim 3,
the first reagent comprises 20-50 g/L of polyhydric alcohol, and the polyhydric alcohol comprises one of sucrose, mannitol or trehalose.
5. The liquid stable β -hydroxybutyrate assay reagent of claim 4,
the first reagent comprises 0.1-1 g/L preservative, and the preservative comprises one of sodium azide, dichloroacetamide or sodium ethyl acetate.
6. The liquid stable β -hydroxybutyrate assay reagent of claim 5,
the buffer solution in the reagent I comprises one of N-trimethyl-3-aminopropane sulfonic acid, trihydroxymethyl glycine or N, N-dihydroxyethyl glycine buffer solution.
7. The liquid stable β -hydroxybutyrate assay reagent of claim 1 or 3,
the reagent II further comprises 2-5 g/L of a metal complexing agent, and the metal complexing agent comprises one of ethylene diamine tetraacetic acid sodium salt, divinyl triamino pentaacetic dianhydride or sodium oxalate.
8. The liquid stable β -hydroxybutyrate assay reagent of claim 7,
the acid buffer solution in the reagent II comprises one of 2-morpholine ethanesulfonic acid-hydrochloric acid, N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid-hydrochloric acid and piperazine-1, 4-diethylsulfonic acid-hydrochloric acid buffer solution.
9. The liquid stable β -hydroxybutyrate assay reagent of claim 1,
the monovalent metal halide salt comprises one of sodium chloride, potassium chloride, sodium bromide, or potassium bromide.
10. The liquid stable β -hydroxybutyrate assay reagent of claim 1,
the water-soluble saturated monohydric alcohol comprises one of methanol, ethanol or isopropanol.
CN201911376198.2A 2019-12-27 2019-12-27 Liquid stable β -hydroxybutyric acid determination reagent Pending CN111020003A (en)

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CN101140279A (en) * 2006-09-04 2008-03-12 上海复星医药（集团）股份有限公司 Reducibility coenzyme composite stabilizer
CN101726463A (en) * 2009-12-24 2010-06-09 张闻 Method for measuring beta-hydroxybutyric acid by using enzyme colorimetric reaction, matched kit and application thereof
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CN102435749A (en) * 2011-09-02 2012-05-02 宁波美康生物科技股份有限公司 Liquid stable kit for measuring beta-hydroxybutyric acid by cyclic enzyme method
CN104198407A (en) * 2014-08-14 2014-12-10 上海睿康生物科技有限公司 Detection kit for detecting content of beta-hydroxybutyrate in serum by adopting stable enzymatic method
CN104730230A (en) * 2013-12-23 2015-06-24 上海复星医药（集团）股份有限公司 Enzymatic method detection kit of D-3-hydroxybutyric acid and preparation method thereof
CN105510255A (en) * 2015-12-24 2016-04-20 江苏迈源生物科技有限公司 Determining method and determining kit for beta-hydroxybutyrate
CN105842437A (en) * 2016-04-28 2016-08-10 安徽伊普诺康生物技术股份有限公司 Kit for detecting D-3-hydroxybutyric acid and preparation method of kit
CN107782679A (en) * 2016-08-26 2018-03-09 山东博科生物产业有限公司 A kind of hydroxybutyric acid detection kits of jamproof D 3

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101140279A (en) * 2006-09-04 2008-03-12 上海复星医药（集团）股份有限公司 Reducibility coenzyme composite stabilizer
CN101825625A (en) * 2009-03-06 2010-09-08 北京中生金域诊断技术有限公司 Kit for detecting urinary lactic acid, creatine and beta-hydroxybutyric acid in human urine simultaneously
CN101726463A (en) * 2009-12-24 2010-06-09 张闻 Method for measuring beta-hydroxybutyric acid by using enzyme colorimetric reaction, matched kit and application thereof
CN102435749A (en) * 2011-09-02 2012-05-02 宁波美康生物科技股份有限公司 Liquid stable kit for measuring beta-hydroxybutyric acid by cyclic enzyme method
CN104730230A (en) * 2013-12-23 2015-06-24 上海复星医药（集团）股份有限公司 Enzymatic method detection kit of D-3-hydroxybutyric acid and preparation method thereof
CN104198407A (en) * 2014-08-14 2014-12-10 上海睿康生物科技有限公司 Detection kit for detecting content of beta-hydroxybutyrate in serum by adopting stable enzymatic method
CN105510255A (en) * 2015-12-24 2016-04-20 江苏迈源生物科技有限公司 Determining method and determining kit for beta-hydroxybutyrate
CN105842437A (en) * 2016-04-28 2016-08-10 安徽伊普诺康生物技术股份有限公司 Kit for detecting D-3-hydroxybutyric acid and preparation method of kit
CN107782679A (en) * 2016-08-26 2018-03-09 山东博科生物产业有限公司 A kind of hydroxybutyric acid detection kits of jamproof D 3

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