CN105842437A - Kit for detecting D-3-hydroxybutyric acid and preparation method of kit - Google Patents

Kit for detecting D-3-hydroxybutyric acid and preparation method of kit Download PDF

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Publication number
CN105842437A
CN105842437A CN201610273294.4A CN201610273294A CN105842437A CN 105842437 A CN105842437 A CN 105842437A CN 201610273294 A CN201610273294 A CN 201610273294A CN 105842437 A CN105842437 A CN 105842437A
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CN
China
Prior art keywords
reagent
buffer
mmol
hydroxybutyric acid
preservative
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN201610273294.4A
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Chinese (zh)
Inventor
蔡晓辉
庄庆华
吴铮
徐运
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Anhui Iprocom Biotechnology Co Ltd
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Anhui Iprocom Biotechnology Co Ltd
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 Anhui Iprocom Biotechnology Co Ltd filed Critical Anhui Iprocom Biotechnology Co Ltd
Priority to CN201610273294.4A priority Critical patent/CN105842437A/en
Publication of CN105842437A publication Critical patent/CN105842437A/en
Pending legal-status Critical Current

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Abstract

The invention discloses a kit for detecting D-3-hydroxybutyric acid and a preparation method of the kit .The kit comprises a kit body composed of two independent liquid components, namely a reagent R1 and a reagent R2 .The reagent R1 is prepared from 20-180 mmol/L of buffer solution, 0.1-4.9 KU/L of D-3-hydroxybutyric acid dehydrogenase, 0.1-6.0 mL/L of polypropylene oxide and polyoxyethylene copolymer and 0.01-0.059 mmol/L of preservative, wherein solvent is purified water; the reagent R2 is prepared from 10-40 mmol/L of oxalate, 0.1-4.9 mmol/L of NAD+ and 0.01-0.059 mmol/L of preservative, wherein solvent is purified water .The invention further discloses the preparation and use method of the kit for detecting D-3-hydroxybutyric acid .The preparation and use method includes the following steps of preparing the reagents according to the contents of the components, mixing the reagent R1 and the reagent R2 with a to-be-detected sample to react sufficiently, detecting the after-reaction light absorption degree differential value through a full-automatic biochemical analyzer, and calculating the concentration of D-3-hydroxybutyric acid in the sample according to the light absorption degree change value .The kit has the advantages of being free of pollution, high in stability and the like.

Description

A kind of test kit measuring D-3-hydroxybutyric acid and preparation method thereof
Technical field
The present invention relates to medical science and technological field of biochemistry, a kind of test kit measuring D-3-hydroxybutyric acid and Preparation method.
Background technology
Diabetes are a kind of commonly encountered diseases, frequently-occurring diseases, and its sickness rate presents ascendant trend year by year.When organism metabolic disorder develops Accelerating to steatolysis, the accumulation of serum ketoboidies is referred to as ketonemia when exceeding normal level, and its clinical manifestation becomes ketosis;Work as keto acid Gather and ketoacidosis occurs to be referred to as during metabolic acidosis, as being in a bad way to when there is stupor, then become " diabetic Stupor ".And the main component of ketoboidies is D-3-hydroxybutyric acid, account for the 78% of ketoboidies total amount, be the main of diabetic ketoacidosis Material, lacks typical clinical symptoms in early days, and traditional ketoboidies detection method is sodium nitroprusside method, and the method is semidefinite Amount, although its detection method has a specificity, but it mainly measures is acetoacetic acid and acetone, and sensitivity is low and can not Quantitatively, in order to judge the state of an illness accurately and in time, it is desirable to have a highly sensitive and index for high specificity, D-3-hydroxybutyric acid is surveyed Determine foundation and the application of method so that it is be increasingly becoming the important means of Diabetic ketosis early diagnosis.
Typically, D-3-hydroxybutyric acid detection method has enzyme process, gas chromatography, radiochemical method etc., and radiochemical method is put Radioactive pollution, gas chromatography operation complexity, and the longest, all quick and substantial amounts of can not be applied to Clinical detection;Enzyme process Reagent stability the best, it is therefore desirable to improve.
Summary of the invention
The technical problem to be solved is to overcome radiochemical method to have radioactive pollution, gas chromatography to grasp Make the defect that complicated and time-consuming longer and enzyme process reagent stability is the best, and a kind of reagent measuring D-3-hydroxybutyric acid is provided Box and preparation method thereof.
The technical scheme that the present invention solves above-mentioned technical problem and provides is: the invention discloses a kind of mensuration D-3-hydroxybutyric acid Test kit, form test kit including reagent R1 independent of each other and reagent R2 biliquid component, including composition and contain accordingly Amount is:
Reagent R1:
Buffer 20 ~ 180mmol/L
D-3-HBD 0.1 ~ 4.9KU/L
Polyoxyethylene polyoxypropylene copolymer 0.1-6.0mL/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 10 ~ 40mmol/L
NAD+ 0.1~4.9mmol/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water.
As preferably, the invention discloses a kind of test kit measuring D-3-hydroxybutyric acid, including reagent R1 independent of each other Form test kit with reagent R2 biliquid component, including composition and corresponding content be:
Reagent R1:
Buffer 100mmol/L
D-3-HBD 2.5KU/L
Polyoxyethylene polyoxypropylene copolymer 3.0mL/L
Preservative 0.035 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 25mmol/L
NAD+ 2.5mmol/L
Preservative 0.035 mmol/L
Its solvent is purified water.
As preferably, in described reagent R1, described buffer is selected from phosphate buffer, trishydroxymethylaminomethane Buffer, Isosorbide-5-Nitrae-piperazine two ethanesulfonic acid buffer, 4-hydroxyethyl piperazine propane sulfonic acid buffer, 4-hydroxyethyl piperazine ethanesulfonic acid buffer Liquid, Pehanorm base propane sulfonic acid buffer, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid buffer, 3- One or many in (N-morpholinyl)-2-hydroxypropyl sulfonic acid buffer, MES buffer, phosphate sodium dihydrogen buffer solution Planting the mixing of arbitrary proportion, in described reagent R1, described preservative is sodium azide, and in described reagent R2, described is anti- Rotten agent is sodium azide.
As preferably, in described reagent R1, the pH value of described buffer is 6~9.
As preferably, the invention also discloses preparation method and the user of the test kit of said determination D-3-hydroxybutyric acid Method, comprises the following steps:
A () prepares reagent according to following component content:
Reagent R1:
Buffer 20 ~ 180mmol/L
D-3-HBD 0.1 ~ 4.9KU/L
Polyoxyethylene polyoxypropylene copolymer 0.1-6.0mL/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 10 ~ 40mmol/L
NAD+ 0.1~4.9mmol/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water.
B sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react;
C () measures reacted absorbance difference with automatic clinical chemistry analyzer;
D () calculates the concentration of D-3-hydroxybutyric acid in sample according to absorbance changing value.
The Cleaning Principle of the present invention is: this reagent by D-3-hydroxybutyric acid under the catalysis of D-3-HBD, D- 3-hydroxybutyric acid is oxidized, generates acetoacetic acid, and NAD+ is reduced into NADH, NADH simultaneously has specific absorption under 340nm wavelength Peak, therefore detects the absorbance rate of change of NADH under 340nm wavelength, calculates the concentration of D-3-hydroxybutyric acid in serum.
D-3-hydroxybutyric acid concentration (mmol/L)=CS ×(mmol/L)
ΔAU/ min is the absorbance changing value of testing sample average minute clock
ΔAB/ min is the absorbance changing value of calibration solution average minute clock
CSFor the concentration of D-3-hydroxybutyric acid in calibration solution
Compared with prior art, the present invention has a following advantageous benefits:
The enzyme process that used of invention, simple to operate, the shortest, and can rapid, high volume be applied to clinical diagnosis, there is no radioactivity The radioactive pollution that chemical element produces, and it is short to improve the enzyme process reagent holding time, and unstable phenomenon, in stability Have and further improved, by adding polyoxyethylene polyoxypropylene copolymer so that D-3-HBD stability It is improved, so that the stability of whole reagent is improved, the whole reagent the most effectively improved Accuracy of measurement.
Detailed description of the invention
Further illustrate below in conjunction with specific embodiment, but the present invention is not limited in these embodiments.
Embodiment 1
The test kit of the present invention includes reagent R1 independent of each other and reagent R2 biliquid component, wherein
Reagent R1:
TRIS buffer 100mmol/L
D-3-HBD 2.5KU/L
Polyoxyethylene polyoxypropylene copolymer 3.0mL/L
Sodium azide 0.035 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 25mmol/L
NAD+ 2.5mmol/L
Sodium azide 0.035 mmol/L
Its solvent is purified water.
Embodiment 2
The test kit of the present invention includes reagent R1 independent of each other and reagent R2 biliquid component, wherein
Reagent R1:
4-hydroxyethyl piperazine ethanesulfonic acid buffer 90mmol/L
D-3-HBD 3.1KU/L
Polyoxyethylene polyoxypropylene copolymer 5.0mL/L
Sodium azide 0.059 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 20mmol/L
NAD+ 3.0mmol/L
Sodium azide 0.059 mmol/L
Its solvent is purified water.
Embodiment 3
The preparation and application of test kit
1, reagent is prepared according to following component content:
Reagent R1:
TRIS buffer 100mmol/L
D-3-HBD 2.5KU/L
Polyoxyethylene polyoxypropylene copolymer 3.0mL/L
Sodium azide 0.035 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 25mmol/L
NAD+ 2.5mmol/L
Sodium azide 0.035 mmol/L
Its solvent is purified water.
2, automatic clinical chemistry analyzer parameter is arranged
(a) detection temperature: 37 DEG C;
(b) detection wavelength: dominant wavelength 340nm, commplementary wave length 700nm;
(c) response time: 8min, wherein, incubation time 5min, measure immediately after adding reagent R2 and read absorbance A 1, Read absorbance A 2 after 3min, calculate absorbance changes delta A=A2-A1;
(d) the Direction of Reaction: negative direction.
3, detecting step
A () takes 200 μ l reagent R1 and the mixing of 200 μ l samples to be tested;
B solution after mixing is hatched 5min under conditions of 37 DEG C by ();
C () adds 50 μ l reagent R2, measure immediately and read absorbance A 1, reads absorbance A 2 after 3min, calculates absorbance change Δ A=A2-A1;
D () is according to D-3-hydroxybutyric acid concentration (mmol/L)=CS ×(mmol/L) D-3-in sample is calculated The concentration of hydroxybutyric acid.
Embodiment 4
The preparation and application of test kit
1, reagent is prepared according to following component content:
Reagent R1:
4-hydroxyethyl piperazine ethanesulfonic acid buffer 90mmol/L
D-3-HBD 3.1KU/L
Polyoxyethylene polyoxypropylene copolymer 5.0mL/L
Sodium azide 0.059 mmol/L
Its solvent is purified water.
Reagent R2:
Oxalates 20mmol/L
NAD+ 3.0mmol/L
Sodium azide 0.059 mmol/L
Its solvent is purified water.
2, automatic clinical chemistry analyzer parameter is arranged
(a) detection temperature: 37 DEG C;
(b) detection wavelength: dominant wavelength 340nm, commplementary wave length 700nm;
(c) response time: 8min, wherein, incubation time 5min, measure immediately after adding reagent R2 and read absorbance A 1, Read absorbance A 2 after 3min, calculate absorbance changes delta A=A2-A1;
(d) the Direction of Reaction: negative direction.
3, detecting step
A () takes 200 μ l reagent R1 and the mixing of 200 μ l samples to be tested;
B solution after mixing is hatched 5min under conditions of 37 DEG C by ();
C () adds 50 μ l reagent R2, measure immediately and read absorbance A 1, reads absorbance A 2 after 3min, calculates absorbance change Δ A=A2-A1;
D () is according to D-3-hydroxybutyric acid concentration (mmol/L)=CS ×(mmol/L) D-3-in sample is calculated The concentration of hydroxybutyric acid.
Mensuration D-3-obtained by the test kit of the table 1 mensuration D-3-hydroxybutyric acid obtained by embodiment 1 and embodiment 2 The result that quality-control product 1 is measured by the test kit of hydroxybutyric acid respectively, wherein the concentration of the D-3-hydroxybutyric acid in quality-control product 1 is 0.29mmol/L, measurement result is shown in Table 1:
Table 1
As shown in Table 1, the test kit of the mensuration D-3-hydroxybutyric acid obtained by the present invention is to the measurement result deviation of quality-control product 1 relatively Little, therefore accuracy of measurement is high, and embodiment 1 is optimum selection.
Mensuration D-3-hydroxyl fourth obtained by the test kit of the table 2 mensuration D-3-hydroxybutyric acid obtained by embodiment 1 and embodiment 2 The result that quality-control product 2 is measured by the test kit of acid respectively, wherein the concentration of the D-3-hydroxybutyric acid in quality-control product 2 is 1.15mmol/L, measurement result is shown in Table 2:
Table 2
1st time (mmol/L) 2nd time (mmol/L) 3rd time (mmol/L) Average (mmol/L) Deviation (%)
Embodiment 1 1.11 1.16 1.16 1.14 0.87%
Embodiment 2 1.12 1.17 1.18 1.16 0.87%
As shown in Table 2, the test kit of the mensuration D-3-hydroxybutyric acid obtained by the present invention is to the measurement result deviation of quality-control product 2 relatively Little, therefore accuracy of measurement is high, and embodiment 1 is optimum selection.
It is the most anti-that same sample to be tested is carried out by the test kit of the table 3 mensuration D-3-hydroxybutyric acid obtained by embodiment 3 The test kit of repetition measurement mensuration D-3-hydroxybutyric acid calmly and obtained by embodiment 4 is surveyed the most repeatedly to what same sample to be tested was carried out Fixed, the result of gained is carried out the calculating of SD and CV, result is as follows:
Table 3
The precision of the test kit of the mensuration D-3-hydroxybutyric acid obtained by the present invention is relatively good as shown in Table 3, and can by table 1 Knowing, embodiment 3 is optimum selection.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any ripe Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage knowing this technology.Cause This, have usually intellectual such as complete with institute under technological thought without departing from disclosed spirit in art All equivalences become are modified or change, and must be contained by the claim of the present invention.

Claims (5)

1. the test kit measuring D-3-hydroxybutyric acid, it is characterised in that: include reagent R1 independent of each other and reagent R2 biliquid Body component composition test kit, including composition and corresponding content be:
Reagent R1:
Buffer 20 ~ 180mmol/L
D-3-HBD 0.1 ~ 4.9KU/L
Polyoxyethylene polyoxypropylene copolymer 0.1-6.0mL/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water
Reagent R2:
Oxalates 10 ~ 40mmol/L
NAD+ 0.1~4.9mmol/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water.
A kind of test kit measuring D-3-hydroxybutyric acid the most according to claim 1, it is characterised in that: include independent of each other Reagent R1 and reagent R2 biliquid component composition test kit, including composition and corresponding content be:
Reagent R1:
Buffer 100mmol/L
D-3-HBD 2.5KU/L
Polyoxyethylene polyoxypropylene copolymer 3.0mL/L
Preservative 0.035 mmol/L
Its solvent is purified water
Reagent R2:
Oxalates 25mmol/L
NAD+ 2.5mmol/L
Preservative 0.035 mmol/L
Its solvent is purified water.
A kind of test kit measuring D-3-hydroxybutyric acid the most according to claim 1 and 2, it is characterised in that: described reagent In R1, described buffer is selected from phosphate buffer, TRIS buffer, Isosorbide-5-Nitrae-piperazine two ethyl sulfonic acid buffering Liquid, 4-hydroxyethyl piperazine propane sulfonic acid buffer, 4-hydroxyethyl piperazine ethanesulfonic acid buffer, Pehanorm base propane sulfonic acid buffer Liquid, glycine buffer, borate buffer, 3-morpholine propane sulfonic acid buffer, 3-(N-morpholinyl)-2-hydroxypropyl sulfonic acid buffering The mixing of one or more arbitrary proportions in liquid, MES buffer, phosphate sodium dihydrogen buffer solution, described reagent In R1, described preservative is sodium azide, and in described reagent R2, described preservative is sodium azide.
A kind of test kit measuring D-3-hydroxybutyric acid the most according to claim 1 and 2, it is characterised in that: described reagent In R1, the pH value of described buffer is 6~9.
The preparation method of a kind of test kit measuring D-3-hydroxybutyric acid the most according to claim 1 and 2 and using method, its It is characterised by: comprise the following steps:
A () prepares reagent according to following component content:
Reagent R1:
Buffer 20 ~ 180mmol/L
D-3-HBD 0.1 ~ 4.9KU/L
Polyoxyethylene polyoxypropylene copolymer 0.1-6.0mL/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water
Reagent R2:
Oxalates 10 ~ 40mmol/L
NAD+ 0.1~4.9mmol/L
Preservative 0.01 ~ 0.059 mmol/L
Its solvent is purified water
B sample to be tested is mixed by () with reagent R1 and reagent R2 so that it is fully react;
C () measures reacted absorbance difference with automatic clinical chemistry analyzer;
D () calculates the concentration of D-3-hydroxybutyric acid in sample according to absorbance changing value.
CN201610273294.4A 2016-04-28 2016-04-28 Kit for detecting D-3-hydroxybutyric acid and preparation method of kit Pending CN105842437A (en)

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Application Number Priority Date Filing Date Title
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106896227A (en) * 2017-04-05 2017-06-27 济南蓄琪生物技术有限公司 A kind of myeloperoxidase enzyme detection kit
CN108680546A (en) * 2018-05-09 2018-10-19 盐城师范学院 A kind of kit for measuring 3-HBA

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1277264A (en) * 1999-05-28 2000-12-20 哈尔滨医科大学第一临床医学院 Reagent kit for enzyme process of determining beta-hydroxy butyric acid
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
CN104988207A (en) * 2015-07-11 2015-10-21 山东博科生物产业有限公司 Stable alpha-hydroxybutyrate dehydrogenase reagent with high interference resistance capacity and detection method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1277264A (en) * 1999-05-28 2000-12-20 哈尔滨医科大学第一临床医学院 Reagent kit for enzyme process of determining beta-hydroxy butyric acid
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
CN104988207A (en) * 2015-07-11 2015-10-21 山东博科生物产业有限公司 Stable alpha-hydroxybutyrate dehydrogenase reagent with high interference resistance capacity and detection method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106896227A (en) * 2017-04-05 2017-06-27 济南蓄琪生物技术有限公司 A kind of myeloperoxidase enzyme detection kit
CN108680546A (en) * 2018-05-09 2018-10-19 盐城师范学院 A kind of kit for measuring 3-HBA

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Application publication date: 20160810