CN104714040A - Method for determining glucose oxidase in serum by adopting double reagents - Google Patents

Method for determining glucose oxidase in serum by adopting double reagents Download PDF

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Publication number
CN104714040A
CN104714040A CN201510168446.XA CN201510168446A CN104714040A CN 104714040 A CN104714040 A CN 104714040A CN 201510168446 A CN201510168446 A CN 201510168446A CN 104714040 A CN104714040 A CN 104714040A
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reagent
glucose
serum
oxidase
double
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CN104714040B (en
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李立和
王彦平
常玉芝
李铮
丁弘
白会仓
郭秋红
闫瑞振
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Tianjin Baodi Hospital
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Tianjin Baodi Hospital
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    • 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
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • 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
    • 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.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Abstract

The invention discloses a method for determining glucose oxidase in serum by adopting double reagents, which belongs to a method for testing a material by utilizing visible light by virtue of the color variation of a test reaction result. According to the technical scheme, a double-reagent internal blank method is adopted for determination, and the interference reaction is inhibited by adopting 3-amino-1,2,4-triazole in the reagent I to inhibit the activity of the heme catalase; after the reagent II is added, the beta-D-type glucose is oxidized by glucose oxidase to generate hydrogen peroxide and D-gluconic acid, the hydrogen peroxide is condensed with N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt and 4-amino-antipyrine to form blue quinone imide to prepare blue quinone imide under the action of the peroxidase, and the detection is carried out by an instrument at the wavelength of 595nm to 610nm. When in detection, the interference of the heme peroxidase in the blood can be avoided. The method is a glucose detection method which is capable of eliminating the hemolysis endogenous interference and higher in accuracy.

Description

Glucose in serum oxidase double reagent assay method
Technical field
The invention belongs to a kind of assay method comprising enzyme; Or utilize visible ray, produce by the result of test reaction the method that color change carrys out test material, particularly relate to the double reagent assay method that a kind of Biochemical Analyzer detects glucose in serum.
Background technology
Clinical labororatory commonly uses GOD-PAP method and measures serum glucose, mensuration wavelength is 520nm, but this method be subject to piarhemia, jaundice blood, haemolysis interference and cause or high or low impact, when haemolysis sample adopts in the reaction of glucose oxidase method single reagent, the hydrogen peroxide that in protoheme, hydrogen peroxidase meeting decomposition glucose oxydasis glucose produces, thus make the red quinone imines of generation not become relation of equal quantity with glucose, and make glucose measurement on the low side; Piarhemia, jaundice, haemolysis are that 520nm produces larger absorbance at middle mensuration wavelength, thus interference glucose assays, the determination influences mainly spectral absorption to serum glucose such as piarhemia, jaundice, haemolysis; When piarhemia, jaundice, haemolysis serum index are larger, the sample limit of instrument designing can be exceeded, instrument can may impact result by alarm, the light absorption of assaying reaction is much smaller than the light absorption of serum index, measured signal " is flooded ", and the subtle change of light signal produces larger fluctuation to measurement result.
In order to overcome the interference of determination of glucose oxidase glucose, particularly haemolysis interference, present invention employs blank determination method in double reagent, uses 3-amino-1,2,4-triazole to suppress disturbance reponse by suppressing protoheme catalase activity; After adding reagent II, glucose oxidase β-D type glucose Hydrogen Peroxide and maltonic acid, hydrogen peroxide is under the effect of peroxidase, with N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS) and 4-AA are condensed into blue look quinone imines, instrument detects at 595-610nm wavelength place, and the protoheme peroxidase do not produced by molten blood hemoglobin when the present invention detects disturbs.Adopt N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt chromogen thing, owing to forming blue look quinone imines, it measures wavelength is 600nm, in the light absorption of the spectral absorption such as piarhemia, jaundice, haemolysis much smaller than the red quinone imines of 520nm, add the ratio of reaction signal and noise, improve the accuracy of mensuration, therefore, both reduce endogenous interference, having the interference eliminating serum index, is the glucose sensing approach that accuracy is higher.
This research adopts new amphyl N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3, 5-dimethoxyaniline sodium salt is the look source of Tinder reaction, chromogenic reaction highly sensitive, and the quinoneimine dye maximum absorption wavelength generated is the methods such as 600nm, haemolysis, jaundice, the interference that piarhemia etc. cause obviously reduces after using double reagent and new chromogen thing, every methodology index meets clinical practice application needs, there is selectivity good, highly sensitive, measure the features such as easy, be applicable to clinical application, the clinical labororatory possessing automatic clinical chemistry analyzer uses.
Summary of the invention
For solving protoheme hydrogen peroxidase interference problem and serum index interference problem in haemolysis sample, the present invention devises double reagent assay method, agent prescription and using method as follows:
Containing 3-amino-1,2,4-triazole 0.80mol, 4-AA 0.60mmol in reagent I: often liter 0.1mol/L phosphate buffer, mutarotase 2KU, Proclin-300 200 μ L.
Containing N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt 1.2mmol, ascorbic acid oxidase 30KU, glucose oxidase (POD) 20KU, peroxidase 15KU, Proclin-300 200 μ L in reagent II: often liter 0.1mol/L phosphate buffer.
Its assay method is: serum first bathes 3 ~ 5 minutes with reagent I in 37 DEG C of temperature, in glucose in serum and reagent I, mutarotase reacts, α type conversion of glucose is β-D type glucose, 3-amino-1,2,4-triazole suppresses protoheme catalase activity, suppresses disturbance reponse as reaction equation (1) (2); Bathe 4 ~ 7 minutes in 37 DEG C of temperature after adding reagent II, glucose oxidase β-D type glucose Hydrogen Peroxide and maltonic acid, hydrogen peroxide is under the effect of peroxidase, with N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt and 4-AA are condensed into blue look quinone imines as reaction equation (3) (4), instrument detects at 595-610nm wavelength place, react for blank with sample and reagent I, react by reagent II the content that the blue look quinone imines produced calculates glucose.Not by serum interference such as haemolysis, piarhemia, jaundice when the present invention detects, not by the impact of the protoheme peroxidase of molten blood hemoglobin generation, do not increase workload and reagent cost, economical convenient and easy, that one can eliminate endogenous interference, haemolysis, piarhemia, jaundice interference can be eliminated again, the glucose sensing approach that accuracy is higher.
The inventive method determination of glucose oxidase reaction formula:
The first step is reacted
Second step reacts
Accompanying drawing explanation
Accompanying drawing is the response curve figure that the present invention measures healthy human body glucose.
Embodiment:
Below by embodiment and accompanying drawing, the present invention is described in further details.
Embodiment 1
The composition of reagent:
A. reagent I: containing 3-amino-1,2,4-triazole 0.80mol, 4-AA 0.60mmol in often liter of 0.1mol/L phosphate buffer, mutarotase 2KU, Proclin-300 200 μ L;
B. reagent II: containing N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt 1.2mmol, ascorbic acid oxidase 30KU, glucose oxidase (POD) 20KU, peroxidase 15KU, Proclin-300 200 μ L in often liter of 0.1mol/L phosphate buffer.
C. titer: 5.55mmol/L D/W.
Wherein, 3-amino-1,2,4-triazole was the catalatic inhibitor of protoheme, and Proclin-300 is efficient liquid antiseptic.
Mensuration program
Double reagent method: on the full-automatic Biochemical Analyzer of Japanese OLYMPUS AU2700,2 μ l samples join in 150 μ l reagent I and mix by instrument automatically, hatch 3 minutes for 37 DEG C, add 50 μ l reagent II mixings, hatch 5.1 minutes for 37 DEG C, fully-automatic analyzer detects at 600nm wavelength place, and instrument calculates Glucose results automatically as Fig. 1, and response parameter is in table 1.
Table 1. robotization Biochemical Analyzer of the present invention test condition
Reaction OD gLUcalculated value=OD 2-OD 1× [(SV+R 1v 1)/(SV+R 1v 1+ R 2v 2)]
Concentration of glucose=F × OD gLU
Wherein OD gLUit is the absorbance that glucose produces; OD 1it is the absorbance recorded after sample adds reagent I reaction; OD 2it is the absorbance recorded after sample adds reagent II reaction; SV is the volume of blood serum sample, R 1v 1it is the volume of reagent I; R 2v 2it is the volume of reagent II; F is correction factor.
Add OD after reagent II 1extension rate be [(SV+R 1v 1)/(SV+R 1v 1+ R 2v 2)], the absorbance added after reagent II is OD 1× [(SV+R 1v 1)/(SV+R 1v 1+ R 2v 2)], so the absorbance of the quinone imines produced by glucose is: OD gLU=OD 2-OD 1× [(SV+R 1v 1)/(SV+R 1v 1+ R 2v 2)], i.e. OD gLU=OD 2– (2+150)/(2+150+50) × OD 1as long as measure OD 1and OD 2the concentration of glucose can be calculated.
Detect the glucose in serum below by the detection reagent that employing two kinds is different, further illustrate good effect of the present invention.
1. detected object: health examination personnel 68 people, the wherein male sex 52 people, 42.5 years old mean age; Women 34 people, 39.5 years old mean age, on an empty stomach blood sampling.
2. reagent
2.1 reagent:
(1) national clinical examination working specification recommend method recommends method preparation by Chinese Medical Association: reagent I: dissolve glucose oxidase 1200U, hydrogen peroxidase 12 00U, 4-AA 10mg in often liter of 0.1mmol (pH7.0) phosphate buffer; Reagent II: dissolve 4-chlorophenol 1g in often liter of 0.1mol/L phosphate buffer.Reagent I during mensuration: reagent II mixed in equal amounts.Sample: reagent=1: 150, reacts 8.1 minutes 520nm wavelength place end-point methods and measures by 37 DEG C.
(2) assay method reagent I of the present invention: containing 3-amino-1,2,4-triazole 0.80mol, 4-AA 0.60mmol in often liter of 0.1mol/L phosphate buffer, mutarotase 2KU, Proclin-300 200 μ L; Reagent II: containing N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt 1.2mmol, ascorbic acid oxidase 30KU, glucose oxidase (POD) 20KU, hydrogen peroxidase 15KU, Proclin-300 200 μ L in often liter of 0.1mol/L phosphate buffer.Sample: reagent I: reagent II=1: 75: 25.37 DEG C, sample and reagent I temperature bathe 3 minutes, add reagent II, react 5.1 minutes, and 600nm wavelength place end-point method measures.
2.2 titers: 5.55mmol/L D/W.
3. instrument: patients serum is divided into two groups by Japanese Olympus AU2700 type automatic clinical chemistry analyzer 4. method, normal serum group adopts biochemical tube blood sampling, and centrifuging measures, and haemolysis group adopts part freeze thawing erythrocyte hemolysis to measure.
3. Comparison of experiment results:
3.1 adopt national clinical examination working specification recommend methods and the inventive method to measure haemolysis group and normal serum group results contrast respectively as table 2:
Table 2 two kinds of glucose assays methods measure haemolysis group and non-haemolysis group blood sugar effects compares (mmol/L)
In table 2, the inventive method and recommend method when measuring normal group patients serum's glucose, measurement result there was no significant difference t=1.03, P>0.05; The inventive method and recommend method are when measuring haemolysis group patients serum's glucose, and measurement result has significant difference t=11.45, P<0.05, and measurement result is on the low side.When using glucose oxidase method measures haemolysis sample, not only be subject to catalatic interference in haemolysis, and be subject to the spectral absorption interference of serum color, and cause comparatively big error, the present invention is by applying blank in double reagent, adding protoheme hydrogen peroxide enzyme inhibitor, changing the methods such as chromogen thing color, add the ratio of reaction signal and noise, decrease disturbance reponse, significantly improve the accuracy of assay method.

Claims (6)

1. a glucose in serum oxidase double reagent assay method, it is characterized in that adopting blank method in double reagent to measure, its assay method is: serum first bathes 3 ~ 5 minutes with reagent I in 37 DEG C of temperature, bathe 4 ~ 7 minutes in 37 DEG C of temperature after adding reagent II, instrument detects at 595-610nm wavelength place, react for blank with sample and reagent I, react by reagent II the content that the blue look quinone imines produced calculates glucose, computing formula is:
OD GLU＝OD 2-OD 1×[(SV+R 1V 1)/(SV+R 1V 1+R 2V 2)]
Concentration of glucose=F × OD gLU
Wherein OD gLUthe absorbance that glucose produces, OD 1the absorbance recorded after sample adds reagent I reaction, OD 2be the absorbance recorded after sample adds reagent II reaction, SV is the volume of blood serum sample, R 1v 1the volume of reagent I, R 2v 2be the volume of reagent II, F is correction factor.
2. a glucose in serum oxidase double reagent assay method, it is characterized in that in reagent I: often liter 0.1mol/L phosphate buffer containing 3-amino-1,2,4-triazole 0.80mol, 4-AA 0.60mmol, mutarotase 2KU, Proclin-300 200 μ L; Containing N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt 1.2mmol, ascorbic acid oxidase 30KU, glucose oxidase 20KU, peroxidase 15KU, Proclin-300 200 μ L in reagent II: often liter 0.1mol/L phosphate buffer.
3. a glucose in serum oxidase double reagent assay method, is characterized in that in reagent I, 3-amino-1,2,4-triazole is protoheme hydrogen peroxide enzyme inhibitor.
4. a glucose in serum oxidase double reagent assay method, is characterized in that in reagent I, chromogen thing is 4-AA; In reagent II, chromogen thing is N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline sodium salts.
5. a glucose in serum oxidase double reagent assay method, is characterized in that the pH value of phosphate buffer in reagent I and reagent II is 7.0 ± 0.2.
6. a glucose in serum oxidase double reagent assay method, is characterized in that the volume ratio measured is: sample: reagent I: reagent II=1: 70 ~ 80: 20 ~ 30.
CN201510168446.XA 2015-04-06 2015-04-06 Glucose in serum oxidase double reagent assay method Expired - Fee Related CN104714040B (en)

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CN106319029A (en) * 2016-09-29 2017-01-11 四川迈克生物科技股份有限公司 Kit and method for testing glucose
CN106367472A (en) * 2016-09-29 2017-02-01 四川迈克生物科技股份有限公司 Kit and method for determining uric acid
CN107884401A (en) * 2017-11-13 2018-04-06 天津市宝坻区人民医院 Eliminate the glucose oxidase assay method of piarhemia interference
CN108007922A (en) * 2017-12-21 2018-05-08 广州市进德生物科技有限公司 A kind of kit using luminol chemiluminescence analysis detection glucose
CN108680569A (en) * 2018-04-11 2018-10-19 天津市宝坻区人民医院 Sucrose-determination method in serum

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106319029A (en) * 2016-09-29 2017-01-11 四川迈克生物科技股份有限公司 Kit and method for testing glucose
CN106367472A (en) * 2016-09-29 2017-02-01 四川迈克生物科技股份有限公司 Kit and method for determining uric acid
CN106319029B (en) * 2016-09-29 2019-01-15 迈克生物股份有限公司 For measuring the kit and method of glucose
CN106367472B (en) * 2016-09-29 2019-01-15 迈克生物股份有限公司 For measuring the kit and method of uric acid
CN107884401A (en) * 2017-11-13 2018-04-06 天津市宝坻区人民医院 Eliminate the glucose oxidase assay method of piarhemia interference
CN107884401B (en) * 2017-11-13 2020-03-24 天津市宝坻区人民医院 Glucose oxidase determination method for eliminating lipemia interference
CN108007922A (en) * 2017-12-21 2018-05-08 广州市进德生物科技有限公司 A kind of kit using luminol chemiluminescence analysis detection glucose
CN108007922B (en) * 2017-12-21 2018-11-13 广州市进德生物科技有限公司 A kind of kit detecting glucose using luminol chemiluminescence analysis
CN108680569A (en) * 2018-04-11 2018-10-19 天津市宝坻区人民医院 Sucrose-determination method in serum

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