CN105671127B - A kind of stable enzyme process serum Mg-ion detection kit - Google Patents
A kind of stable enzyme process serum Mg-ion detection kit Download PDFInfo
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- CN105671127B CN105671127B CN201610126969.2A CN201610126969A CN105671127B CN 105671127 B CN105671127 B CN 105671127B CN 201610126969 A CN201610126969 A CN 201610126969A CN 105671127 B CN105671127 B CN 105671127B
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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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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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
- C12Q1/26—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
Abstract
The present invention provides a kind of stable enzyme process serum Mg-ion detection kits; by using glycerine, D- glucopyranose, polyethylene glycol and glucan substance; a kind of membrane substance with stronger insulation blocking effect is formed by the combination of different proportion; insulation blocking enzyme is achieved the purpose that; after reagent R1 and R2 mixing, segregate enzyme is released, and then is reacted with substrate; reagent long term storage is achieved the purpose that allow, has solved the problems, such as that enzyme is unstable in reagent.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, it is related specifically to utilize the serum in enzyme process detection serum
The kit of magnesium ion content.
Background technique
Serum Mg-ion detection method is mainly include the following types: ion chromatography, enzyme process, colorimetric method, isotope method, fluorescence
Method etc..
The most authoritative detection method of serum Mg-ion is isotope method, but isotope method detection serum Mg-ion
Equipment is complex, and price costly, is not suitable for automatically analyzing.
Colorimetric method mainly includes titan yellow method, calmagite method, azo method and methyl thymol blue method.Relative to same position
Plain method, colorimetric method is cheap, easy to operate, operates convenient for routine experiment.But it is larger to apparatus damage, it is red to can not rule out gallbladder
The problem of element interference, stability is also very poor.
Ion-chromatographic determination serum Mg-ion, it is complicated for operation.It needs to be effectively treated sample early period, but handles
It just will appear result inaccuracy accidentally, it is larger by artifical influence factor, it is unsuitable for automated analysis.
Enzyme process detection serum Mg-ion is recently having very big breakthrough, also achieves rapid progress.Enzyme process
Testing result is more accurate, can be used for automatic analyzer, and compared to isotope method, enzyme process is cheap, can satisfy hospital
It examines and laboratory routine operation.But equally there is also some problems, it is to pass through enzymatic reaction that enzyme process, which detects serum Mg-ion,
It carries out, the absorbance change under certain wavelength is caused by substrate, calculates serum Mg-ion content indirectly.But enzyme process by
In wherein there is many enzymes to participate in biochemical reaction, stability is also the key of reagent success or failure, the enzyme process to circulate currently on the market
Serum Mg-ion checkout and diagnosis kit is not accomplished on the basis of guaranteeing enzyme activity, and reagent storage time, therefore reagent are extended
It is highly prone to the influence and destruction of temperature.
Summary of the invention
Against the above technical problems, the present invention provides a kind of stable enzyme process serum Mg-ion checkout and diagnosis kit,
The different enzymes in reagent are protected by certain protective agent, reduces or enzyme is inhibited to react, are guaranteeing enzyme activity
On the basis of, it extends the storage of reagent and using the time, kit of the present invention mainly solves enzyme process serum Mg-ion reagent
Unstable and anti-bilirubin interference problem.
Testing principle of the invention is as follows:
This method is that the variation of magnesium ion is detected by the variation of reduced coenzyme, by reaction principle it is seen that main
If magnesium ion activates hexokinase, reaction generates adenosine diphosphate, adenosine diphosphate and enol pyruvic acid and swashs in pyruvic acid
Pyruvic acid is generated under enzyme effect, pyruvic acid is aoxidized under ammonium ion and reduced coenzyme existence condition by alanine dehydrogenase
Effect generates pyruvic acid, l-Alanine and reduced coenzyme.Reducibility coenzyme has maximum light absorption value in 340nm wavelength, passes through prison
Absorbance change at 340nm is surveyed, to calculate the content that reduced coenzyme content indirectly calculates magnesium ion.
It is as follows using kit measurement Magnesium in Serum ion concentration method of the present invention: Detection wavelength 340nm, sample size:
Reagent R1: reagent R2=10ul:150ul:150ul.
The present invention is a kind of stable serum Mg-ion checkout and diagnosis kit, and kit of the present invention is by reagent R1 and examination
The liquid-type double reagent checkout and diagnosis reagent of two kinds of reagents of agent R2 composition, wherein reagent R1 is formed are as follows:
Glycine-HCI buffer 0.1mol/L
NADH 25mmol/L
Glucose 0.1-0.5g/L
Adenosine triphosphate 2mmol/L
Phosphoenolpyruvate 2mmol/L
CTAB 5mmol/L
Chloroform 0.1ml/L
Glycerine 0.1-0.75ml/L
Polyethylene glycol 0.3-0.5g/L
Glucan 1-2.34g/L
D- glucopyranose 0.05-0.1g/L；
Reagent R2 composition are as follows:
TOOS buffer 0.1mol/L
Alanine dehydrogenase 1000-5000U/L
Hexokinase 2 500-4000U/L
Pyruvate kinase 3000U/L
Polyethylene glycol 1-1.5g/L
Glucan 0.7-1.5g/L
D- glucopyranose 0.25-0.5g/L.
In stable serum Mg-ion monitoring, diagnosing kit provided by the invention, glycine-HCI buffer is mainly
Buffer function guarantees the stabilization of external environment when reaction, testing result is caused not will receive fluctuation and influence.NADH reduced coenzyme
Various forms of coenzyme are generated in enzymatic reaction, cause absorbance change.Glucose, adenosine triphosphate and phosphoenolpyruvate
Pyruvic acid is used as reaction substrate, participates in enzymatic reaction.CTAB cetyl trimethylammonium bromide is as reaction cosolvent, acceleration
Agent, guarantee is good coordinating, provides ammonium ion.Chloroform is for removing bilirubin caused interference when detecting.The third three
Alcohol, polyethylene glycol, glucan and D- glucopyranose combine to form certain protective film by different ratio, under certain condition
Enzyme is wrapped up, reaction is inhibited, but under the conditions of working as a certain existing for chaotropic agent, it is anti-that the enzyme r e lease of package is come out into participation
It answers.TOOS buffer is mainly buffer function, guarantees the stabilization of external environment when reaction, cause testing result not and will receive fluctuation and
It influences.Alanine dehydrogenase acts on the reaction of coarse language pyruvate dehydrogenase and generates alanine.Hexokinase is catalyzed glucose and generates grape
Sugar -6-P.Pyruvate kinase is catalyzed enol pyruvic acid and generates pyruvic acid.
It is automated in the full-automatic analyzer for the series such as auspicious, Mai Rui, Hitachi, Toshiba that present invention can apply to enlightening
Detection.
Using the present invention carry out in vitro sample diagnosis detection when, operating method is as follows, setting wavelength be 340nm, end-point method,
Sample size: reagent R1: reagent R2=10ul:150ul:150ul.Specimen needle is incubated in reagent R1 after drawing sample, to protect
The stabilization of reaction environment external condition is demonstrate,proved, then reading absorption photometric value is Δ A1, and reaction certain time reads after reagent R2 is added
Take Δ A2.Serum Mg-ion content=(Δ A2- Δ A1) standard substance concentration.
It is compared with the enzyme process serum Mg-ion detection kit that market is circulated at present, stability of the present invention is good, anti-bilirubin
Ability is strong, reproducible, easy to operate, convenient for storage and transport.Suitable for automatic clinical chemistry analyzer, clinical use value compared with
Greatly.
Detailed description of the invention
Fig. 1 is contrast agents box property figure related to the present invention.
Specific embodiment
We will in conjunction with chart, examples illustrate the present invention.
Embodiment 1
The present invention in specific implementation, the reagent R1 are as follows:
Glycine-HCI buffer 0.1mol/L
NADH 25m mol/L
Glucose 0.5g/L
Adenosine triphosphate 2mmol/L
Phosphoenolpyruvate 2mmol/L
CTAB 5mmol/L
Chloroform 0.1ml/L
Glycerine 0.75ml/L
Polyethylene glycol 0.5g/L
Glucan 2.34g/L
D- glucopyranose 0.1g/L；
Reagent R2 composition are as follows:
TOOS buffer 0.1mol/L
Alanine dehydrogenase 5000U/L
Hexokinase 2 500U/L
Pyruvate kinase 3000U/L
1.5 g/L of polyethylene glycol
0.7 g/L of glucan
0.5 g/L of D- glucopyranose
Embodiment 2
The present invention in specific implementation, the reagent R1 are as follows:
Glycine-HCI buffer 0.1mol/L
NADH 25m mol/L
0.25 g/L of glucose
Adenosine triphosphate 2m mol/L
Phosphoenolpyruvate 2m mol/L
CTAB 10m mol/L
0.1 ml/L of chloroform
1 ml/L of glycerine
0.75 g/L of polyethylene glycol
1.75 g/L of glucan
0.2 g/L of D- glucopyranose
Reagent R2 composition are as follows:
TOOS buffer 0.1mol/L
Alanine dehydrogenase 5000U/L
Hexokinase 2 500U/L
Pyruvate kinase 3000U/L
1.5 g/L of polyethylene glycol
0.8 g/L of glucan
0.2 g/L of D- glucopyranose
Embodiment 3
The present invention in specific implementation, the reagent R1 are as follows:
Glycine-HCI buffer 0.1mol/L
NADH 25m mol/L
0.1 g/L of glucose
Adenosine triphosphate 2m mol/L
Phosphoenolpyruvate 2m mol/L
CTAB 5m mol/L
0.1 ml/L of chloroform
0.55 ml/L of glycerine
0.25 g/L of polyethylene glycol
2 g/L of glucan
0.1 g/L of D- glucopyranose
Reagent R2 composition are as follows:
TOOS buffer 0.1mol/L
Alanine dehydrogenase 5000U/L
Hexokinase 2 500U/L
Pyruvate kinase 3000U/L
2.5 g/L of polyethylene glycol
1 g/L of glucan
0.5 g/L of D- glucopyranose
Kit performance detection test of the present invention:
Stability test detection:
The kit to circulate with kit of the invention real (formula of embodiment 1 is prepared) and currently on the market compares,
Calibration solution and quality-control product are Landau 926UN and 1005UN respectively, the use of instrument are automatic clinical chemistry analyzer BS-800, and parameter is set
As follows, wavelength 340nm is set, the Direction of Reaction is opposite direction, and reading point is set as 14-16 point and 31-33 point, sample size: reagent
R1: reagent R2=10ul:150ul:150ul, instrument BS-800.Testing result is as shown in table 1:
The reagent of the present invention of table 1 and contrast agents Detection of Stability result
Remarks: quality-control product 1005UN target value is 0.89m mol/L
By this experiment, it has been found that kit of the present invention is appointed after lasting 4 months under the conditions of 4 DEG C almost without generation
What changes, but contrast agents have reduced 10% at the 60th day under the conditions of 4 degree DEG C, should be 30-60 days and do not count in detail
According to cannot extrapolate contrast agents box is when to start decaying after 30 days；We have found that reagent of the present invention under the conditions of 37 DEG C
Box contrast agents box is after there is a decaying after 120 days, but kit of the present invention decaying, less than 5%, contrast agents box is but more than
10%；Kit of the present invention was decayed at the 30th day under the conditions of 45 degree has been over 10%, and contrast agents box was decayed at the 9th day
It has been more than 10%.Testing result under comprehensive different temperatures, it has been found that kit of the present invention and the reagent to circulate at present in the market
Box has very strong superiority, this kit of the present invention, which puts goods on the market, has established certain basis.
Bilirubin interference experiment
Enzyme process serum Mg-ion detection kit is highly prone to bilirubin influence, causes result error.It is red to buy sterling gallbladder
Element is configured to the bilirubin sample containing various concentration with 1005UN quality-control product.Maximum concentration is 200uM, remaining is followed successively by
150uM, 100 uM, 75 uM, 50uM, 25 uM and 0 uM stay a gradient.Using kit of the present invention (formula of embodiment 2 is prepared)
With contrast agents box, both detections concentration, 1005UN quality-control product target value are 0.89 mM respectively.Detecting instrument is BS-800, instrument
Parameter setting and operation are not repeating.Testing result is as shown in table 2:
The kit of the present invention of table 2 and contrast agents box interference--free experiments result
Remarks: 100UN quality-control product target value is 0.89m M
By table 2 we it is not difficult to find that the serum magnesium enzyme process detection kit to circulate currently on the market, in content of bilirubin
When 50uM, testing result is partial to target value 20% or so, when bilirubin be up to 150uM it is even higher when, it has been found that contrast agents box
It can not be used to detect, monitoring result is not only to affect clinical judgment, and be easy to mislead doctor to patient's
The state of an illness makes erroneous diagnosis.Testing result will not be generated when sample content of bilirubin is 150uM compared to two speech kits of the present invention
Any erroneous judgement, but when bilirubin concentration is 200uM Lower result 10% or so.People's content of bilirubin generally in 0-20uM or so,
But work as liver, when there is lesion, content of bilirubin is horizontal much higher than this, and contrast agents cannot accurately measure such disease
The serum Mg-ion content of people, but kit of the present invention can be competent at this work completely.
Clinical sample detection test
Kit of the present invention is tested with contrast agent box with into N=40 sample, and test result is in table form
It is embodied, the numerical value recorded by the table 3, and then is calculated, the reagent of kit of the present invention and market circulation in following table
The correlation of box.Kit of the present invention is detected using the formula of embodiment 3.Testing result is as shown in table 3:
The kit of the present invention of table 3 and contrast agents box clinical sample testing result
Contrast agents box and kit clinical correlation map of the present invention are as shown in Figure 1.Illustrate this hair in conjunction with table 3 and Fig. 1
Bright kit has good correlation with contrast agents box when detecting General Clinical sample, correlation coefficient r=0.9995.
In conclusion kit of the present invention has passed through clinical verification, when detecting 40 clinical samples, contrast agents box with
Kit correlation of the present invention is higher, but kit of the present invention is much better than contrast agents box in terms of stability, for the present invention
Kit, which puts goods on the market, lays a good foundation.On treating bilirubin jamming performance, a kind of stable enzyme process serum gallbladder of the present invention is red
Plain detection kit is able to detect the sample of content 150uM bilirubin, and guarantees the accuracy of result.Kit of the present invention is steady
Qualitative good, cost is relatively low, will not generate corrosion impact to instrument, and easy to operate, to environment almost without pollution, feasibility is strong,
Economic and social benefit is huge, for later developing target market, expands occupation rate of market and provides strong technical support and science branch
It holds.
Claims (1)
1. a kind of stable enzyme process serum Mg-ion detection kit, which is characterized in that including equivalent reagent R1 and reagent R2,
Middle reagent R1 composition are as follows:
Glycine-HCI buffer 0.1mol/L
NADH 25mmol/L
Glucose 0.1-0.5g/L
Adenosine triphosphate 2mmol/L
Phosphoenolpyruvate 2mmol/L
CTAB 5mmol/L
Chloroform 0.1ml/L
Glycerine 0.1-0.75ml/L
Polyethylene glycol 0.3-0.5g/L
Glucan 1-2.34g/L
D- glucopyranose 0.05-0.1g/L；
Reagent R2 composition are as follows:
TOOS buffer 0.1mol/L
Alanine dehydrogenase 1000-5000U/L
Hexokinase 2 500-4000U/L
Pyruvate kinase 3000U/L
Polyethylene glycol 1-1.5g/L
Glucan 0.7-1.5g/L
D- glucopyranose 0.25-0.5g/L.
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| CN201610126969.2A CN105671127B (en) | 2016-03-07 | 2016-03-07 | A kind of stable enzyme process serum Mg-ion detection kit |
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| CN201610126969.2A CN105671127B (en) | 2016-03-07 | 2016-03-07 | A kind of stable enzyme process serum Mg-ion detection kit |
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| CN105671127A CN105671127A (en) | 2016-06-15 |
| CN105671127B true CN105671127B (en) | 2019-01-04 |
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| CN111118137A (en) * | 2018-11-01 | 2020-05-08 | 广东中科康仪生物技术有限公司 | Gene detection method and kit for thrombocytasthenia |
| CN113528499A (en) * | 2021-05-29 | 2021-10-22 | 北京华宇亿康生物工程技术有限公司 | Buffer system capable of stably storing Hexokinase (HK) |
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| CN1778957A (en) * | 2004-11-23 | 2006-05-31 | 苏州艾杰生物科技有限公司 | Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit |
| CN101464375A (en) * | 2007-12-19 | 2009-06-24 | 苏州艾杰生物科技有限公司 | Magnesium (ion) diagnosis/measuring reagent kit and magnesium (ion) concentration determination method |
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| CN104459164A (en) * | 2014-11-28 | 2015-03-25 | 山东博科生物产业有限公司 | Serum creatinine detecting reagent |
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2016
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| CN1227920A (en) * | 1998-12-09 | 1999-09-08 | 天津市医药科学研究所 | Reagent for testing serum Mg-ion with enzyme |
| CN1778957A (en) * | 2004-11-23 | 2006-05-31 | 苏州艾杰生物科技有限公司 | Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit |
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