CN104726536A - Stable reagent for determining magnesium ions by enzyme process - Google Patents
Stable reagent for determining magnesium ions by enzyme process Download PDFInfo
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- CN104726536A CN104726536A CN201310723541.2A CN201310723541A CN104726536A CN 104726536 A CN104726536 A CN 104726536A CN 201310723541 A CN201310723541 A CN 201310723541A CN 104726536 A CN104726536 A CN 104726536A
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- magnesium ion
- enzymatic assays
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Abstract
The invention provides a reagent for determining magnesium ions in a serum or plasma sample. The reagent is composed of 4 parts of reagent 1 and 1 part of reagent. The reagent 1 is composed of 0.01-1.5 mmol/L NAD+(H)/NADP+(H) derivative, 0.01-1.0 mmol/L buffer solution, 0.1-1000 mmol/L substrate, 0.1-100 mmol/L stabilizer and 0.1-10 mmol/L bacteriostatic agent. The reagent 2 is composed of 0.01-1.0 mmol/L buffer solution, 0.1-150 KU/L enzyme, 0.1-100 mmol/L stabilizer and 0.1-10 mmol/L bacteriostatic agent. The pH value of the reagent is 5.5-9.5. The reagent has the advantages of high stability, high determination accuracy and high cross contamination resistance, overcomes the defects in the prior art, can perform detection on a biochemical analysis meter, and has great application value.
Description
Technical field
The present invention relates to biological reagent, be specifically related to a kind of reagent and application thereof of stable enzymatic assays magnesium ion.
Background technology
At present, the method measuring blood plasma or Magnesium in Serum ion content is clinically a lot, mainly contain enzyme process, colorimetry, atomic absorption spectrophotometry (atomic-absorption-spectrophotometry, AAS), isotopic dilution-inductively coupled plasma mass spectrometry method (isotope dilution-inductively coupled plasma-mass spectrometry, ID-ICP-MS) etc.In the above-mentioned methods, method is ID-ICP-MS the most accurately, is secondly AAS, but the equipment that these two kinds of methods use is complicated, somewhat expensive, is difficult in the universal use of Routine Test Lab and clinical labororatory.Colorimetry is that current clinical labororatory is most widely used, it comprises titan yellow method, methyl thymol blue method (methyl thymol blue, MTB), the purple method (Xylylazo Violet) of Calmagite method, arsenazo I method, arsenazo Ⅲ method, xylene azoic etc.Colorimetry is simple to operate, expense is low, be applicable to Clinical Laboratory to use, but colorimetry can be subject to other cationic interference in various degree, and in order to increase the stability of reagent, highly toxic substance prussiate can be added in working reagent, have potential injury dangerous to operator.Colorimetry is according under alkaline environment (pH is greater than 11.0), and dyestuff and magnesium ion sequestration change color and set up, but can absorb the carbonic acid gas in air in the process that uses in uncork of reagent, makes the pH of reagent change and cause the instability of reagent.In addition, enzymatic assays blood plasma or Magnesium in Serum ion interference is little, result is accurate, dependency is good is adopted.The method that enzyme process detects magnesium ion mainly contains isocitric enzyme, pyruvate kinase coupling serum lactic dehydrogenase, glucokinase coupling glucose-6-phosphate dehydrogenase (G6PD), phosphoglucomutase coupling glucose-6-phosphate dehydrogenase (G6PD), glycerol kinase coupling phospho-glycerol enzyme and peroxidase etc., these methods all can use NADH or NADPH, indirectly calculate the concentration of magnesium ion by the change of absorbancy under 340nm wavelength.NADH and NADPH is only just relatively stable when pH value of water solution is greater than 9 under normal conditions, but reagent pH value is all lower in above-mentioned enzymatic assays, such as the reagent pH of pyruvate kinase coupling lactic dehydrogenase enzyme process is 7.4, and the reagent pH of glucokinase coupling glucose-6-phosphate dehydrogenase (G6PD) enzyme process is 7.8.Therefore, existing enzymatic assays magnesium ion reagent stability is also not enough to the needs meeting clinical assays sample, and the research that enzyme process detects magnesium ion also needs further improvement and bring new ideas.Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned weak point, the enzymatic assays magnesium ion reagent of research and design good stability.
The invention provides a kind of reagent of stable enzymatic assays magnesium ion.
Described reagent is made up of by 4 parts: 1 part reagent one and reagent two.
Described reagent one is by NAD
+(H)/NADP
+(H) derivative 0.01 ~ 1.5mmol/L, damping fluid 0.01 ~ 1.0mol/L, substrate 0.1 ~ 1000mmol/L, stablizer 0.1 ~ 100mmol/L and fungistat 0.1 ~ 10mmol/L form;
Described reagent two is by damping fluid 0.01 ~ 1.0mol/L, and enzyme 0.1 ~ 150KU/L, stablizer 0.1 ~ 100mmol/L and fungistat 0.1 ~ 10mmol/L form.
Reagent one and reagent two use according to the ratio that volume ratio is 4:1.
NAD described in reagent of the present invention
+(H)/NADP
+(H) derivative is 3-acetylpyridine-NADH, 3-acetylpyridine-NADPH, sulfo--NADH, sulfo--NADPH, 3-acetylpyridine-NAD
+, 3-acetylpyridine-NADP
+, sulfo--NAD
+or sulfo--NADP
+, preferred sulfo--NADH or sulfo--NAD
+.
Described damping fluid is Tutofusin tris-hcl buffer, N, N'-bis-(2-hydroxyethyl) glycine buffer, imidazoles-hcl buffer, N-bicine N-damping fluid or two (2-hydroxyethyl) imido grpup three (methylol) aminomethane buffer.
Described substrate is glycerine, glucose or pyruvic acid, preferred glucose.
Described enzyme is glycerol kinase, 3-phosphate dehydrogenase, hexokinase, glucokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase or serum lactic dehydrogenase, preferred glucokinase or glucose-6-phosphate dehydrogenase.
Described stablizer is that albumin, sphaeroprotein, sodium-chlor, thiocarbamide, methylthiourea, halfcystine, N-acetylcystein, gsh, mercaptoethanol, dithiothreitol (DTT), 2,2'-thiodiethanol acid, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), polyvinyl alcohol, polyoxyethylene glycol or polyoxyethylene glycol are to isooctyl phenyl ether.
Described fungistat is sodiumazide, 2-chlor(o)acetamide or ProClin300.
ProClin300 is the mixing solutions of MIT and CMIT, and ProClin300 is trade(brand)name.
The reagent pH of a kind of stable enzymatic assays magnesium ion of the present invention is within the scope of 5.5-9.5.
The application of the reagent of a kind of stable enzymatic assays magnesium ion of the present invention comprises the following steps:
Method one: blood plasma or serum sample and the solution containing glucose, adenosine triphosphate, oxidized coenzyme, glucokinase and glucose-6-phosphate dehydrogenase mix by (1), make it following reaction occurs:
(2), under reaction mixture being placed in ultraviolet/visible light spectrophotometer, the ascensional range of the absorbancy of predominant wavelength 398nm is detected;
Or under reaction mixture is placed in semi-automatic/automatic clinical chemistry analyzer, detects the ascensional range of the absorbancy of predominant wavelength 405nm, and then extrapolate the magnesium ion content in sample.
Method two: blood plasma or serum sample and the solution containing glycerine, adenosine triphosphate, oxidized coenzyme, glycerol kinase and glycerol-3-phosphate mix by (1), make it following reaction occurs:
(2), under reaction mixture being placed in ultraviolet/visible light spectrophotometer, the ascensional range of the absorbancy of predominant wavelength 363nm is detected;
Or under reaction mixture is placed in semi-automatic/automatic clinical chemistry analyzer, detects the ascensional range of the absorbancy of predominant wavelength 340nm, and then extrapolate the magnesium ion content in sample.
NADH is different with the maximum absorbance of coenzyme derivative, and the maximum absorption band of NADH/NADPH is 340nm, and the maximum absorption band of 3-acetylpyridine-NADH is 363nm, and the maximum absorption band of sulfo--NADH is 398nm.Although; automatic biochemistry analyzer there is no the light of 363nm and 398nm two wavelength; but all can there is the light of the 340nm wavelength for measuring NADH/NADPH; the light of this and 363nm wavelength closely, can detect with Biochemical Analyzer so measure reagent with the magnesium ion that 3-acetylpyridine-NADH prepares.Same, most automatic biochemistry analyzer all can have the light of 405nm wavelength, this light with 398nm wavelength closely, so the magnesium ion mensuration reagent using sulfo--NADH of the present invention to be mixed with also can use Biochemical Analyzer to detect.
The temperature of reacting in mensuration process controls at 20 ~ 50 DEG C, and on ultraviolet/visible light spectrophotometer, the reaction times controlled at 1 ~ 30 minute, and on Biochemical Analyzer time controling at 2 ~ 5 minutes.
The present invention's application magnesium ion activates the feature of kinase activity, when Triphosaden is excessive, the size of kinase activity is directly proportional with the concentration of magnesium ion in the blood plasma measured or serum sample, then by coupling desaturase by oxidized coenzyme sulfo--NAD (P)
+or 3-acetylpyridine-NAD (P)
+be reacted into reduced coenzyme sulfo--NAD (P) H or 3-acetylpyridine-NAD (P) H.Reducibility coenzyme has obviously absorption peak at 340nm or 405nm, and the change of the absorbancy produced before and after reaction is directly proportional to the content of magnesium ion in sample.Therefore, measured the change of 340nm or 405nm place absorbancy by Biochemical Analyzer, just indirectly can record the content of magnesium ion in sample.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, but is not limited only to this.
Embodiment uses raw material to be obtained by commercially available.
Embodiment 1:
Be configured to (1L) magnesium ion by following composition and measure reagent:
Reagent one (1L)
Tutofusin tris-hcl buffer 0.1mol/L, glucose 50mmol/L, bovine serum albumin 0.5%(w/v), thiocarbamide 15mmol/L, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) 0.13mmol/L, Triphosaden 1mmol/L, glucokinase 1000U/L, sodiumazide 15mmol/L, the pH value of reagent is 7.8.
Reagent two (1L)
Tutofusin tris-hcl buffer 0.1mol/L, bovine serum albumin 0.5%(w/v), polyoxyethylene glycol is to isooctyl phenyl ether 0.05%(w/v), sulfo--NAD (P)
+1mmol/L, glucose-6-phosphate dehydrogenase 4200U/L, sodiumazide 30mmol/L, the pH value of reagent is 7.8.
Hitachi 7100 automatic biochemistry analyzer sets: temperature of reaction is 37 DEG C, reagent one and sample are first hatched 5 minutes (37 DEG C), add reagent two afterwards and start reaction, reaction times is 5 minutes, test predominant wavelength is 405nm, reagent one and reagent two use according to the ratio that volume ratio is 4:1, volume ratio between determined sample and reagent is 1:30, the Direction of Reaction is positive reaction (reaction that absorbancy rises), analytical procedure is rate method, contrasts the content that corresponding typical curve calculates magnesium ion in sample.
Embodiment 2:
Be configured to magnesium ion by following composition and measure reagent:
Reagent one (1L)
Two (2-hydroxyethyl) imido grpup three (methylol) aminomethane buffer: Tutofusin tris-hcl buffer 0.1mol/L, glucose 50mmol/L, N-acetylcystein 15mmol/L, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA) 0.13mmol/L, sulfo--NAD (P)
+0.5mmol/L, Triphosaden 1mmol/L, sodiumazide 15mmol/L, the pH value of reagent is 8.0.
Reagent two (1L)
Two (2-hydroxyethyl) imido grpup three (methylol) aminomethane buffer: Tutofusin tris-hcl buffer 0.1mol/L, bovine serum albumin 0.5%(w/v), polyoxyethylene glycol is to isooctyl phenyl ether 0.05%(w/v), glucokinase 4500U/L, glucose-6-phosphate dehydrogenase 4200U/L, sodiumazide 30mmol/L, the pH value of reagent is 7.5.
Hitachi 7100 automatic biochemistry analyzer sets: temperature of reaction is 37 DEG C, reagent one and sample are first hatched 5 minutes (37 DEG C), add reagent two afterwards and start reaction, reaction times is 5 minutes, test predominant wavelength is 405nm, reagent one and reagent two use according to the ratio that volume ratio is 4:1, volume ratio between determined sample and reagent is 1:30, the Direction of Reaction is positive reaction (reaction that absorbancy rises), analytical procedure is rate method or end-point method, contrasts the content that corresponding typical curve calculates magnesium ion in sample.
The stability study of embodiment 3 reagent of the present invention
Carry out Performance Evaluation with reagent described in embodiment 1, calibration object is Roche c.f.a.s. caliberator, and lot number is 16951201.
Reagent of the present invention (embodiment 1) is measured with commercially available magnesium ion mensuration reagent (enzyme process) (purchased from Ningbo Pu Ruibai Bioisystech Co., Ltd) and magnesium ion the reagent trough interior (2 ~ 10 DEG C) that test kit-Mg (the blue colorimetry of xylidene(s)) (purchased from Beijing Leadman Biochemistry Technology Co., Ltd.) is placed on Hitachi 7100 automatic biochemistry analyzer, calibrate as calibration object with Roche c.f.a.s. caliberator, the outlier clinical chemistry measured from the multiple star Long March medical science and technology company limited in Shanghai controls serum (lot number is: 467UE), then Continuous Tracking measures this sample, until this sample data measured with the 0th day survey data relative deviation and exceed ± 10%, finally infer and obtain reagent stability number of days.In table 1, result shows that reagent of the present invention uncork can stablize 24 days, is better than commercially available similar-type products and magnesium ion mensuration reagent (xylidene(s) blue laws)
The uncork stability test result of table 1 this law
The accuracy studies of embodiment 4 reagent of the present invention
Reagent of the present invention (embodiment 1) is placed on the reagent trough interior (2 ~ 10 DEG C) of Hitachi 7100 automatic biochemistry analyzer, calibrate as calibration object with Roche c.f.a.s. caliberator, 16833101) and pathology value quality control product (lot number: 16841101) Roche Holding Ag normal value quality control product (lot number: is measured respectively, each replication 10 times, the results are shown in Table 2.
The accuracy determination result of table 2 reagent of the present invention
The precision research of embodiment 5 reagent of the present invention
Reagent of the present invention (embodiment 1) is placed on the reagent trough interior (2 ~ 10 DEG C) of Hitachi 7100 automatic biochemistry analyzer, calibrate as calibration object with Roche c.f.a.s. caliberator, 16833101) and PreciControl ClinChem Multi2(lot number Roche PreciControl ClinChem Multi1(lot number is measured respectively:: 16841101), each replication 20 times, calculate its average, standard deviation and the variation coefficient, the results are shown in Table 3.Day to day precision: respectively measured once morning and afternoon every day by above-mentioned Roche quality controlled serum, measures 10 days altogether, calculates its average, standard deviation and the variation coefficient, the results are shown in Table 4.
Batch interior replication result of table 3 reagent of the present invention
Above-mentioned experiment proves the method adopting mensuration magnesium ion content of the present invention, the magnesium ion content in blood plasma or serum sample can be determined completely by ultraviolet/visible light analyser or full-automatic/semi-automatic biochemical analyzer, test result is highly sensitive, precision good, uncork good stability, not by the pollution of interior allogenic material.Magnesium ion provided by the invention measures test kit, uncork good stability, and still accurately can detect the magnesium ion content in various sample after long storage time.
Claims (10)
1. a reagent for stable enzymatic assays magnesium ion, is characterized in that, described reagent is made up of by 4 parts: 1 part reagent one and reagent two;
Described reagent one is by NAD
+(H)/NADP
+(H) derivative 0.01 ~ 1.5mmol/L, damping fluid 0.01 ~ 1.0mol/L, substrate 0.1 ~ 1000mmol/L, stablizer 0.1 ~ 100mmol/L and fungistat 0.1 ~ 10mmol/L form;
Described reagent two is by damping fluid 0.01 ~ 1.0mol/L, and enzyme 0.1 ~ 150KU/L, stablizer 0.1 ~ 100mmol/L and fungistat 0.1 ~ 10mmol/L form; Described reagent pH is 5.5-9.5.
2. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, is characterized in that, described NAD
+(H)/NADP
+(H) derivative is 3-acetylpyridine-NADH, 3-acetylpyridine-NADPH, sulfo--NADH, sulfo--NADPH, 3-acetylpyridine-NAD
+, 3-acetylpyridine-NADP
+, sulfo--NAD
+or sulfo--NADP
+.
3. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 2, is characterized in that, described NAD
+(H)/NADP
+(H) derivative is sulfo--NADH or sulfo--NAD
+.
4. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described damping fluid is Tutofusin tris-hcl buffer, N, N'-bis-(2-hydroxyethyl) glycine buffer, imidazoles-hcl buffer, N-bicine N-damping fluid or two (2-hydroxyethyl) imido grpup three (methylol) aminomethane buffer.
5. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described substrate is glycerine, glucose or pyruvic acid.
6. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 5, it is characterized in that, described substrate is glucose.
7. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described enzyme is glycerol kinase, 3-phosphate dehydrogenase, hexokinase, glucokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase or serum lactic dehydrogenase.
8. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described enzyme is glucokinase or glucose-6-phosphate dehydrogenase.
9. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described stablizer is that albumin, sphaeroprotein, sodium-chlor, thiocarbamide, methylthiourea, halfcystine, N-acetylcystein, gsh, mercaptoethanol, dithiothreitol (DTT), 2,2'-thiodiethanol acid, ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA), polyvinyl alcohol, polyoxyethylene glycol or polyoxyethylene glycol are to isooctyl phenyl ether.
10. the reagent of a kind of stable enzymatic assays magnesium ion according to claim 1, it is characterized in that, described fungistat is sodiumazide, 2-chlor(o)acetamide or ProClin300.
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|---|---|---|---|
| CN201310723541.2A CN104726536A (en) | 2013-12-24 | 2013-12-24 | Stable reagent for determining magnesium ions by enzyme process |
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|---|---|---|---|
| CN201310723541.2A CN104726536A (en) | 2013-12-24 | 2013-12-24 | Stable reagent for determining magnesium ions by enzyme process |
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|---|---|
| CN104726536A true CN104726536A (en) | 2015-06-24 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278679A (en) * | 2021-04-28 | 2021-08-20 | 深圳市锦瑞生物科技有限公司 | Preparation method of magnesium determination reagent ball, reagent ball and microfluidic chip |
| CN114354524A (en) * | 2021-03-02 | 2022-04-15 | 北京九强生物技术股份有限公司 | Stable liquid detection kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778956A (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 |
| CN1778960A (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 |
| WO2009135035A1 (en) * | 2008-05-02 | 2009-11-05 | Specialty Assays, Inc. | Enzymatic determination of potassium ions using stable nad(p)h analogs. |
| WO2011013464A1 (en) * | 2009-07-28 | 2011-02-03 | 日東紡績株式会社 | Method and kit for measurement of dehydrogenase or substrate for the dehydrogenase |
-
2013
- 2013-12-24 CN CN201310723541.2A patent/CN104726536A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778956A (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 |
| CN1778960A (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 |
| WO2009135035A1 (en) * | 2008-05-02 | 2009-11-05 | Specialty Assays, Inc. | Enzymatic determination of potassium ions using stable nad(p)h analogs. |
| WO2011013464A1 (en) * | 2009-07-28 | 2011-02-03 | 日東紡績株式会社 | Method and kit for measurement of dehydrogenase or substrate for the dehydrogenase |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114354524A (en) * | 2021-03-02 | 2022-04-15 | 北京九强生物技术股份有限公司 | Stable liquid detection kit |
| CN113278679A (en) * | 2021-04-28 | 2021-08-20 | 深圳市锦瑞生物科技有限公司 | Preparation method of magnesium determination reagent ball, reagent ball and microfluidic chip |
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