CN1778956A - Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit - Google Patents
Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit Download PDFInfo
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- CN1778956A CN1778956A CN 200410065549 CN200410065549A CN1778956A CN 1778956 A CN1778956 A CN 1778956A CN 200410065549 CN200410065549 CN 200410065549 CN 200410065549 A CN200410065549 A CN 200410065549A CN 1778956 A CN1778956 A CN 1778956A
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Abstract
The invention is about the enzyme method of measuring magnesium ion and its diagnosis reagent box. Making use of the peculiarity that magnesium ion in the sample of plasma or serum and so on can activate the activation of hexokinase, and producing glucose-6-phosphate by reacting glucose under the existence of adenosine triphosphate, and then causing coupled reaction with phosphoglucose dehydrogenase and transferring oxidized coenzyme to reduced coenzyme. Testing the ascending range of dominant wave-length340nm absorbance and finally measuring the content of magnesium ion in the sample. This method has high specificity and would not be contaminated by material of internal and exogenous sources, and the result is precise and accurate. Diving the diagnosis reagent box into double-dose or three-dose can reduces the cross interaction of each element, keeps the stability of the reagent and deposits chronically. Using this method can realize the fast testing in common ultraviolet/ visible light analyzer or semiautomatic/automatic analyzer and doesn't require special or additional apparatus, so the cost is low. Thus, this method can be easily promoted and applied in the whole industry.
Description
Technical field
The present invention relates to the method for magnesium ion content in the samples such as enzymatic assays blood plasma, serum, and use the formulated magnesium ion diagnostic kit of this method, belong to medical test determination techniques field.
Background technology
The means of medically measuring magnesium ion content (abbreviation magnesium determination) in the samples such as blood plasma, serum at present are many, summarize to get up to have: colorimetry, fluorescent method, ion chromatography, ion specific electrode method (ISE), enzyme process, atomic absorption spectrophotometry (AAS), isotopic dilution mass spectrometry (ID-MS) etc.Wherein conclusive method is ID-MS, secondly is AAS, though these two kinds of method results are accurate, equipment complexity, expense costliness are not suitable for Routine Test Lab and analysis automatically.
Colorimetry is most widely used method, comprising: methyl thymol blue method (MTB), titan yellow method, OCPC method (OCPC), calmagite method (Calmagite) and 2-(8 '-hydroxyquinoline-5 '-sulfonic acid-7 '-azo)-variable color acid system (8Q5SAC) of reporting recently.Colorimetry is easy and simple to handle, expense is low, is fit to Routine Test Lab and uses, but have reagent blank absorbancy height, be subject to bilirubin and other positively charged ion disturbs, reagent stability is poor, and contain in the reagent and be corrosive or shortcoming such as toxic component.Ion specific electrode method (ISE) can be measured magnesium ion activity in the physiological solution, adopts neutral carrier (ETH7025) liquid film ion selective electrode, and the accuracy of mensuration is higher, but still exists the calcium in the physiological range to disturb Ca
2+Maximum interference reaches 10%.Ion chromatography needed sample is carried out pre-treatment before measuring, and comprised acidifying dilution and filtration.This method of uses such as Thienpont is analyzed with the definite value serum of atomic absorption spectrophotometry five kinds " international standard and technology meetings ", average deviation is 0.35% only as a result, thinks that ion chromatography can be used as the valuable reference method of measuring the magnesium elements total amount.
Magnesium ion (mainly is Mg in the samples such as enzymatic assays blood plasma, serum
2+) research very active in recent years, obtained bigger progress.Be applied to Mg at present
2+The Enzymology method of measuring has: 1. hexokinase and glucose-6-phosphate dehydrogenase coupling method; 2. glycerol kinase, GPO and superoxide enzyme coupling method; 3. glucokinase and glucose-6-phosphate dehydrogenase coupling method; 4. enzyme joins chemoluminescence method; 5. phosphoglucomutase and glucose-6-phosphate dehydrogenase coupling method; 6. isocitric enzyme method; 7. pyruvate kinase and lactic dehydrogenase enzyme coupling method.
On the whole, employing enzyme process detection magnesium ion result is accurate, interference is little, be suitable for automated analysis.But, use different detection reagent and test route, the sensitivity of detection, the accuracy of detected result have very big difference, directly have influence on the use and the popularization of this method, and this is the people reason place constantly studying in the field, develop and innovate just.Because extraordinary application prospect arranged, the research that enzyme process is detected magnesium ion has become the focus in this field.
Summary of the invention
The objective of the invention is: a kind of method of enzymatic assays magnesium ion content is provided, and uses the formulated magnesium ion diagnostic kit of this method.Adopt the reagent in this test kit, can utilize ultraviolet analyser or semi-automatic/automatic clinical chemistry analyzer that the content of magnesium ion in the samples such as blood plasma, serum is carried out fast, accurately measures, so that promote the use of method and corresponding diagnostic kit that enzyme process detects magnesium ion, make this field in detection means obtain further abundant and develop.
For realizing purpose of the present invention, a kind of method of enzymatic assays magnesium ion content, adopt following steps to carry out:
At first,, make it to take place following reaction with needs such as blood plasma, serum sample of measuring and the reagent mixing that contains glucose, adenosine triphosphate, hexokinase, G6P-DH and oxidized coenzyme,
(wherein " 6-phosphogluconolactone " is 6-phospho-D-gluconolactone)
Then, reaction mixture is placed under ultraviolet analyser or the semi-automatic/automatic clinical chemistry analyzer, detect the ascensional range of the absorbancy of predominant wavelength 340nm, and then calculate the content of magnesium ion in the sample.
In the mensuration process, the usage ratio of sample and reagent by volume was controlled at 1: 10～1: 500, and temperature of reaction is controlled at 20 ℃～50 ℃, and the reaction times was controlled at 2～30 minutes, set commplementary wave length during detection more than 405nm.
The magnesium ion diagnostic kit of realizing the inventive method can be single agent, is grouped into by following one-tenth:
Damping fluid 40～200mmol/l,
Glucose 1～40mmol/l,
Adenosine triphosphate 0.2～20mmol/l,
Oxidized coenzyme 0.2～5mmol/l,
Hexokinase 4000～50000U/l,
G6P-DH 4000～50000U/l,
Stablizer/reagent cumulative volume 10～80%.
Also the various compositions in above single agent can be carried out formulated in combination and become two agent, be beneficial to eliminate the pollution of inside and outside source G-6-P, such as:
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: oxidized coenzyme, G6P-DH etc. can be placed on reagent II; Composition among the reagent II: glucose, adenosine triphosphate, hexokinase etc., also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.
Reagent can also be made into following three reagent, not only more help eliminating the pollution of inside and outside source G-6-P, it is more stable to also help reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the oxidized coenzyme among the reagent I can be placed among reagent II or the reagent III, G6P-DH among the reagent II can be placed among reagent I or the reagent III, glucose among the reagent III, adenosine triphosphate, hexokinase also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.
In the middle of the composition of above-mentioned diagnostic kit, selecting the basic demand of damping fluid is that pH value is within 6.0～11.0 scopes, can be: " Tutofusin tris～hydrochloric acid (Tris-HCl) damping fluid ", " trolamine (Triethanolamine) damping fluid ", " 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid ", " imidazoles～hydrochloric acid (Imidazole-HCl) damping fluid ", " glycylglycine (Glycylglycine) damping fluid ", " citric acid～sodium citrate buffer solution ", " Veronal sodium～hydrochloride buffer ", " boric acid～borate buffer solution ", " glycine～sodium hydrate buffer solution ", " borax～sodium hydrate buffer solution ", at least a in " phosphoric acid (Phosphate) damping fluid " or " phosphoric acid salt (Phosphate-Sodium Chloride; PBS) damping fluid ", but range of choice be not subjected to these enumerate limit.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, its consumption accounts for 10～80% (perhaps concentration is within 10～50mmol/l scopes) of place reagent cumulative volume.
Material with used as stabilizers can be: at least a in ethylene glycol, propylene glycol, glycerine, ammonium sulfate, thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glutaminate, reduced glutathion, lactose, N.F,USP MANNITOL, succinate or the sodium-chlor.
In the middle of the reagent composition of above-mentioned magnesium ion diagnostic kit, described oxidized coenzyme is---NAD (NAD
+), NADP+ (NADP
+), thio-NAD+ (thio-NAD
+) or thio-NADP+ (thio-NADP
+); Described reduced coenzyme is---nicotinamide adenine dinucleotide reduced (NADH), NADPH (NADPH), Thio-NADH (thio-NADH) or thio-NADPH (thio-NADPH).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent, two agent or three doses, and the magnesium ion diagnostic kit of following system component relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 80～120mmol/l,
Glucose 10～20mmol/l,
Adenosine triphosphate 2～10mmol/l,
Oxidized coenzyme 1～5mmol/l,
Hexokinase 1 0000～30000U/l,
G6P-DH 10000～30000U/l,
Stablizer/reagent cumulative volume 20～50%.
The present invention uses the characteristics that magnesium ion activates hexokinase activity, the degree that hexokinase activity intensifies is directly proportional with the content of magnesium ion in the samples such as blood plasma, serum, in the presence of adenosine triphosphate, generate G-6-P with glucose response, the coupling G6P-DH is reacted into reduced coenzyme with oxidized coenzyme again.Because reduced coenzyme has very significantly absorption peak at 340nm, and the oxidized coenzyme of their correspondences does not have absorption peak at 340nm, the variation of the absorbancy that reaction is produced is directly proportional with the content of magnesium ion in the sample.Therefore, in Fixed Time Interval, measure the ascensional range of predominant wavelength 340nm place absorbancy, just can reflect the content of magnesium ion in the sample well.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes Enzymology method fully, and enzyme digestion reaction has the high characteristics of specificity, and by oxidized coenzyme is reacted into reduced coenzyme, quantitative response goes out the content of magnesium ion in the sample, and test result is accurate;
(2) composition of participation enzyme linked reaction all adds, and is not subjected to the pollution of inside and outside source material, test process tolerance range height;
(3) this method is easy, easy to operate, can obtain detected result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just rapid detection on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that measuring method provided by the invention can be made various ways such as liquid reagent, powdered reagent, dried reagent, be mainly used in the content of measuring magnesium ion in human body and other animal body, also can be, supplementary means such as concentrate and measure magnesium ion in other sample in conjunction with dilution;
(6) liquid magnesium ion diagnostic kit provided by the invention, good stability, the three-D space structure that is present in wherein each kind of enzyme is kept perfectly, and has guaranteed the application testing effect well.Make after two agent or three doses, can further reduce the cross influence between the various compositions, detected result is more credible, and reagent is more stable, can store for a long time.
Embodiment
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare magnesium ion diagnostic kit in the serum by following composition and consumption:
Glycylglycine damping fluid 80mmol/l,
Glucose 10mmol/l,
Adenosine triphosphate 2mmol/l,
thio-NAD
+ 1mmol/l，
Hexokinase 1 0000U/l,
G6P-DH 10000U/l,
Ethylene glycol 50% (accounting for the reagent cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of sample and reagent is 1: 25, the Direction of Reaction is positive reaction (absorbancy rises, down together).
Adding serum sample and the single agent that is made into, both detect, write down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.Detect altogether and read a little 31 times, approximately every 20 seconds record one secondary data, according to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of magnesium ion in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment two (two agent)
Prepare magnesium ion diagnostic kit in the serum by following composition and consumption:
Reagent I---
Imidazoles～hydrochloride buffer 100mmol/l,
Glucose 15mmol/l,
Adenosine triphosphate 6mmol/l,
NADP
+ 3mmol/l，
G6P-DH 20000U/l,
Glycerine 50% (accounting for reagent I cumulative volume);
Reagent II---
Imidazoles～hydrochloride buffer 100mmol/l,
Hexokinase 2 0000U/l,
Ethylene glycol 50% (accounting for reagent II cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 30 ℃ of temperature, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I and reagent II, reagent I is 4: 1 with the ratio of the consumption of reagent II, and the Direction of Reaction is positive reaction.
Adding serum sample and reagent I add reagent II after 5 minutes earlier, and the three detects, writes down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of magnesium ion in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment three (three doses)
Prepare magnesium ion diagnostic kit in the serum by following composition and consumption:
Reagent I---
Trolamine damping fluid 120mmol/l,
Glucose 20mmol/l,
Adenosine triphosphate 10mmol/l,
thio-NADP
+ 5mmol/l，
Propylene glycol 20mmol/l;
Reagent II---
Trolamine damping fluid 120mmol/l,
G6P-DH 30000U/l,
Propylene glycol 50% (accounting for reagent II cumulative volume);
Reagent III---
Trolamine damping fluid 120mmol/l,
Hexokinase 30000U/l,
Ethylene glycol 50% (accounting for reagent III cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I, reagent II and reagent III, the consumption of reagent I, reagent II and reagent III is 8: 1: 1, and the Direction of Reaction is positive reaction.
Adding serum sample and reagent I, reagent II add reagent III after 5 minutes earlier, and they detect, write down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of magnesium ion in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment four (two agent preference)
Prepare the serium inorganic phosphorus diagnostic kit by following composition and consumption:
Reagent I---
Tris-HCl damping fluid 100mmol/l,
NAD
+ 4mmol/l，
G6P-DH 15000U/l,
Glycerine 50% (accounting for reagent I cumulative volume);
Reagent II---
Tris-HCl damping fluid 100mmol/l,
Glucose 20mmol/l,
Adenosine triphosphate 5mmol/l,
Hexokinase 1 5000U/l,
Glycerine 50% (accounting for reagent II cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I and reagent II, reagent I is 4: 1 with the ratio of the consumption of reagent II, and the Direction of Reaction is positive reaction.
Add serum sample and reagent I earlier, add reagent II after 5 minutes, following principle reaction takes place at the inner mixing automatically of analyser in the three:
Detect, write down the rising situation of 340nm absorbancy.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of magnesium ion in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
One of striking features of the present invention is the pollution that can eliminate inside and outside source G-6-P.The effect of eliminating inside and outside source G-6-P occurs in the first half of entire reaction time period, in time second half section, contaminated inside and outside source G-6-P is consumed totally, and the needed G-6-P of second half section time detecting all is to be derived from the magnesium ion that contains in the sample.
In a word, experimental results show that and adopt measuring method of the present invention, can pass through ultraviolet analyser or semi-automatic/automatic clinical chemistry analyzer device fully, determine the content of magnesium ion in the samples such as blood plasma, serum, measurement sensitivity height, tolerance range are good, are not subjected to the pollution of inside and outside source material.And, magnesium ion diagnostic kit provided by the invention, good stability still can accurately detect the magnesium ion content in all kinds sample after the long storage time.
Claims (9)
1. the method for an enzymatic assays magnesium ion content may further comprise the steps:
1. with testing sample and the reagent mixing that contains glucose, adenosine triphosphate, hexokinase, G6P-DH and oxidized coenzyme, make it to take place following reaction,
2. reaction mixture is placed under ultraviolet analyser or the semi-automatic/automatic clinical chemistry analyzer, detect the ascensional range of the absorbancy of predominant wavelength 340nm, calculate the content of magnesium ion in the sample.
2. the method for enzymatic assays magnesium ion content according to claim 1 is characterized in that: the usage ratio of testing sample and reagent by volume was controlled at 1: 10～1: 500.
3. the method for enzymatic assays magnesium ion content according to claim 1 and 2 is characterized in that: temperature of reaction is controlled at 20 ℃～50 ℃, and the reaction times was controlled at 2～30 minutes, sets commplementary wave length during detection more than 405nm.
4. magnesium ion diagnostic kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 40～200mmol/l,
Glucose 1～40mmol/l,
Adenosine triphosphate 0.2～20mmol/l,
Oxidized coenzyme 0.2～5mmol/l,
Hexokinase 4000～50000U/l,
G6P-DH 4000～50000U/l,
Stablizer/reagent cumulative volume 10～80%.
5. magnesium ion diagnostic kit according to claim 4 is characterized in that: the PH scope of described damping fluid is 6.0～11.0.
6. magnesium ion diagnostic kit according to claim 5 is characterized in that: described damping fluid is " Tutofusin tris～hydrochloride buffer ", " trolamine damping fluid ", " 2-amino-2-methyl-1-propanol damping fluid ", " imidazoles～hydrochloride buffer ", " glycylglycine damping fluid ", " citric acid～sodium citrate buffer solution ", " Veronal sodium～hydrochloride buffer ", " boric acid～borate buffer solution ", " glycine～sodium hydrate buffer solution ", " borax～sodium hydrate buffer solution ", at least a in " phosphoric acid buffer " or " phosphate buffered saline buffer ".
7. magnesium ion diagnostic kit according to claim 4 is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, ammonium sulfate, thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glutaminate, reduced glutathion, lactose, N.F,USP MANNITOL, succinate or the sodium-chlor.
8. magnesium ion diagnostic kit according to claim 4 is characterized in that: described oxidized coenzyme is---NAD NAD
+, NADP+ NADP
+, thio-NAD+ thio-NAD
+Perhaps thio-NADP+ thio-NADP
+Described reduced coenzyme is---nicotinamide adenine dinucleotide reduced NADH, NADPH NADPH, Thio-NADH thio-NADH or thio-NADPH thio-NADPH.
9. any magnesium ion diagnostic kit in the claim 4～8 is characterized in that: described reagent is made into single agent, two agent or three doses.
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|---|---|---|---|
| CN 200410065549 CN1778956A (en) | 2004-11-23 | 2004-11-23 | Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit |
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|---|---|---|---|
| CN 200410065549 CN1778956A (en) | 2004-11-23 | 2004-11-23 | Determination of magnesium ion content from enzyme method and magnesium ion diagnostic reagent kit |
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ID=36769411
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726536A (en) * | 2013-12-24 | 2015-06-24 | 上海复星医药（集团）股份有限公司 | Stable reagent for determining magnesium ions by enzyme process |
| 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 |
-
2004
- 2004-11-23 CN CN 200410065549 patent/CN1778956A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726536A (en) * | 2013-12-24 | 2015-06-24 | 上海复星医药（集团）股份有限公司 | Stable reagent for determining magnesium ions by enzyme process |
| 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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