CN1108527C - Reagent for testing serum Mg-ion with enzyme - Google Patents

Abstract

The present invention relates to an enzymology detection technique, particularly to an enzyme method agent for detecting magnesium ions in serum by coupling pyruvate kinase (EC2.7.1.40, PK) and lactate dehydrogenase (EC1.1.1.27, LDH). Via research proof, the agent has the advantages of high precision and good accuracy, the average inaccuracy in the batch is lower than 2.45%, the average inaccuracy on the day is lower than 3.1%, the detection range is from 0.1 to 2.0mol/l, and the interference resistance is strong. Compared with an atomic absorption method and a colorimetric method, the detection technique has good correlativity, the agent is stabilized for at least 5 days after redissolution at 4 DEG C and has the low price of 0.5 yuan/ml, and then the agent is suitable for various automatic biochemistry analyzers, semi-automatic biochemistry analyzer and manual detection. Owing to the low price, the agent is domestically produced and is superior to various existing foreign enzyme method agents.

Description

Reagent for testing serum Mg-ion with enzyme

The present invention relates to the zymetology detection technique, provide particularly that a kind of (EC2.7.1.40, PK) (EC 1.1.1.27 LDH) is used to measure the enzyme process reagent of serum Mg-ion to the coupling lactic dehydrogenase with pyruvate kinase.

Magnesium ion is intracellular main kation, and content is only second to potassium ion, and its concentration in extracellular fluid is 0.7-1.10mmol/L.Magnesium ion participates in the many physiological functions of body, can with many important component in the cell, as formation compounds such as ATP, activate the plurality of enzymes system, thereby to formation such as glucose glycolysis, fat, protein, nucleic acid, coenzyme, contraction of muscle, the transmethylations in the antibody metabolic process etc. play regulating action; It is again the important accessory factor of oxidative phosphorylation, and mitochondrial function is had material impact, and is special relevant with energetic supersession; In protein building-up process, magnesium ion also participates in mRNA and protein and mechanism such as transcribes; In addition, magnesium ion also plays an important role to keeping nervimuscular excitability.Magnesium ion lacks or too much, neuromuscular system often is one of early symptom.Therefore the clinical meaning of serum Mg-ion concentration change is more and more paid attention to by physicians.

The detection method of serum Mg-ion is a lot.As: flame photometry, fluorescence method, atomic absorption spectrophotometry (AAS), chromatography, the chromatography of ions, dyestuff colourimetry and enzyme process etc.Wherein the most accurate, responsive with the AAS method, specificity is also best, but because the instrument price is very expensive, is difficult for automation mechanized operation and must uses the potential danger that inflammable gas brought, popularize and be difficult in clinical labororatory.Though flame photometry, fluorescence method, chromatography, ion-chromatographic determination magnesium ion have report, clinical practice is used actually rare.The dyestuff colourimetry is according under alkaline environment, and dyestuff and magnesium ion huge legendary turtle are closed and change color this basis foundation, comprises dimethylaniline blue laws (XLB), reaches the yolk method, Calmagite method and methyl thymol blue method (MTB).The blank value that reaches the yolk method is higher, it is big to disturb, specificity is relatively poor, the error at measurment rate surpasses 30%, remaining several colourimetry because of easy to use, economical, be leveraged to that the present, especially Calmagite method and methyl thymol blue method (MTB) are existing to be recommended as the conventional method that serum Mg-ion is measured by the Ministry of Public Health.But these class methods can be subjected to kation or bilirubinic interference in various degree, pH is changed and unstable, fresh preparation of palpus of work reagent or correction repeatedly are to increase reagent stability, need to add the supertoxic agent prussiate, thereby operating personnel are had potential danger.

Nineteen fifty-nine, Baum and Czok have just proposed that (EC1.1.1.42 ICD) measure the method for serum Mg-ion, but for various reasons, the method is not accepted by clinical always with isocitric dehydrogenase.Surplus nearly ten year, enzyme process is just further being developed aspect the serum Mg-ion measuring.According to statistics, have 300 plurality of enzymes to be activated by magnesium ion approximately, according to this principle, the various countries scholar has reported employing enzyme coupling technology in succession, measures the different Enzymology methods of serum Mg-ion.As: Tabata equals 1985, report hexokinase (EC 2.7.1.1, HK) coupling glucose-6-phosphate dehydrogenase (G6PD) (EC1.1.1.49, method G6PDH); 1986, they reported that again (EC2.7.1.2 Gluk) replaces HK, and the method for coupling G6PDH with glucokinase; The same year, Wimmer etc. reported glycerokinase (EC 2.7.1.30, GK) the coupling glycerol-3-phosphate oxidase (EC1.1.3.21, GPO) and peroxidase (EC 1.11.1.7, method POD); 1989, Fossati etc. reported the method according to the Gluk coupling G6PDH of Tabata method improvement again; 1991, Suzuki etc. reported with phosphoglucomutase (EC5.4.2.2, PGM) method of coupling G6PDH.

Above-mentioned four kinds of methods are except that the glycerokinase method, all be that catalysis glucose Cori's eater Cori generates the 6-glucose 1-phosphate1-, coupling G6PDH catalysis 6-glucose 1-phosphate1-and NADP generate 6-phosphogluconic acid lactone and NADPH again, the increase of the absorbance that NADPH causes during by the 340nm wavelength, the concentration of coming the indirect calculation serum Mg-ion.On expense, the method for coupling G6PDH, the expense of one liter of reagent of preparation is approximately 4000 to 7000 yuans; Send out by glycerokinase, the expense of one liter of reagent of preparation is just higher, and only the expense of import toolenzyme is more than 18,000 yuans, and the method is the interference problem of unresolved calcium ion also.These all become the reason that hinders the further popularization of enzyme process.

Nineteen ninety-five, the people such as Fujita of Japan have reported the method with different lemon ldh assay magnesium ion, and use biochemical technology that ICD is modified, to improve the stability of this enzyme in aqueous solution, 1996, people such as Englishman Stone etc. according to the also successful foundation of the article that the Japanese delivers this kind method, and make enzyme stability after the modification before the unmodified extended in two weeks all around more than.One liter of reagent place of single preparation needs the expense of ICD and NADP at least also to want more than 17,000 yuan.

The object of the present invention is to provide the enzyme process reagent that pyruvate kinase coupling lactic dehydrogenase is used to measure serum Mg-ion, and the accuracy height, precision is good, and measurement range is wide, and antijamming capability is strong, correlativity, good stability.

The invention belongs to enzymatic assays research to inorganic ions, based on magnesium ion in the research rabbit flesh pyruvate kinase there is this discovery of tangible activation, pyruvate kinase (Pyruvate Kinase, PK, EC 2.7.1.40) being atriphos pyruvate phosphate transferase again, is to regulate one of glucolytic three rate-limiting enzymes.PK is having manganese ion and magnesium ion to have catalysis phosphoenolpyruvic acid generation pyruvic acid under the situation, and potassium and ammonium ion can make the PK activity strengthen greatly there being magnesium ion to exist under the situation, and this principle is used for measuring magnesium ion concentration by this research.Because the reaction product pyruvic acid can not directly be indicated the degree of PK catalytic reaction, need coupling LDH system to indicate PK activity or product growing amount indirectly.Lactic dehydrogenase (Lactate Dehydrogenase, LDH, EC 1.1.1.27) is one of maximum enzyme of animal body intensive amount, extensively is present in animal tissue's internal organs.As toolenzyme, oxidation and reduction reaction between main catalysis pyruvic acid and the lactic acid, simultaneously with the conversion between NADH and the NAD, the maximum absorption band of NADH is at 340nm, so its concentration change can make the absorbance under the 340nm wavelength change, and LDH is coupled to the mensuration that the PK system carries out serum Mg-ion.PK and LDH coupling reaction principle are as follows:

Reaction principle: phosphoenolpyruvic acid (PEP) and adenosine diphosphate (ADP) (ADP) generate pyruvic acid (Pyruvate) and atriphos (ATP) under pyruvate kinase (PK) catalysis, pyruvic acid again with the coupling of lactic dehydrogenase enzyme system, generate lactic acid (Lactate) through lactic dehydrogenase (LDH) catalysis, NADH is oxidized to NAD simultaneously, the existence of magnesium ion can make the active significantly enhancing of pyruvate kinase, so NADH can reflect the concentration of serum Mg-ion indirectly in the variation of 340nm wavelength absorbance.

Based on above-mentioned reaction principle, agent prescription of the present invention is made up of R1, the R2 of following density component, R1 wherein: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 110.00mmol/L, potassium chloride (KCL) 40.00-50.00mmol/L, lactic dehydrogenase (LDH) 1500-2500U/L, pyruvate kinase (PK) 500-750U/L, adenosine diphosphate (ADP) (ADP) 1.50-3.00mmol/L, reducibility coenzyme I (NADH) 0.40-0.50mmol/L; R2: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.491.80mmol/L, phosphoenolpyruvic acid (PEP) 2.00-4.00mmol/L, ethylene glycol (b-aminopyrine) tetraacethyl (EGTA) 15.00-20.00mmol/L.

Mentioned reagent source: trishydroxymethylaminomethane (Tris, AR, Shanghai reagent three factories); Pyruvate kinase (PK, Sigma, specific activity: 40U/mg freeze-dried powder, Shanghai reagent three factories); Lactic dehydrogenase (LDH, specific activity: 30U/mg freeze-dried powder, Shanghai reagent three factories); Phosphoenolpyruvic acid (PEP, AR, Shanghai reagent three factories); Reducibility coenzyme I (NADH, Shanghai reagent three factories); Adenosine diphosphate (ADP) (ADP, the biochemical factory of Shanghai east wind); Potassium chloride (KCL, AR, the biochemical factory of Shanghai east wind); Ethylene glycol (b-aminopyrine) tetraacethyl (EGTA, extraordinary chemical reagent development centre, North China).

The present invention adopts the conventional method preparation, the wherein preparation of Tris-HCL damping fluid: accurately take by weighing Tris 1.21g and be dissolved in water, transfer pH to 7.4 (25 ℃) with 1mol/L HCL, be diluted to 100ml with deionized water again, place 4 ℃ of preservations.

Determining of the automatic analytical parameters of the present invention.The location parameter of Cobas Mira automatic biochemistry analyzer is as follows: mensuration mode, absorbance; Reactive mode, test (R)-sample (S)-startup reagent (SRI), calibrating mode: reference material is non-linear, reagent blank: reagent/water, wavelength 340nm, decimal is counted, 2 significant digits, unit: mmol/L, sample circulation: 1, volume 15 μ L, dilution liquid measure: 5 μ L, reagent circulation: 1, amount of reagent: 160 μ L, start the reagent circulation: 2, start amount of reagent 40 μ L, dilution: 0 μ L, the Direction of Reaction: reduce, aligning step: end-point method, read first a little: 3, read at last a little: 8, proofread and correct at interval: each operation, standard 1:0.00mmol/L, standard 2:0.40mmol/L, standard 3:0.80mmol/L, standard 4:140mmol/L, standard 5:2.00mmol/L, repetitive mode: 1 time, difference percent: 10%.

The detected parameters of Kedy III Snijders semi-automatic biochemical analyzer is as follows: detection mode: fixed time, wavelength; 340nm, temperature: 37 ℃, unit: mmol/L, the pathology low value: 0.700, the high value of pathology: 1.100, draw liquid measure: 500 μ L, sample size: 60 μ L, reagent 1 amount: 480 μ L, reagent 2 amounts: 120 μ L, time delay: 40 seconds, minute: 60 seconds, standard 1:0.40, standard 2:0.80, standard 3:1.40, standard 4:2.00.

Experimental study of the present invention

1 kation is to the influence of pyruvate kinase activity: instrument: Tianjin, island UV-240, Ciba-Corning2800 spectrophotometer and HHW21-CR420 type electric heating constant temperature water bath.Reagent: 0.1mol, pH7.4, Tris-HCL damping fluid, ADP, PEP, NADH, LDH and PK; Sample (S) is potassium, sodium, calcium, lithium, manganese, ammonia and the magnesium ion solution of 50mmol/L; Experimental water is deionized water.Reagent (R1) is made up of ADP, LDH, PK and NADH, and reagent (R2) is mainly PEP.Location parameter: temperature: 37 ℃, 340nm, R1:1.0ml, R2:0.1ml, S:0.025ml, dilution: 0.125ml.Determination step: R1 and sample are added in the test tube, and 37 ℃ of water-baths are more than 5 minutes, and behind the adding R2 (continuing in 37 ℃ of water-baths), the absorbance in the immediate record unit interval changes (Δ A/min).

Conclusion: manganese and magnesium ion are the direct agonists of PK, and potassium and ammonium ion in fact only are its indirect activators, ammonium ion to the indirect activation effect of PK significantly greater than potassium ion.The inhibitor of calcium ion PK, proof thus: use the pyruvate kinase method to measure magnesium ion, must solve the interference problem of calcium ion earlier.

2 study on accuracy:

(1) with the comparison of definite value serum: Boehreiger Mannheim (BM) the company definite value serum that adopts double (high, normal value) import, high value serum: lot number is Lot/187891-187894No.651265, normal value serum: lot number is Lot/189636-189639No.651257.See Table one, the result shows that accuracy is quite satisfied.

(2) linear research: calcium acetate and the Adlerika of preparation 0.1mol/L, the dilution preparation becomes the series standard liquid that contains magnesium ion 0,0.2,0.4,0.6,0.8,1.2,1.6,2.0mmol/L respectively, and the calcium ion concentration in the titer is 2.5mmol/L.Above-mentioned titer is measured twice on Cobas Mira automatic analyzer, each every part of sample is surveyed three times, regression equation: Y=0.181x+0.011, r=0.9998, figure one is seen in P＜0.001, and the result shows, magnesium ion concentration is in the 0-2.0mmol/L scope time, and absorbance and concentration have good linear relationship.The normal serum magnesium ion concentration is generally at 0.7-1.1mmol/L, so this range of linearity can meet clinical needs.

(3) reclaim experiment: with the magnesium ion concentration is the basic value of 0.2mmol/L, adds magnesium ion 0.6,0.8,1.0 and 1.2mmol/L respectively again, makes the theoretical value of potassium ion become 0.8,1.0,1.2 and 1.4mmol/L respectively, reclaims experiment.See Table two, the recovery average out to 100.76% by calculating magnesium ion in the table shows that the recovery of this method is quite satisfied.

3 precision researchs: withinrun precision: be worth serum at one time with normal value and height respectively, replication is 15 times simultaneously, and calculates its average, standard deviation and the coefficient of variation, the results are shown in Table three; Day to day precision: above-mentioned serum every day is measured once, measured eight days altogether continuous twice, calculate its average, standard value and the coefficient of variation, the results are shown in Table four; The result shows batch interior day to day precision that reaches on average only 2.01 and 3.12%, and the result of this result and other enzyme process authors report is closely similar, therefore measures serum Mg-ion with this Enzymology method, meets repeated requirement fully.

4 interference tests: select the endogenous interfering material: potassium, ammonia, calcium, manganese, copper, zinc, ferric ion, cholerythrin, triglyceride and haemoglobin carry out interference experiment.When carrying out the kation interference experiment, set up control group to compare with adding the cationic experimental group of variable concentrations, magnesium ion concentration is all identical, calculates the average and the standard deviation of the control group magnesium ion concentration of measuring 10 times simultaneously, each experimental group is then measured three times simultaneously, and computation of mean values.Whether estimate annoyance level then (in the scope of means ± 2S), exceeds this scope and has been interference in mean value 2 standard deviations of control group according to the experimental group average.

The magnesium ion concentration of control group and experimental group is 0.80mmol/L.Measurement result: control group x ± S is 0.811 ± 0.020mmol/L, and control group scope x ± 2S is 0.771-0.851mmol/L.

Experimental group: A. potassium ion: the potassium chloride that adds variable concentrations, make the potassium ion reaction density be respectively 6.0,8.0 and 10.0mmol/L, three groups magnesium ion average is respectively 0.789,0.773 and 0.782mmol/L after measured, so the potassium ion of 10mmol/L is noiseless to this method.

B. ammonium ion: serum ammonium ion upper limit of concentration is 0.056mmol/L.The sulfate of ammoniac concentration-response that adds in the interference test sample is respectively 0.5,0.75 and 1.0mmol/L, is 8.9,13.4 and 17.9 times of upper limits of normal, and the magnesium ion measured value is 0.839,0.802 and 0.821mmol/L, and is noiseless.

The C calcium ion: calcium ion is the inhibitor of PK, confirmed by the present invention, add calcium acetate 6.0,8.0 and 10.0mmol/L in the interference test respectively, the magnesium ion measured value is respectively 0.826,0.845 and 0.844mmol/L, do not see the interference of calcium ion, and Wimmer etc. are when measuring magnesium ion with the glycerokinase method, and calcium ion promptly produces significant negative interference effect to measurement result in the sample when 5.0mmol/L.

The D manganese ion: manganese ion is the same with magnesium ion, and many enzymes are had activation, and manganese ion belongs to trace element in serum, be limited to 0.013mmol/L on its serum-concentration.Add 1.0mmol/L in this interference experiment, do not see interference.

The E copper ion: the upper limits of normal of serum copper ion concentration is 0.025mmol/L, adds copper sulphate 0.05,0.075 and 0.10mmol/L in this test respectively, and the magnesium ion measured value is respectively 0.801,0.837 and 0.792mmol/L, does not see the interference of copper ion.

The F zinc ion: the upper limits of normal 0.025mmol/L of serum zinc ion concentration, add zinc oxide 0.05,0.07 and 0.10mmol/L respectively in this interference test, the magnesium ion measured value is respectively 0.804,0.823 and 0.823mmol/L, does not see that zinc ion disturbs.

The G ferric ion: the upper limits of normal of serum levels of iron ion concentration is 0.03mmol/L, and when the adding ferric trichloride reached 0.10mmol/L in this interference test, the magnesium ion concentration measured value was 0.848mmo l/L, does not see the interference of ferric ion.

Cholerythrin, triglyceride and haemoglobin interference test are used determination of serum, and press following formula and calculate interference percentage: p=[(AM-CM)/CM] * 100, p is for disturbing percentage in this formula, AM is the practical measurement value, CM here is a theoretical value, when p≤| during 3.0| (| 3.0| represents absolute value, be equivalent to ± 3%), be regarded as noiseless.

A cholerythrin interference test: add cholerythrin 100mmol/L (5.85mg/dl) in high value serum, making its magnesium ion theoretical value is 1.62mmol/L, then, is determined as 1.637mmol/L with this method, and p is 1.049%, and is noiseless.

B triglyceride interference test: the high triglyceride serum that in serum, adds different amounts, make triglyceride concentration be respectively 4.86 and 7.3mmol/L, the magnesium ion theoretical concentration is respectively 0.949 and 1.243mmol/L, this method measured value is 0.947 and 1.230mmol/L, disturb percentage p to be respectively-0.211% and-1.046%, do not see interference.

C haemoglobin interference test: use haemoglobin standard liquid (141g/L) to carry out interference test.Add 0.1,0.2,0.4,0.6,0.8 and the 1.0G/L haemoglobin respectively, make the serum Mg-ion theoretical concentration be followed successively by 0.986,0.972,0.944,0.916,0.888 and 0.860mmol/L, and practical measurement concentration is 1.002,0.970,0.963,0.934,0.865 and 0.854mmol/L, disturb percentage p to be respectively 1.623 ,-0.206,2.013,1.965 ,-2.59 and-0.698%, all less than ± 3.0%, therefore, judge that haemoglobin is noiseless to the method.

5 with the methodology of other method relatively: clinical labororatory's colourimetry commonly used is dimethylaniline blue laws (XLB), Calmagite method (CAL) and methyl thymol blue method (MTB) at present, and CAL method and MTB method now still are recommended as the conventional method of serum Mg-ion mensuration by China Ministry of Public Health.The spy makes correlation research.

(1) methodology with atomic absorption method (AAS) compares: the AAS method is by Tianjin City No.1 Central Hospital, atomic absorption spectrophotometer (AAS) (Smith-Hieftje-22 is used in the trace element chamber, Termo Jarrall Ash, USA) and Beijing chemical industry metallurgical research institute of Ministry of Nuclear Industry produce, lot number is 080132, concentration is the national secondary magnesium mark liquid GBW (E) of 100mg/ml, the dilution back is measured, sample is provided by the biochemical chamber in sky and hospital, get the definite value serum (height and normal value) of 8 parts of patients serums and two parts of BM companies at random, measure with two kinds of methods, data are learned processing: AAS by statistics: means standard deviation is 0.936 ± 0.249mmol/L; This law is: 0.905 ± 0.254mmol/L, t=0.277, the average there was no significant difference of p＞0.5, two kind of method; Regression equation: this law: Y=0.999x-0.03, r=0.980, n=10, p＜0.01, see figure two, two kinds of method height correlations, bias (deviation)=0.031mmol/L, Sy/x=0.054mmol/L, Sd=0.051mmol/L, t=1.824, so p＞0.1 is the deviation not statistically significant of two kinds of methods.

(2) methodology of sending out with MTB relatively: instrument is 721 spectrophotometers (Shanghai the 3rd analytical instrument factories), reagent is that the Beijing Chemical Plant produces azoviolet box (MTB), lot number: 970411, measure according to the kit instructions, sample is provided by the biochemical chamber in sky and hospital, get the definite value serum (high value) of 30 parts of fresh patients serums and a BM company at random, mensuration is seen figure three simultaneously.The regression equation Y=0.011+1.172x of this law and MTB method, r=0.922, n=31, p＜0.01.Carry out statistical procedures according to measurement result, computing method average, standard deviation and t check: the MTB method is 0.648 ± 0.142mmol/L; This law is 0.77 ± 0.18mmol/L, t=2.979, p＜0.005, though as seen the correlativity of these two kinds of methods is good, but a significant methodology difference is arranged, this error is because the lot number that employed Beijing Chemical Plant produces is 970411 azoviolet box (MTB method), make measurement result on the low side due to, this also is confirmed by BM definite value serum result, high value is the definite value serum of 1.62 (1.43-1.81) mmol/L, the MTB method only is measured as 1.289mmol/L (out of control), and absolute error reaches 0.351mmol/L (21.40%), and this law is that the 1.60mmol/L absolute error is only for 0.02mmol/L (1.23%).After it is identical repeatedly to repeat conclusion.

(3) with the methodological comparison of XLB method: instrument: 721 spectrophotometers (Shanghai the 3rd analytical instrument factory); Reagent: middle living bioengineering High-tech company produces the azoviolet box, lot number: 970905, measure to specifications; Sample is provided by the biochemical chamber in sky and hospital, gets the definite value serum (high value and normal value) of 45 parts of patients serums and two parts of BM companies at random, measures simultaneously, sees figure four.The regression equation of this law and XLB method: Y=0.118+0.827x, r=0.933, n=47, p＜0.01.Unite according to measurement result and 2 learn to handle, computing method average, standard deviation and t check: the XLB method is 0.796=0.179mmol/L, and this law is 0.777 ± 0.159mmol/L, t=0.543, p＞0.5, the correlativity of these two kinds of methods is fine, between the deviation not statistically significant.

(4) methodology with the CAL method compares: the CAL method is used Beckman CX5 automatic biochemistry analyzer and Beckman titer and kit measurement by the biochemical chamber of No.1 Hospital Attached to Tianjin College of Traditional Chinese Medicine, sample is got the definite value serum (high value and normal value) of 31 parts of patients serums and two parts of BM companies at random also from the biochemical chamber of first affiliated hospital of the college of traditional Chinese medicine.Above sample uses two kinds of methods to measure on two instruments respectively.Data are learned processing by statistics: computation of mean values ± standard deviation, CAL method are 0.852 ± 0.224mmol/L; This law is 0.800 ± 0.229mmol/L, t=0.929, and p＞0.3 illustrates the average there was no significant difference of two kinds of methods; Regression equation: Y=0.982x-0.037, r=0.960, n=33, five, two kinds of method height correlations of figure are seen in p＜0.01, the deviation not statistically significant between two kinds of methods.

6 reagent stabilities are observed: the application reagent that will prepare places 4 ℃ of refrigerator cold-storages, and is used for measuring in continuous 7 days the standard items of two kinds of concentration.The result shows that this reagent after being formulated as application liquid, places 4 ℃ of stored refrigerated, can stablize at least 5 days, during this measurement result is not had influence, and the stable fine of reagent is described.

Embodiment: this reagent is checked accuracy on Kedy III semi-automatic biochemical analyzer.Check the definite value serum of two lot numbers of accuracy use BM company.The preparation of titer: the storage reagent that is mixed with 100mmol/L respectively with dry calcium acetate and magnesium sulphate, accurately be diluted to the titer that contains magnesium ion 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8 and 2.0mmol/L with deionized water again before the test, the calcium ion concentration in the titer is 2.5mmol/L.Agent prescription is: R1: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 110.00mmol/L, potassium chloride (KCL) 41.25mmol/L, lactic dehydrogenase (LDH) 2250U/L, pyruvate kinase (PK) 750U/L, adenosine diphosphate (ADP) (ADP) 2.28mmol/L, reducibility coenzyme I (NADH) 0.41mmol/L; R2: phosphoenolpyruvic acid (PEP) 3.85mmol/L, ethylene glycol (b-aminopyrine) tetraacethyl (EGTA) 16.50mmol/L, trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 91.80mmol/L.Operation steps: accurately take by weighing R1 480 μ l and inject the 2ml test tube, add 60 μ l titer and samples more successively respectively, mixing places 37 ℃ of constant temperature water tanks to hatch more than 5 minutes, accurately adds to start reagent R2 120 μ l again, mixing immediately, measure by aforementioned parameters, the results are shown in Table five, so this reagent can be widely used in the laboratory of domestic various scales, various automatic or semi-automatic biochemical analyzers more help promoting the use of because of it is with low cost.

Clinic trial of the present invention is observed (clinical laboratory of No.3 Central Hospital of Tianjin City): instrument and method: instrument is the ToshibaTBA-40FR automatic biochemistry analyzer; The reagent that uses, titer provides by the present invention's preparation, and definite value serum is from BM company, and the A. height is worth serum: lot number: 185259, magnesium ion: atomic absorption method (AAS) target value: 1.65 (1.45-1.85) mmol/L; Xylidin blue laws (XLB) target value: 1069 (1.49-1.89) mmol/L; B. normal value serum: lot number: 18968802, AAS method target value: 0.88 (0.77-0.99) mmol/L, XLB method target value 0.85 (0.75-0.95) mmol/L.Carry out evaluation of the accuracy by measuring definite value serum, and select two parts of patients serums to carry out precision immediately and measure.The results are shown in Table six and table seven, accuracy: with two kinds of method target values of definite value serum relatively, the measurement result of this method in the high value in the middle of two kinds of methods, at normal value, then more approaching with the AAS method.Precision: select two parts of patients serums to carry out 20 times at random and measure, and calculate batch interior imprecision, low value (0.485mmol/L) only is 0.829% with the average imprecision of normal value (0.799mmol/L) as a result.

Table one. measure as a result definite value serum target value of import definite value serum, (scope) measured value deviation, (%) AAS, (H) 1.62, (1.43-1.81) 1.637 0.017, (1.05) XLB, (H) 1.64, (1.44-1.84) 1.637-0.003, (0.18) AAS, (N) 0.88, (0.77-0.99) 0.858-0.022, (2.50) XLB, (N) 0.85, (0.75-0.95) 0.858 0.008, (0.94)

Table two. recovery test as a result basic value adds the pH-value determination pH value alluvial rate of recovery (%) 0.20 0.60 0.817 0.617 102.830.20 0.80 1.003 0.803 100.380.20 1.00 1.180 0.980 98.000.20 1.20 1.422 1.222 101.83

Table three. replication result in crowd

Normal value high value average C.V (%) Mean 0.858 1.637S, 0.021 0.026CV (%) 2.448 1.588 2.006

Table four. replication result in the daytime

Normal value high value average C.V (%) Mean 0.870 1.627S, 0.032 0.042CV (%) 3.664 2.568 3.116

Table five. the accuracy of semi-automatic mensuration is definite value serum target value as a result, (scope) measured value deviation, (%) AAS, (H) 1.62, (1.43-1.81) 1.661 0.041, (2.53%) XLB, (H) 1.64, (1.44-1.84) 1.661 0.021, (1.28%) AAS, (N) 0.88, (0.77-0.99) 0.902 0.022, (2.50%) XLB, (N) 0.85, (0.75-0.95) 0.902 0.052, (6.12%)

Table six. use accuracy measurement result definite value serum target value of the present invention (scope) measured value deviation (%)

(mmol/L) (PK)185259AAS 1.65(1.45-1.85) 1.68 0.03(1.82％)XLB 1.69(1.49-1.89) 1.68 -0.01(0.59％)18968802AAS 0.88(0.77-0.99) 0.90 0.02(2.27％)XLB 0.85(0.75-0.95) 0.90 0.05(5.88％)

Table seven. imprecision measurement result in batch

The low value normal value is average

(mmol/L) (mmol/L) CV(％)Mean 0.485 0.799S 0.005 0.005CV(％) 1.031 0.626 0.829

Claims (2)

1. reagent for testing serum Mg-ion with enzyme, it is characterized in that being formed by R1, the R2 of following concentration component, R1 wherein: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 110.00mmol/L, potassium chloride (KCL) 40.00-50.00mmol/L, lactic dehydrogenase (LDH) 1500-2500U/L, pyruvate kinase (PK) 500-750U/L, adenosine diphosphate (ADP) (ADP) 1.50-3.00mmol/L, reducibility coenzyme I (NADH) 0.40-0.50mmol/L; R2-trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 91.80mmol/L, phosphoenolpyruvic acid (PEP) 2.00-4.00mmol/L, ethylene glycol (b-aminopyrine) tetraacethyl (EGTA) 15.00-20.00mmol/L.

2. reagent as claimed in claim 1, it is characterized in that being formed by R1, the R2 of following density component, R1 wherein: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 110.00mmol/L, potassium chloride (KCL) 41.25mmol/L, lactic dehydrogenase (LDH) 2250U/L, pyruvate kinase (PK) 750U/L, adenosine diphosphate (ADP) (ADP) 2.28mmol/L, reducibility coenzyme I (NADH) 0.41mmol/L; R2: trishydroxymethylaminomethane-hcl buffer (Tris-HCL) pH7.4 91.80mmol/L, phosphoenolpyruvic acid (PEP) 3.85mmol/L, ethylene glycol (b-aminopyrine) tetraacethyl (EGTA) 16.50mmol/L.