CN1869652A - Investigating agent and method of serum sodium ion enzyme method - Google Patents

Abstract

The invention discloses an in-serum sodium ion enzyme method determining reagent and method, finding a relatively good concentration by applying different reagent proportions on complexing ability of crown ether which has characters of cavity ether and sodium ions, and it is the same as the cavity ether in the aspects of sensitivity, accuracy, and precision. And the invention has determining effect similar to the determining effect obtained by the method adopting cavity ether, but the invention is simple and convenient and largely reduces cost.

Description

Serum sodium ion by enzymatic method is measured reagent and method

Technical field

The invention belongs to the clinical biochemical analytical approach, be used for the mensuration of serum sodion.

Background technology

In biochemical clinical trial, it is analysis project conventional in the hospital that serum sodium ion is measured.The normal value of sodium is 136-145mmol/L, and common clinically electrolyte disturbance is a hyponatremia.

The analytical approach of sodion is a flame photometry the earliest.The principle of this method is that some sodium atom to be measured is excited by adding thermostimulation, sends the light with characteristic wavelength when getting back to ground state.The characteristic wavelength that produces by the sodium atom to be measured that is excited in the flame, the intensity of radiation energy directly is directly proportional with the quantity of the sodium atom to be measured that is excited in the flame, and the sodium atom quantity to be measured that is excited directly is directly proportional with the concentration of sodium atom to be determined in the sample.The device that this method needs is complicated and expensive, and will use inflammable gas.

The sodion method for measuring of generally using in the hospital is an ion selective electrode method at present.Ideal situation is that each electrode all has single ion selectivity, and electrode is only responded to a kind of ion.Actual conditions but are not that all ion-selective electrodes all have interfering ion like this.Moreover although can proofread and correct ion specificity electrode, usually specificity still is not absolute, so the result who records with the electrode method neither be very accurate.And cost of determination is also than higher, and especially auxiliary material and instrument are relatively more expensive.

The enzymatical detection method that had occurred serum sodium ion in recent years again.The theoretical foundation of this assay method is the effect that many ions all have kinase.The measuring principle of sodion is the enzyme kinetics reaction detection sodium by sodium dependence beta galactosidase catalytic substrate O-nitro pyranoside, and its product O-nitrophenols rises proportional with na concn at the light absorption value of 405nm.Because Na ion concentration is too high in the serum, is unsuitable for direct mensuration, needs to add ionophore complexing part sodion, thereby sodion is adjusted to a suitable concentration.Enzyme is the most responsive to the subtle change of sodion in this concentration range, so just can obtain measurement result more accurately.

Enzymatic assays is not only accurate, and convenient, can reinstate fully-automatic analyzer with other conventional sense project one and detect, and has broken away from the instrument of original electrode method, has also reduced the detection cost simultaneously.Electrode method originally not only will be bought instrument in addition, but also to often change electrode, and the consumption of electrolytic solution is also than comparatively fast, and these reasons make sodion cost in the determination of electrode serum than higher, and adopt enzymatic assays just not have these problems, so cost of determination reduces a lot.

Summary of the invention

The purpose of this invention is to provide a kind of reagent with sodion in the enzymatic assays serum.

Another object of the present invention provides the assay method of this reagent.

It is composed as follows that serum sodium ion by enzymatic method provided by the invention is measured reagent:

R 1Reagent	Concentration (MMOL/L)
		TRIS buffer pH8.7 (37 ℃)	500mmol/L
3-mercaptoethanol	5mmol/L
		MgSO 4	6mmol/L
LiCl	50-100mmol/L
		Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium	0.5-5mmol/L
15-is preced with-5 ethers	10-25mmol/L
		β-galactosidase (beta galactosidase)	1000-4000U/L

R 2Reagent	Concentration (MMOL/L)
		O-nitro-β-D-pyranoside	10-20mmol/L

Reagent of the present invention is made up of dilution and dried frozen aquatic products.Because the time that some component is preserved in aqueous solution is very short, so want freeze-drying to preserve.Amount ratio when R1 and R2 test is: 2.5: 1, so the amount of preparation ratio is 2.5: 1.Process for preparation is as follows:

R ₁：

(1) dilution: (1000ml)

Tris-Hcl buffr pH 8.7 (25 ℃) 500mmol/L 1000ml accurately adds following material successively:

MgSO ₄

LiCl

Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium

15-is preced with-5 ethers

(2) dried frozen aquatic products: (100ml, 10 times of concentrates)

Tris-Hcl buffer pH8.7 (25 ℃) 500mmol/L 100ml accurately adds following material successively:

3-mercaptoethanol

β-galactosidase

Sucrose 18 grams (forming agent)

R ₂：

(1) dilution (400ml)

Tris buffer pH8.7(25℃)10.0mmol/L 400ml

(2) dried frozen aquatic products (100ml, 4 times of concentrates)

The new H that steams ₂O 100ml accurately adds following material successively:

O-nitro-β-D-pyranoside

Sucrose 15 grams (forming agent)

Except that ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium, can buy.Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium be spent glycol (alpha-amido ethyl) ether tetraacethyl (be called for short: EGTA) and lithium hydroxide (LiOH) prepare.Method is as follows: take by weighing 1.902gEGTA and be dissolved in the 5ml deionized water, the LiOH solution that adds 4mol/L more all dissolves to EGTA, is diluted to 50ml with deionized water again.(pH should be 8.7,25 ℃).Take according to the consumption in the prescription during preparation finished product reagent.

The present invention utilizes crown ether to have the characteristics of cave ether and the ability of sodion complexing, adopts the proportioning of different reagent to seek out the above serum sodium ion by enzymatic method that is used for and measures reagent.

Key of the present invention has been to use a kind of novel ionophore, this carrier low price, and it belongs to the crown ether-like material.What be that they use with the method difference of bibliographical information is a kind of cave ethers carrier Kryptofix-2.2.1 (being called for short K221), and its chemical molecular formula is C ₁₆H ₃₂N ₂O ₅, and cave ether is very expensive, about 4000 yuans of 1ml.The crown ether-like carrier that we use is very cheap.Screening through prescription has obtained reasonable measurement result.The chemical formula of this material is as follows: 15-crown-5 is called: 15-is preced with-5 ethers, molecular formula: C ₁₀H ₂₀O ₅Bibliographical information (Lin Zili, the application progress of crown compound colorimetric estimation potassium sodium are arranged.) cave ether and crown ether be as follows to the complexing power of sodion:

	With Na +The complexing stability constant	Complexing power is (cave ether/crown ether) relatively
			15-is preced with-5 ethers	1.2×10 6	3×10 4
Cave ether K221	3.6×10 10

Owing to the hole is arranged in their macrocyclic structure, in addition Nei Bu oxygen atom have share electron pair can with complexing of metal ion, so according to the size in hole, the metallic ion of alternative complexing different-diameter.Though the ability of crown ether-like material complexing sodion is nothing like the cave ether material, we have screened a reasonable concentration through a large amount of experiments, are obtaining aspect sensitivity, accuracy, the precision and are using the identical effect of cave ether.

The typical concentration scope of the main agents that serum sodium ion is measured is as follows:

R 1Reagent	Concentration (MMOL/L)
		TRIS buffer pH8.7 (37 ℃)	500mmol/L
3-mercaptoethanol	5mmol/L
		MgSO 4	6mmol/L
LiCl	50-100mmol/L
		Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium	0.5-5mmol/L
15-is preced with-5 ethers	10-25mmol/L
		β-galactosidase (beta galactosidase)	1000-4000U/L

R 2Reagent	Concentration (MMOL/L)
		O-nitro-β-D-pyranoside	10-20mmol/L

Subordinate list:

Measure reagent and measure the reagent performance relatively with crown ether preparation sodion with cave ether preparation sodion:

	Crown ether preparation sodion is measured reagent	Cave ether preparation sodion is measured reagent
			Two kinds of reagent correlations of linear repeated degree of accuracy Sensitivity Stability	Be in the 100-160mmol/L scope in linear batch and can stablize 1 year Y=1.0672X-10.284 under 2-8 ℃ of environment of (n=20) CV (%)＜2 ± 5% OD/mmol/L=0.003	Be in the 100-160mmol/L scope in linear batch and can stablize 1 year r=0.9806 under 2-8 ℃ of environment of (n=20) CV (%)＜3 ± 5% OD/mmol/L=0.005

Description of drawings

Fig. 1 measures reagent mensuration clinical sample (n=26) correlativity relatively with crown ether preparation sodion mensuration reagent with using cave ether preparation sodion:

The x axle is for measuring the result of clinical sample with the reagent of cave ether preparation;

The y axle is the result who measures clinical sample with the reagent of crown ether preparation.

Beneficial effect

The crown ether that use value of the present invention is more much lower than cave ether, and the mensuration effect reaches similar to cave ether, the characteristics that assay method is easy.

Embodiment

Embodiment:

1. serum sodium ion is measured the reagent composition:

R 1Reagent	Concentration (MMOL/L)
		TRIS buffer pH8.7 (37 ℃)	500mmol/L
3-mercaptoethanol	5mmol/L
		MgSO 4	6mmol/L
LiCl	100mmol/L
		Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium	0.5mmol/L
15-is preced with-5 ethers	25mmol/L
		β-galactosidase (beta galactosidase)	2000U/L

R 2Reagent	Concentration (MMOL/L)
		O-nitro-β-D-pyranoside	15mmol/L

Test procedure:

Instrument: full-automatic or semi-automatic biochemical analyzer;

Temperature: 37 ℃; Cuvette 1ml;

Predominant wavelength 405nm; Commplementary wave length: 660nm;

Method two point velocity methods;

Operation;

	Blank	Standard	Sample
				Distilled water standard model R 1	40μL 1000μL	40μL 1000μL	40μL 1000μL
Mixing is hatched 5min in 37 ℃, adds then
				R 2	400μL	400μL	400μL

Fully mix, hatch 0.5min in 37 ℃, read A ₁, read A after the 2min ₂, the changes delta A=A of absorbance ₂-A ₁

Calculate:

Use two standards of height, with the changing value of absorbance the concentration value of corresponding standard items is made typical curve, the concentration of the sample changing value of absorbance is per sample found from typical curve.

Standard items are the standard solution with sodium chloride and potassium chloride preparation, in order to make standard items and clinical sample close, so will add potassium chloride.Compound method is as follows: prepare two base soln: 200mmol/LNaCl earlier, 4mmol/LKCl solution (1#) 1400ml and 4mmol/LKCl solution (2#) 600ml, according to following table two solution are mixed according to certain volume then, just can obtain the height standard items of sodium reagent box.

NaCl concentration mmol/L (theoretical concentration)	120	160
			1# liquor capacity (ml)	600	800
2# liquor capacity (ml)	400	200

Because by the concentration of calculating gained is not very accurate, need measure accurate concentration with the electrode method, and then with the quality-control product certificate of inspection of Randox.

2. serum sodium ion is measured the reagent composition:

R 1Reagent	Concentration (MMOL/L)
		TRIS buffer pH8.7 (37 ℃)	500mmol/L
3-mercaptoethanol	5mmol/L
		MgSO 4	6mmol/L
LiCl	50mmol/L
		Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium	2.0mmol/L
15-is preced with-5 ethers	20mmol/L
		β-galactosidase (beta galactosidase)	4000U/L

R 2Reagent	Concentration (MMOL/L)
		O-nitro-β-D-pyranoside	10mmol/L

3. serum sodium ion is measured the reagent composition:

R 1Reagent	Concentration (MMOL/L)
		TRIS buffer pH8.7 (37 ℃)	500mmol/L
3-mercaptoethanol	5mmol/L
		MgSO 4	6mmol/L
LiCl	70mmol/L
		Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium	5.0mmol/L
15-is preced with-5 ethers	10mmol/L
		β-galactosidase (beta galactosidase)	1000U/L

R 2Reagent	Concentration (MMOL/L)
		O-nitro-β-D-pyranoside	20mmol/L

Above assay method is with embodiment 1.

Claims (4)

1. a serum sodium ion by enzymatic method is measured the composed as follows of reagent: R ₁Reagent Concentration (MMOL/L) TRIS buffer pH8.7 (37 ℃) 500mmol/L 3-mercaptoethanol 5mmol/L MgSO ₄ 6mmol/L LiCl 50-l00mmol/L Ethylene glycol (alpha-amido ethyl) ether tetraacethyl lithium 0.5-5mmol/L 15-is preced with-5 ethers 10-25mmol/L β-galactosidase (beta galactosidase) 1000-4000U/L R ₂Reagent Concentration (MMOL/L) O-nitro-β-D-pyranoside 10-20mmol/L

2. mensuration reagent according to claim 1 is characterized in that crown ether is 15-hat-5 ethers.

3. mensuration reagent as claimed in claim 1 is characterized in that adopting two point velocity methods, and running program is as follows: Blank Standard Sample Distilled water standard model R ₁ 40μL 1000μL 40μL 1000μL 40μL 1000μL Mixing is hatched 5min in 37 ℃, adds then R ₂ 400μL 400μL 400μL

Fully mix, hatch 0.5min in 37 ℃, read A ₁, read A after the 2min ₂, the changes delta A=A of absorbance ₂-A ₁

Calculate:

Use two standards of height, with the changing value of absorbance the concentration value of corresponding standard items is made typical curve, the concentration of the sample changing value of absorbance is per sample found from typical curve.

4. the purposes of mensuration reagent as claimed in claim 1 is used for measuring the sodion of serum.

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