CN102994612A - Liquid monomer reagent for determining content of ammonia in blood serum, preparation method and application thereof - Google Patents
Liquid monomer reagent for determining content of ammonia in blood serum, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a liquid monomer reagent for determining the content of ammonia in blood serum, a preparation method and an application thereof. The liquid monomer reagent comprises the following components: buffering solution, glutamate dehydrogenase, alpha-oxoglutarate, reduction type nicotinamide adenine dinucleotide, glucose, glucose dehydrogenase and a stabilizing agent, wherein the enzyme activity ratio of the glutamate dehydrogenase and the glucose dehydrogenase is (3-3000):1. Shown by accelerating destruction test at the temperature of 37 DEG C and 42 DEG C, the blood-ammonia liquid monomer reagent is good in stability, solves the fussy problem that a lyophilized agent needs to be lyophilized and redissolved, can be prepared and used at any time, and can be stable for 18 mouths at the temperature of 2-8 DEG C; and shown by clinic correlation test, the liquid monomer reagent is stable in blood-ammonia determination, is high in determined-value accuracy, is applicable to manual, semi-automatic and automatic detection system and is easily promoted and used in all hospitals.
Description
Technical field
The present invention relates to a kind of reagent that detects ammonia content, relate in particular to the monomer reagent of measuring ammonia content in the serum, the invention further relates to the preparation method of this monomer reagent and the application of ammonia content in detecting serum, belong to the detection field of ammonia content in the serum.
Background technology
Free Blood Ammonia Concentration is extremely low in the normal human, and normal value is (10-47) μ mol/L.Blood ammonia can be divided into endogenous ammonia and exogenous ammonia by its source.Endogenous ammonia is mainly derived from amino acid that the body internal protein produces and decomposes and the ammonia that comes through deamination in metabolic process, exogenous ammonia is mainly derived from the bacterial degradation of protein-based food in enteron aisle.Liver is the main organ that participates in except ammonia, therefore, the ammonia metabolism obstacle disease occurs, and such as hepatic coma, hepatogenic encephalopathy, hepatitis gravis, uremia etc., causes the blood ammonia rising because ammonia can not eubolism excretes; Hyperammonemia content has neurotoxicity, and the mensuration of blood ammonia is significant for diagnosis, observation and the Prognosis of the diseases such as some congenital metabolism disorders of Cirrhotic Portal Hypertension, hepatic coma, Reye's syndromes and paediatrics.
The experimental technique of measuring ammonia content in the serum has microdiffusion, colorimetry, ion exchange method, ammonia electrode method and enzyme process etc. usually.The glutamate dehydrogenase method is easy and simple to handle than additive method, is applicable to automatic biochemistry analyzer, and its reaction principle is: ammonia reacts with α-KG and NADH under the effect of glutamate dehydrogenase, generates Pidolidone and NAD
+, by the fall off rate of monitoring NADH, with the calibration solution comparison of same processing, can calculate the content of ammonia in the serum.
The AMM test kit that is applied to clinical diagnosis mostly is the glutamate dehydrogenase method, has plurality of liquid formulation and the dry plate preparations such as single, double, three reagent.The Major Difficulties of test kit is the stability problem of the stability of reagent, especially NADH.NADH has the strongest absorption peak as reducibility coenzyme commonly used at 340nm, often is used as chromogen in external diagnosis reagent.But it is unstable in the aqueous solution, and redox reaction occurs easily, thereby the reagent effect phase is shortened, and is difficult to promote the use of in hospital.Blood ammonia dry plate preparation has solved the shortcoming of liquid dosage form poor stability well, but reagent needs freeze-drying process when preparing, and need to redissolve process during application, and is comparatively loaded down with trivial details, also increased reagent cost.
Application number is that 200610030784.8 Chinese invention patent discloses a kind of reducibility coenzyme composite stabilizer, relate to the one package stabilizers such as damping fluid, stablizer, organic solvent, sequestrant, sanitas and inorganic salt, reducibility coenzyme NADH and NADPH are had high stability; Disclose the reagent of stabilizing reducing cozymase and NADPH but application number is 00111788.2 Chinese invention patent, added metal ion chelation agent, can under wider pH value condition, stop the oxidation of NADH and NADPH.
But in liquid reagent that blood ammonia detects especially single reagent, the adding of depending merely on these stablizers can not guarantee the stable of NADH in the aqueous solution or NADPH long-time (at least 18 months), is difficult to promote the use of at various big hospital.Application number is the powdered reagent that 200410043900.0 Chinese invention patent application discloses a kind of Determination of Ammonia in Plasma with Colorimetry, reaches 2 years stationary phase; Application number is that 200410041963.2 Chinese invention patent application discloses a kind of liquid reagent, adopts different enzyme reaction principles, but does not relate to the validity period of reagent.Therefore lack at present a kind of long blood ammonia tracer liquid single reagent of effect phase of preserving.
Summary of the invention
Main purpose of the present invention provides good stability, preserves long blood ammonia tracer liquid single reagent of effect phase;
Another object of the present invention provides a kind of method for preparing described blood ammonia tracer liquid single reagent.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of blood ammonia tracer liquid single reagent comprises: damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase, stablizer.
Determination of blood ammonia is the reaction of falling of NADH, and glutamate dehydrogenase is controlled the speed of response of this main reaction; Use the glutamate dehydrogenase reactive system, reducibility coenzyme (NADH) is oxidized to oxidisability coenzyme (NAD+), by measuring the rate of descent direct reaction Blood Ammonia Concentration of reducibility coenzyme under main ripple 340nm.Bad because of reducibility coenzyme stability in solution, easily be oxidized to NAD+, it is very fast to cause the reagent blank absorbancy to descend, and the effect phase shortens, and is difficult to promote the use of clinically; Have in view of that, the present invention detects at blood ammonia and introduces the NADH regeneration system rapidly in the single reagent, make NAD+ oxidized in the monomer reagent again be converted into NADH, live ratio and reaction times by the enzyme of regulating glutamate dehydrogenase and Hexose phosphate dehydrogenase, can make this regenerative response not affect main reaction carries out, the reagent measured value is accurate, and can keep stable for a long time.The enzymatic reaction meeting of Hexose phosphate dehydrogenase causes the rising of NADH concentration in the regeneration system rapidly, and Hexose phosphate dehydrogenase has certain restraining effect to glutamate dehydrogenase, therefore inappropriate enzyme affects detected result than regular meeting, and the concentration ratio of two kinds of enzymes of control is to prepare measured value accurately and the key of the reagent of good stability.The present invention finally determines by a large amount of tests; in the enzyme of monomer reagent Glutamic Acid desaturase and Hexose phosphate dehydrogenase is lived the scope of ratio at 3-3000:1; can effectively guarantee reducibility coenzyme stability in solution, and then guarantee the accuracy of final measurement result.
In order to reach better technique effect, consumption or the concentration of each component are preferably in per 1 liter of monomer reagent: damping fluid 50-200mM, glutamate dehydrogenase 500-40000 U, α-ketoglutaric acid 2-30 mM, nicotinamide adenine dinucleotide reduced (NADH) 0.1-0.4 mM, glucose 1-500 mM, Hexose phosphate dehydrogenase 5-1000 U, stablizer account for the 0.05-5% of monomer reagent cumulative volume; Surplus is distilled water.
Described damping fluid includes but are not limited at least a in Tutofusin tris (Tris) damping fluid, 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid (CAPSO) damping fluid, 3-(cyclohexylamino) propanesulfonic acid (CAPS) damping fluid, morpholino propane sulfonic acid (MOPS) damping fluid or N-three (methylol) methyl-3-amino propane sulfonic acid (TAPS) damping fluid; The concentration of described damping fluid is preferably 50-200mM; The pH of described damping fluid is preferably 7.0-11.0, more preferably 8.0-9.6.
Stablizer described in the monomer reagent of the present invention comprise but be not limited among bovine serum albumin, ethylene glycol, glycerine, EDETATE SODIUM/sylvite or the TritonX-100 any one or multiple.
Another object of the present invention provides a kind of method for preparing described blood ammonia tracer liquid single reagent, may further comprise the steps:
Damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stablizer are dissolved in the distilled water, wherein, consumption or the concentration of each component is controlled to be in per 1 liter of monomer reagent: damping fluid 50-200mM, glutamate dehydrogenase 500-40000 U, α-ketoglutaric acid 2-30 mM, nicotinamide adenine dinucleotide reduced (NADH) 0.1-0.4 mM, glucose 1-500 mM, Hexose phosphate dehydrogenase 5-1000 U, stablizer account for the 0.05-5% of monomer reagent cumulative volume; Surplus is distilled water.
Another object of the present invention has provided a kind of method that described blood ammonia tracer liquid single reagent detects Blood Ammonia Concentration in the serum of using, and comprising: detected sample is joined blood ammonia tracer liquid single reagent react; Reactant is placed on ultraviolet/visible spectrophotometer or half, the automatic clinical chemistry analyzer, detect the decline of absorbancy under the 340nm wavelength, calculate Blood Ammonia Concentration.
Wherein detected sample and blood ammonia tracer liquid single reagent ratio are between 1/100-1/10; The described reaction times is preferably 1-20 minute, can detect in 20-45 ℃ of scope, and Optimal Temperature is 37 ℃.
For example on Olympus AU400 full automatic biochemical apparatus, setup parameter is: sample and ratio of reagents are 20:200, main ripple 340nm, and complementary wave 410nm, the Direction of Reaction is for falling reaction, and read point is made as the 1-10 point, and methodology is end-point method.
37 ℃ and 42 ℃ accelerate the failure test and clinical correlations are tested and to be shown, the prepared blood ammonia liquid single-reagent of the present invention has good stability, solved the freeze-dried loaded down with trivial details problem that needs freeze-drying and redissolution, can be now with the current, (2-8) ℃ can stablize 18 months, measured value is accurate, is applicable to manual, semi-automatic, automatic checkout system, is easy to promote the use of at various big hospital.
Can find out that from the clinical correlation curve blood ammonia liquid single-reagent that the present invention is prepared and Dialab reagent measured value dependency are good, so the prepared determination of blood ammonia liquid single-reagent of the present invention is stablized measured value accuracy height.
Description of drawings
The blood ammonia liquid single-reagent that Fig. 1 embodiment of the invention 1 is prepared and the clinical correlation curve of Dialab reagent.
The blood ammonia liquid single-reagent that Fig. 2 embodiment of the invention 2 is prepared and the clinical correlation curve of Dialab reagent.
The blood ammonia liquid single-reagent that Fig. 3 embodiment of the invention 3 is prepared and the clinical correlation curve of Dialab reagent.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
The preparation of embodiment 1 blood ammonia tracer liquid single reagent
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 1 in per 1 liter of monomer reagent.
Table 1
| Component | Concentration |
| Tris damping fluid (pH8.5) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 8000U |
| Hexose phosphate dehydrogenase | 200U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 40 |
The preparation of embodiment 2 blood ammonia tracer liquid single reagents
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 2 in per 1 liter of monomer reagent.
Table 2
| Component | Concentration |
| Tris damping fluid (pH9.0) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 600U |
| Hexose phosphate dehydrogenase | 200U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 3 |
The preparation of embodiment 3 blood ammonia tracer liquid single reagents
CAPSO damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 3 in per 1 liter of monomer reagent.
Table 3
| Component | Concentration |
| CAPSO（pH9.2） | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 15000U |
| Hexose phosphate dehydrogenase | 5U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 3000 |
The preparation of embodiment 4 blood ammonia tracer liquid single reagents
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 4 in per 1 liter of monomer reagent.
Table 4
| Reagent R component | Concentration |
| Tris damping fluid (pH8.2) | 100mM |
| α-ketoglutaric acid | 6mM |
| NADH | 0.29mM |
| Glucose | 10mM |
| Glutamate dehydrogenase | 10000U |
| Hexose phosphate dehydrogenase | 100U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.4% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase | 100 |
The preparation of embodiment 5 blood ammonia tracer liquid single reagents
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase, bovine serum albumin and glycerine are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 5 in per 1 liter of monomer reagent.
Table 5
| Reagent R component | Concentration |
| Tris damping fluid (pH8.6) | 150mM |
| α-ketoglutaric acid | 10mM |
| NADH | 0.32mM |
| Glucose | 20mM |
| Glutamate dehydrogenase | 15000U |
| Hexose phosphate dehydrogenase | 10U |
| Bovine serum albumin | 0.1% |
| Glycerine | 2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase | 1500 |
The comparative example 1
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH) and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 6 in per 1 liter of monomer reagent.
Table 6
| Component | Concentration |
| Tris damping fluid (pH8.5) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glutamate dehydrogenase | 8000U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
The comparative example 2
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH) and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 7 in per 1 liter of monomer reagent.
Table 7
| Reagent R component | Concentration |
| Tris damping fluid (pH9.0) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 025mM |
| Glutamate dehydrogenase | 600U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
The preparation of comparative example's 3 blood ammonia tracer liquid single reagents
CAPS damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH) and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 8 in per 1 liter of monomer reagent.
Table 8
| Component | Concentration |
| CAPS（pH9.6） | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glutamate dehydrogenase | 15000U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
The preparation of comparative example's 4 blood ammonia tracer liquid single reagents
CAPSO damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH) and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 9 in per 1 liter of monomer reagent.
Table 9
| Component | Concentration |
| CAPSO damping fluid (pH9.2) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glutamate dehydrogenase | 9000U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
The comparative example 5
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 10 in per 1 liter of monomer reagent.
Table 10
| Component | Concentration |
| Tris damping fluid (pH8.5) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 80U |
| Hexose phosphate dehydrogenase | 40U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 2 |
The comparative example 6
Tris damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 11 in per 1 liter of monomer reagent.
Table 11
| Component | Concentration |
| Tris damping fluid (pH8.8) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 40U |
| Hexose phosphate dehydrogenase | 40U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 1 |
The preparation of comparative example's 7 blood ammonia tracer liquid single reagents
CAPSO damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 12 in per 1 liter of monomer reagent.
Table 12
| Component | Concentration |
| CAPSO damping fluid (pH9.6) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 9300U |
| Hexose phosphate dehydrogenase | 3U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 3100 |
The preparation of comparative example's 8 blood ammonia tracer liquid single reagents
CAPSO damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced (NADH), glucose, Hexose phosphate dehydrogenase and stabilizer T ritonX-100 are dissolved in the distilled water; The consumption of each component or concentration are shown in the table 13 in per 1 liter of monomer reagent.
Table 13
| Component | Concentration |
| CAPSO damping fluid (pH9.2) | 50mM |
| α-ketoglutaric acid | 20mM |
| NADH | 0.25mM |
| Glucose | 5mM |
| Glutamate dehydrogenase | 10500U |
| Hexose phosphate dehydrogenase | 3U |
| Bovine serum albumin | 0.1% |
| TritonX-100 | 0.2% |
| Glutamate dehydrogenase/Hexose phosphate dehydrogenase hydrogen enzyme | 3500 |
The stability test of test example 1 blood ammonia tracer liquid monomer reagent of the present invention
1, test materials
(1) for the examination material: the blood ammonia tracer liquid monomer reagent that embodiment 1-5 is prepared;
(2) control material: the reagent that comparative example 1-8 is prepared;
2, test method
To place separately 37 ℃ and the 42 ℃ experiments that accelerate the failure for examination material and control material, regularly take out and measure Quality Control, compare reagent stability, the Quality Control of two levels is all according to the preparation rules autogamy of the registered blood ammonia quality control product in Beijing Leaderman Biochemistry Co., Ltd, the empirical tests Quality Control has good stability, and validity period was at 24 months.Quality Control ℃ is closed the lid preservation in (2-8) within two weeks of the test that accelerates the failure, and has good stability.
3, test-results
Test-results sees Table 14-16.
Table 14
Table 15
Table 16
From the table 14-16 data as seen: 37 ℃, 42 ℃ accelerated the failure respectively 14 days, 3 days, it is more stable that embodiment 1-3 reagent is measured the Quality Control value, and the prepared reagent of comparative example carries out middle measured value appearance decline in various degree 37 ℃, 42 ℃ rupture tests, can judge also that from the lowering speed of Blank the NADH degradation speed is slower the embodiment 1-3 reagent, well below the degradation speed of its contrast agents.According to common rule, biological reagent was whenever stablized 1 day at 37 ℃, was equivalent to ℃ stablize 1.5 months at (2-8), whenever stablized 1 day for 42 ℃, was equivalent at (2-8) ℃ stable 5-6 month, and therefore liquid single-reagent of the present invention ℃ can be stablized 18 months at (2-8) at least.
Tested by 37 ℃ and 42 ℃ accelerate the failure test and clinical correlations and to show, blood ammonia liquid single-reagent according to preparation method's preparation provided by the invention has good stability, solved the freeze-dried loaded down with trivial details problem that needs freeze-drying and redissolution, can be now with the current, (2-8) ℃ can stablize 18 months, measured value is accurate, is applicable to manual, semi-automatic, automatic checkout system, is easy to promote the use of at various big hospital.
The clinical correlation test of test example 2 blood ammonia tracer liquid monomer reagent
1, test materials
(3) for the examination material: the blood ammonia tracer liquid monomer reagent that embodiment 1-5 is prepared;
(4) control material: outsourcing Dialab determination of blood ammonia reagent;
2, test method
To measure simultaneously 50 routine clinical patient samples for examination material and control material, carry out the clinical correlation test.
3, test-results
Test-results sees Table 17 and Fig. 1-3.
Table 17
Can find out from the measured value of table 4 and the clinical correlation curve of Fig. 1-3, the determination of blood ammonia liquid single-reagent that embodiment 1-3 is prepared and Dialab reagent measured value dependency are good, therefore the prepared determination of blood ammonia liquid single-reagent of the present invention is stablized, and the measured value accuracy is high.
Claims (10)
1. a liquid single-reagent of measuring ammonia content in the serum is characterized in that, comprising: damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced, glucose, Hexose phosphate dehydrogenase and stablizer.
2. according to liquid single-reagent claimed in claim 1, it is characterized in that: the enzyme of glutamate dehydrogenase and the Hexose phosphate dehydrogenase ratio of living is 3-3000:1.
3. according to liquid single-reagent claimed in claim 1, it is characterized in that consumption or the concentration of each component are in per 1 liter of monomer reagent: damping fluid 50-200mM, glutamate dehydrogenase 500-40000 U, α-ketoglutaric acid 2-30 mM, nicotinamide adenine dinucleotide reduced 0.1-0.4 mM, glucose 1-500 mM, Hexose phosphate dehydrogenase 5-1000 U, stablizer account for the 0.05-5% of monomer reagent cumulative volume; Surplus is distilled water.
4. according to claim 1 or 3 described liquid single-reagents, it is characterized in that: described damping fluid include but are not limited in tris buffer, 3-(hexahydroaniline)-2-hydroxyl-1-propanesulfonic acid damping fluid, 3-(cyclohexylamino) propanesulfonic acid damping fluid, morpholino propane sulfonic acid damping fluid or N-three (methylol) methyl-3-amino propane sulfonic acid damping fluid any one or multiple.
5. according to liquid single-reagent claimed in claim 5, it is characterized in that: the concentration of described damping fluid is 50-200mM.
6. according to claim 4 or 5 described liquid single-reagents, it is characterized in that: the pH of described damping fluid is 7.0-11.0, is preferably 8.0-9.6.
7. according to claim 1 or 3 described liquid single-reagents, it is characterized in that: described stablizer comprise but be not limited among bovine serum albumin, ethylene glycol, glycerine, EDETATE SODIUM/sylvite or the TritonX-100 any one or multiple.
8. method for preparing any one described liquid single-reagent of claim 1-3, may further comprise the steps: damping fluid, glutamate dehydrogenase, α-ketoglutaric acid, nicotinamide adenine dinucleotide reduced, glucose, Hexose phosphate dehydrogenase and stablizer are dissolved in the distilled water, and get final product.
9. it is characterized in that in accordance with the method for claim 8: consumption or the concentration of each component are controlled to be in per 1 liter of monomer reagent: damping fluid 50-200mM, glutamate dehydrogenase 500-40000 U, α-ketoglutaric acid 2-30 mM, nicotinamide adenine dinucleotide reduced 0.1-0.4 mM, glucose 1-500 mM, Hexose phosphate dehydrogenase 5-1000 U, stablizer account for the 0.05-5% of monomer reagent cumulative volume; Surplus is distilled water.
10. any one described liquid single-reagent of claim 1-3 detects the purposes in the ammonia content reagent in the serum in preparation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103543117A (en) * | 2013-10-21 | 2014-01-29 | 大连市沙河口区中小微企业服务中心 | Measurement method of ammonia content in serum |
| CN105784614A (en) * | 2016-03-10 | 2016-07-20 | 威海威高生物科技有限公司 | Blood ammonia determination reagent and preparation method thereof |
| CN107782683A (en) * | 2016-08-27 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of glutamate dehydrogenase enzyme detection kit |
| CN114773416A (en) * | 2022-04-01 | 2022-07-22 | 深圳瑞亚力集团有限公司 | NADH protectant and its prepn |
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| CN105784614A (en) * | 2016-03-10 | 2016-07-20 | 威海威高生物科技有限公司 | Blood ammonia determination reagent and preparation method thereof |
| CN107782683A (en) * | 2016-08-27 | 2018-03-09 | 山东博科生物产业有限公司 | A kind of glutamate dehydrogenase enzyme detection kit |
| CN114773416A (en) * | 2022-04-01 | 2022-07-22 | 深圳瑞亚力集团有限公司 | NADH protectant and its prepn |
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