CN1500884A - Nicotinamide agent and preparation method thereof - Google Patents
Nicotinamide agent and preparation method thereof Download PDFInfo
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- CN1500884A CN1500884A CNA021392986A CN02139298A CN1500884A CN 1500884 A CN1500884 A CN 1500884A CN A021392986 A CNA021392986 A CN A021392986A CN 02139298 A CN02139298 A CN 02139298A CN 1500884 A CN1500884 A CN 1500884A
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Abstract
The present invention is reducing nicotine amide coenzyme as enzyme testing reagent for testing body fluid. The enzyme testing reagent of the present invention includes oxidizing nicotine amide coenzyme, corresponding enzyme and substrate. The oxidizing coenzyme is reduced into reducing nicotine amide coenzyme slowly to test the tested matter. The reagent includes alanine aminotransferase reagent, aspartic acid aminotransferase reagent, urea reagent, ammonia reagent, creatinine reagent, CO2 reagent, etc. Owing to constant creation of nicotine amide coenzyme, the present invention has greatly raised reagent stability and greatly lowered reagent cost.
Description
Technical field
The present invention relates to adopt reduced form nicotinamide coenzyme (NADH or NADPH) to measure reagent and method for making thereof.
Background technology
The analyte that adopts reduced form nicotinamide coenzyme (NADH or NADPH) to measure in the body fluid sample is a widely used method, and the degree of oxidation of NADH or NADPH in the reagent is directly proportional with the concentration of analyte in the sample.According to analytes different in the sample, adopt corresponding enzyme reaction mechanisms, through the enzyme-to-substrate reaction, NADH or NADPH are oxidized in the reagent, the absorbancy of reactive system changes, and by the absorbancy changing value of assaying reaction system, just can calculate the concentration of analyte in this.
The mensuration of ammonia concentration in the plasma sample for example, the ammonia in the blood plasma generate glutaminate with the α-Tong Wuersuan reaction under the effect of L-glutamic acid desaminase, NADPH is oxidized to NADP simultaneously
+, reactive system descends at the absorbance at 340nm place, by measuring the drop-out value of 340nm place absorbancy.Just can calculate the concentration of ammonia in the sample.
The mensuration of alanine aminotransferase concentration in the serum sample for example again, alanine aminotransferase catalysis L-Ala amino is converted to α-Yang Daiwuersuan in the sample, generates pyruvic acid and L-L-glutamic acid; The pyruvic acid that generates is reduced to L-lactic acid by the serum lactic dehydrogenase in the reagent, and NADH is oxidized to NAD simultaneously, and reactive system descends at the absorbance at 340nm place.By measuring the drop-out value of 340nm place absorbancy, just can calculate the concentration of alanine aminotransferase in the sample.
L-third presses acid+α-ammonia for pentanedioic acid → pyruvic acid+L-L-glutamic acid
Pyruvic acid+NADH → L-lactic acid+NAD
+
But because NADH or NADPH are unstable in this fermentoid reagent, powdered reagent or the liquid double reagents of adopting in the practical application more.Powdered reagent is by keeping the drying regime of reagent, and the moisture content of reagent generally is lower than 20%, keeps stablizing of NADH or NADPH; Liquid double reagent contains NADH or NADPH reagent pH value partly by adjustment, and this part reagent pH value generally between 7.5-11.5, keeps stablizing of NADH or NADP.
NADH or NADPH are in the application of liquid single agents, Joseph De Giorgio etc. are (referring to United States Patent (USP): 5804402 and 5705356) invented the regeneration enzyme system that adds NADH or NADPH in measuring reagent, as glucose-6-phosphate dehydrogenase (G6PD) and non-specificity substrate glucose thereof, with NADH or NADPH in the reagent preservation process, the NAD of oxidized generation or NADP slowly are reduced to NADH or NADPH again, thereby keep measuring effective NADH or NADPH concentration in the reagent.In their patent, do not add NAD or NADP when measuring reagent production or preparation,, add NADH or NADPH still according to traditional reagent production or compound method, the regeneration enzyme system that embeds only is used for the unidirectional regeneration after NADH or the NADPH oxidation, does not generate new NADH or NADPH.
Richard A, Kaufman etc. are (referring to United States Patent (USP): 5589438) utilize glucose-6-phosphate dehydrogenase (G6PD) and specificity substrate G-6-P thereof, original position generates reduced coenzyme fast in reagent mensuration process, the mensuration that is used for analyte, the speed of its generation need be greater than the speed that is reoxidized by the analyte enzyme system.The mensuration of analyte is when generating reduced coenzyme or carries out later on, the volumetric molar concentration of G-6-P is lower than the volumetric molar concentration of the oxidized coenzyme that is used to generate reduced coenzyme in the reagent, therefore after measuring reagent and being mixed with single liquid reagent, the same instability in the NADH of generation or NADPH and the common mensuration reagent.This method is used for liquid double reagent more.In their patent, the speed that coenzyme after will reducing with respect to the analyte enzyme system reoxidizes, the speed that generates reduced coenzyme can not be finished with absolute fast speed, therefore, the standard samples that need measure concentration known when measuring analyte content in the sample contrasts calculating, thereby obtains the content of analyte in the sample.Adopt this method to be difficult in actual applications the enzyme content in the body fluid sample is measured.
Summary of the invention
Of the present inventionly be to provide a kind of good stability in purpose, nicotinamide coenzyme reagent and method for making thereof that cost is low.
Mensuration reagent of the present invention adds enzyme-substrate-NAD or enzyme-substrate-NADP system when producing or preparing, effective NADH or NADPH content are generated by this system in the mensuration reagent.The present invention uses the enzymic measuring reagent that reduced form nicotinamide coenzyme (NADH or NADPH) is measured analyte in the body fluid sample, be characterized in production or process for preparation, the enzymic measuring reagent of indication of the present invention does not add this reduced form nicotinamide coenzyme, but add oxidized form nicotinamide coenzyme (NAD or NADP), by the slow circulation of enzyme-substrate-NAD embedded in the reagent or enzyme-substrate-NADP system oxidized coenzyme is reduced to the reduced form nicotinamide coenzyme, when the reduced form nicotinamide coenzyme that is reduced reached certain concentration, the enzyme process reagent of indication of the present invention just can be applicable to measure the concentration of analyte.Because the speed of embedded enzyme and substrate system reduction NAD or NADP significantly is lower than analyte and mensuration enzyme or substrate system thereof the rate of oxidation to NADH or NADPH, therefore this embedded enzyme and substrate system do not influence the assaying reaction of analyte.
The present invention discloses a kind of preparation of mensuration reagent and production method of saving and improveing, do not use NADH or NADPH during owing to reagent production preparation, thereby greatly reduce the raw materials cost of reagent.Adopt the reagent of this production method preparation to comprise alanine aminotransferase reagent, aspartate amino transferase reagent, urea reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent etc.
The present invention discloses a kind of mensuration reagent of improvement, this class reagent comprises that the phenylalanine ammonia base changes enzyme reagent, amino enzyme reagent, urea reagent, ammonia reagent, creatinine reagent and the carbonic acid gas reagent etc. of changeing of aspartic acid.
The generation system of adoptable NADH and NADPH comprised Hexose phosphate dehydrogenase-glucose-NAD system, serum lactic dehydrogenase-pyruvic acid-NAD system, glycerol dehydrogenase-glycerine-NAD system and (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-NAD or (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-NADP system etc. during reagent was measured.In order to reduce the generating rate of NADH or NADPH effectively.Do not influence the mensuration performance of reagent, can adjust the concentration of substrate in the above-mentioned generation system, the speed that makes its generation (reduction) NADHA or NADPH does not influence the rate of oxidation of NADH or NADPH when measuring analyte.For example, Hexose phosphate dehydrogenase-glucose-NAD system can be adjusted to serum lactic dehydrogenase-Hexose phosphate dehydrogenase-wood sugar-NAD system, serum lactic dehydrogenase-pyruvic acid-NAD system can be adjusted to lactic dehydrogenase desaturase-α-Tong Wuersuan-NAD system, glycerol dehydrogenase-glycerine-NAD system can be adjusted to glycerol dehydrogenase-ethylene glycol-NAD system, and (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-NAD or (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-NADP system can be adjusted to (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NAD or (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-NADP system can be adjusted to (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NAD or (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NADP system etc.Consider that the substrate material that may contain in the sample when measuring sample has influence on the generating rate of NADH or NADPH, and then influencing the assaying reaction of analyte, the system of preferred usefulness is (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NAD or (glucose-phosphate dehydrogenase)-glucose-NADP system in above-mentioned each system.
The reduced coenzyme that reduced coenzyme generation system among the present invention generates is after reaching certain concentration, can carry out the concentration determination of analyte with other mensuration composition of reagent, this moment, the reduced coenzyme generation system can become the formation reaction of reduced coenzyme fully, also can still continue to generate reduced coenzyme.These characteristics have guaranteed to adopt the present invention can prepare or produce the liquid reagent of single stable.The reduced coenzyme generation system generates the speed that the speed of reduced coenzyme can be any not interference measurement reaction among the present invention, the speed of effective NADH of the generation that can adopt or NADPH concentration in 0.01-365 days between, the absorbancy of the reduced coenzyme that generates is within the 0.5-5.0 scope, wavelength 340nm, optical path 10mm; Generating rate was less than 0.01 day, and generation system can influence the mensuration performance of mensuration system, and generating rate was greater than 365 days, and the preparation of measuring reagent is very difficult.More suitable generating rate is within 1-30 days, and the absorbancy of the reduced coenzyme of generation is 0.8-3.0, wavelength 340nm, optical path 10mm, the preferential generating rate that adopts is in 1-7 days, the absorbancy of the reduced coenzyme of generation is 1.1-2.5, wavelength 340nm, optical path 10mm.The concentration range of glucose-6-phosphorus desaturase is between 100-100000U/L, and more suitable scope is between 1,000 one 10000U/L, and the scope of preferentially selecting for use is between the 2000-8000U/L scope.The range of choice of glucose concn is bigger, and comparatively suitable scope is between 1-500mM, and the scope of preferentially selecting for use is between 0.20-0.50mM.
The mensuration reagent of the present invention's preparation, NADH wherein or the content of NADPH also can adopt the reduced coenzyme generation system among the present invention partly to generate, and other parts still adopt traditional method to add, to shorten the rise time of reduced coenzyme, help the market supply of flexible adaptation agent.
The reduced coenzyme generation system can add damping fluid, sanitas, heavy metal complexing agent, tensio-active agent and enzyme stabilizers in conjunction with measuring reagent.Damping fluid commonly used comprises Tris damping fluid, imidazole buffer, di-alcohol damping fluid, phosphate buffered saline buffer, Good ' s damping fluid: as HEPES, TES, BES, TAPS, TAPSO, POPSO, DIPSO, MOPS, MOPSO, PIPES, HEPPSO, TRICINE, AMPD, CHES, AMPSO, CAPSO etc., wherein the application of Tris damping fluid is comparatively general, the damping fluid of preferentially selecting for use among the present invention is the Tris damping fluid, the concentration of damping fluid is typically chosen within the 10-200mM scope, and the 50-100mM scope is more suitable.The sanitas that can preferentially adopt is a sodium azide, adopts the other biological sanitas then better.Heavy metal complexing agent comprises EDTA, EGTA, HEDTA etc., and the heavy metal complexing agent of preferentially selecting for use among the present invention is EDTA.Enzyme stabilizers adopts bovine serum albumin among the present invention, and mannitol, phosphoric acid salt are used for stablizing glucose-6-phosphate dehydrogenase (G6PD) and improve its reactive behavior.In case of necessity, can add tensio-active agent in the reagent, as TritonX-100 etc.
When adopting the present invention to prepare enzyme process reagent, single liquid reagent can be mixed with, also liquid double reagent can be mixed with as required.Owing to do not use NADH or NADPH, the reagent preparation requires simple, can not consider the dissolving order of raw material.Suggestion reagent is when preparation, earlier the weighing of generalization compound is finished and add and prepare container, add the required pure water of preparation again, be stirred to dissolving fully gently, add other activeconstituents and zymoprotein again, be stirred to dissolving fully gently, seal rearmounted 2-8 ℃, allow the reduced coenzyme generation system slowly generate NADH or NADPH.When the growing amount of NADH or NADPH reached certain concentration, reagent just can be used for measuring.During the obtaining liq double reagent, the reduced coenzyme generation system of employing should be in same reagent part as (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NAD or (glucose-6-phosphate dehydrogenase (G6PD))-glucose-NADP.
Give an example 1: prepare alanine nitrogen based transferase reagent, form by every liter content by following component,
7.15,37 ℃ of Tris buffer solution phs, 10～158g
L～L-Ala 34.0～45.0g
Serum lactic dehydrogenase 2000～5000u
α～ketoisocaproic 0.5～3.5g
EDTA.Na
2.2H
2O 1.0～0.3g
NAD 0.1～0.3g
D～glucose 15.0～21.0g
Glucose～6～phosphate dehydrogenase (leuconos toc), 1000～4000u
Dipotassium hydrogen phosphate 0.3～1.3g
Sodium azide 0.1～1.0g
According to the method for above-mentioned reagent preparation solution, after above alanine aminotransferase reagent preparation is finished, put 2～8 ℃, 1～3 day, reagent blank absorbancy 〉=1.10,340nm, 10mm optical path. when measuring sample, adopt rate method, sample reagent is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Give an example 2: preparation aspartate amino transferase reagent, form by every liter content by following component,
7.50,37 ℃ of Tris buffer solution phs, 10-15g
L～aspartic acid 34.0-43.0g
Malate dehydrogenase (malic acid dehydrogenase) 100-600u
Lactic dehydrogenase 10 00-4000u
α～ketoisocaproic 0.5-4.5g
EDTA.Na
2.2H
2O 1.0-3.0g
NAD 0.1-0.3g
D～glucose 15.0-21.0g
Glucose～6～phosphate dehydrogenase (leuconos toc) 1000-4500u
Dipotassium hydrogen phosphate 0.3-2.0g
Sodium azide 0.1-1.0g
According to the method for above-mentioned reagent preparation solution, after above aspartate amino transferase reagent preparation is finished, put 2～8 ℃, 1～5 day, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path. when measuring sample, adopt rate method, sample reagent is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Give an example 3: the preparation urea reagent, form by every liter content by following component,
7.50,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum diacid 0.05～5.0
Adenosine diphosphate potassium salt 0～0.8
Glutamate dehydrogenase (microbe-derived) 500～6000u
Urase 5000～15000u
NAD 0.1～0.3g
D～glucose 3～21g
Glucose～6～phosphate dehydrogenase (leuconos toc) 1000～4000m
Dipotassium hydrogen phosphate 0.3～2.0g
EDTA.Na
2.2H
2O 0.1～0.1g
Sodium azide 0.1～1.0g
According to the method for above-mentioned reagent preparation solution, after above urea reagent preparation is finished, put 2-8 ℃, 1-7 days, reagent blank absorbancy 〉=1.20,340nm, 10mM optical path.When measuring sample or calibration object, adopt two-point method, the sample reagent ratio is 1: 100,37 ℃ of temperature, and 30 seconds time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Give an example 4: the preparation ammonia reagent, form by every liter content by following component,
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum albumin 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000u
NADP 0.1～0.35g
D～glucose 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 1000～4000u
Potassium primary phosphate 0.5～2.0g
EDTA.Nα
2.2H
2O 0.1～1.0g
Sodium azide 0.1～1.0g
According to the method for above-mentioned reagent preparation solution, absorbancy behind the reagent sky 〉=1.20,340nm, 10mM optical path were finished in above ammonia reagent preparation rearmounted 2-8 ℃ 1-7 days.When measuring sample or calibration object, adopt two-point method, the sample reagent ratio is 1: 10,37 ℃ of temperature, and 0 second time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Give an example 5: preparation creatinine liquid double reagent, form by every liter content by following component,
Reagent 1:
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic, 0.5～3.5g
Bovine serum albumin, 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000u
NADP 0.1～0.5g
D～glucose 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 1000～4000u
Potassium primary phosphate 0.3～2.0g
EDTA.Na
2.2H
2O 0.1～1.0g
Sodium azide 0.1～1.0g
According to the method for above-mentioned reagent preparation solution, after the preparation of above ammonia reagent is finished, put 2-8 ℃ 1-7 days, absorbancy behind the reagent sky 〉=1.20,340nm, 10mm optical path.
Reagent 2:
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum albumin 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000
EDTA.Na
2.2H
2O 0.1～1.0g
Creatinine imino-desaturase 500～4000
Mannitol 3～15g
Sodium azide 0.1～1.0g
Method according to above-mentioned reagent preparation solution, configuration creatinine reagent 2 is put 2-8 ℃ of preservation, when measuring sample, adopt two-point method, reagent 1: sample: the ratio of reagent 2 is 200: 20: 50,37 ℃ of temperature, and reagent 1 adds sample or hatched 300 seconds in the mensuration temperature calibration product back, adding reagent 2 begins to measure, 0 second time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Creatinine reagent preferably selects for use the liquid double reagent type to help getting rid of the interference of ammonia in the sample.According to principle of the present invention, creatinine reagent also can be mixed with the liquid single agents, and this moment, the interference of ammonia was left in the basket in the sample when measuring.
Give an example 6: preparation carbonic acid gas reagent, form by every liter content by following component,
The Tris damping fluid, PH8.0,37 ℃, 10.0～15.0g
Phosphoenolpyruvic acid 0.1～0.5g
Phosphoric acid enol pyruvic acid carboxylase 200～1000u
Malate dehydrogenase (malic acid dehydrogenase) 500～3000u
Bovine serum albumin 0.05～5.0g
Sal epsom 0.5～3.0g
Sodium oxamate 0.1～0.4g
NADH 0.1～0.35g
NAD 0.1～0.35g
D～glucose 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 2000～10000u
Potassium primary phosphate 0.3～20g
Sodium azide 0.1～1.0g
According to the method for above-mentioned reagent preparation solution, after above carbonic acid gas reagent preparation is finished, put absorbancy behind the reagent sky 〉=1.20,340nm, 10mm optical path the 2-8 ℃ of airtight 1-7 of leaving standstill days.When measuring sample or calibration object, adopt end-point method, the sample reagent ratio is 1: 200,37 ℃ of temperature, and minute 300 seconds, body fluid sample or calibration product read the 1st absorbance after adding reagent at once, read the 2nd absorbance in the time of 300 seconds.
Specific embodiment
Embodiment 1: prepares alanine nitrogen based transferase reagent, forms by every liter content by following component,
Tris damping fluid 100mM PH7.15,37 ℃,
L～L-Ala 500mM,
Serum lactic dehydrogenase 2.7ku/L,
α～ketoisocaproic 15mM,
EDTA.Na
2.2H
2O 60mM
NAD 0.27mM
D～glucose 50mM.
Glucose～6～phosphate dehydrogenase (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM
Sodium azide 1.5mM
According to the method for mentioned reagent solution, after above alanine aminotransferase reagent preparation is finished, put 2～8 ℃, 1～3 day, reagent blank absorbancy 〉=1.10,340nm, 10mm optical path.When measuring sample, adopt rate method, sample reagent is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Embodiment 2: preparation aspartate amino transferase reagent, form by every liter content by following component,
Tris damping fluid 80mM, PH7.50,37 ℃,
L～aspartic acid 240mM,
Malate dehydrogenase (malic acid dehydrogenase) 600U/L
Lactic dehydrogenase 10 00U/L
A～ketoisocaproic 12mM,
EDTA.Na
2.2H
2O 6mM
NAD 0.28mM
D～glucose 80mM.
Glucose～6～phosphate dehydrogenase (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM
Sodium azide 1.5mM
According to the method for above-mentioned reagent preparation solution, after above aspartate amino transferase reagent preparation is finished, put 2～8 ℃, 1～5 day, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path.When measuring sample, adopt rate method, sample reagent is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Embodiment 3: the preparation urea reagent, form by every liter content by following component,
7.50,37 ℃ of Tris buffer solution phs, 14.54g
α～ketoisocaproic 1.43g
Bovine serum diacid 5.0g
Adenosine diphosphate potassium salt 0.65
Glutamate dehydrogenase (microbe-derived) 550u
Urase 12000u
NAD 0.192g
D～glucose 18.0g
Glucose～6～phosphate dehydrogenase (leuconos toc) 3700u
Dipotassium hydrogen phosphate 0.87g
EDTA.Na
2.2H
2O 0.37g
Sodium azide 0.26g
According to the method for above-mentioned reagent preparation solution, after the preparation of above urea reagent is finished, put 2-8 ℃ 1-7 days, reagent blank absorbancy 〉=1.20,340nm, 10mM optical path.When measuring sample or calibration object, adopt two-point method, the sample reagent ratio is 1: 100,37 ℃ of temperature, and 30 seconds time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Embodiment 4: the preparation ammonia reagent, form by every liter content by following component,
8.0,37 ℃ of Tris buffer solution phs, 12.0g
α～ketoisocaproic 1.43g
Bovine serum albumin 5.0g
Adenosine diphosphate potassium salt 1.25g
Glutamate dehydrogenase (beef liver source) 4500u
NADP 0.29g
D～glucose 7.21g
Glucose～6 phosphate dehydrogenases (leuconos toc) 3700u
Potassium primary phosphate 0.68g
EDTA.Na
2.2H
2O 0.37g
Sodium azide 0.455g
According to the method for above-mentioned reagent preparation solution, absorbancy behind the reagent sky 〉=1.20,340nm, 10mm optical path were finished in above ammonia reagent preparation rearmounted 2-8 ℃ 1-7 days.When measuring sample or calibration object, adopt two-point method, the sample reagent ratio is 1: 10,37 ℃ of temperature, and 0 second time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Embodiment 5: preparation creatinine liquid double reagent, form by every liter content by following component,
Reagent 1:
8.0,37 ℃ of Tris buffer solution phs, 12.0g
α～ketoisocaproic 1.43g
Bovine serum albumin 5.0g
Adenosine diphosphate potassium salt 1.0g
Glutamate dehydrogenase (beef liver source) 3500u
NADP 0.36g
D～glucose 7.21g
Glucose～6 phosphate dehydrogenases (leuconos toc) 3700u
Potassium primary phosphate 0.68g
EDTA.Na
2.2H
2O 0.372g
Sodium azide 0.455g
According to the method for above-mentioned reagent preparation solution, after the preparation of above ammonia reagent is finished, put 2-8 ℃ 1-7 days, absorbancy behind the reagent sky 〉=1.20,340nm, 10mm optical path.
Reagent 2:
8.0,37 ℃ of Tris buffer solution phs, 12.1g
α～ketoisocaproic 1.43g
Bovine serum albumin 5.0g
Adenosine diphosphate potassium salt 1.0g
Glutamate dehydrogenase (beef liver source) 3500u
EDTA.Na
2.2H
2O 0.372g
Creatinine imino-dehydrogenase 1 100u
Mannitol 10.02g
Sodium azide 0.455g
Method according to above-mentioned reagent preparation solution, configuration creatinine reagent 2 is put 2-8 ℃ of preservation, when measuring sample, adopt two-point method, reagent 1: sample: the ratio of reagent 2 is 200: 20: 50,37 ℃ of temperature, and reagent 1 adds sample or hatched 300 seconds in the mensuration temperature calibration product back, adding reagent 2 begins to measure, 0 second time of lag, minute 120 seconds, reading are selected 2 available points in minute.
Creatinine reagent preferably selects for use the liquid double reagent type to help getting rid of the interference of ammonia in the sample.According to principle of the present invention, creatinine reagent also can be mixed with the liquid single agents, and this moment, the interference of ammonia was left in the basket in the sample when measuring.
Embodiment 6: preparation carbonic acid gas reagent, form by every liter content by following component,
8.0,37 ℃ of Tris buffer solution phs, 6.06g
Phosphoenolpyruvic acid 0.17g
Phosphoric acid enol pyruvic acid carboxylase 500u
Malate dehydrogenase (malic acid dehydrogenase) 1250u
Bovine serum albumin 5.0g
Sal epsom 2.46g
Sodium oxamate 0.28g
NADH 0.08g
NAD 0.3g
D～glucose 18.0g
Glucose～6 phosphate dehydrogenases (leuconos toc) 7500u
Potassium primary phosphate 0.68g
Sodium azide 0.5g
According to the method for above-mentioned reagent preparation solution, after above carbonic acid gas reagent preparation is finished, put absorbancy behind the reagent sky 〉=1.20,340nm, 10mm optical path the 2-8 ℃ of airtight 1-7 of leaving standstill days.When measuring sample or calibration object, adopt end-point method, the sample reagent ratio is 1: 200,37 ℃ of temperature, and minute 300 seconds, body fluid sample or calibration product read the 1st absorbance after adding reagent at once, read the 2nd absorbance in the time of 300 seconds.
After the foregoing description 1-6 reagent preparation was finished, its application method was identical with general reagent application method in the association area, does not repeat them here.
The compound method of the foregoing description 1-6 reagent only is used to illustrate principle of the present invention and application thereof, the present invention not the office in above-mentioned range of application of giving an example; In addition, the professional and technical personnel in association area of the present invention can make similar with it various mensuration reagent according to principle of the present invention and method, but not detach principle of the present invention and range of application.
Claims (8)
1, a kind of nicotinamide coenzyme reagent, it is characterized in that: embed enzyme-substrate-NAD or enzyme-substrate-NADP, slowly generate the enzymatic reaction system of NADH or NADPH, and the NADH that utilize to generate or NADPH enzymic measuring reagent that analyte concentration in the body fluid sample is measured, the reduced coenzyme generation system can be in conjunction with measuring reagent, add damping fluid, sanitas, heavy metal complexing agent, tensio-active agent and enzyme stabilizers, damping fluid comprises the Tris damping fluid, imidazole buffer, the di-alcohol damping fluid, phosphate buffered saline buffer, Good ' s damping fluid, the concentration of damping fluid is typically chosen within the 10-200mM scope, the sanitas that adopts is a sodium azide, heavy metal complexing agent comprises EDTA, EGTA, HEDTA, enzyme stabilizers adopts bovine serum albumin, mannitol.
2, a kind of method for making of nicotinamide coenzyme reagent, it is characterized in that: embed enzyme-substrate-NAD or enzyme-substrate-NADP, slowly generate the enzymatic reaction system of NADH or NADPH, and with the method for this system applies in enzymic measuring reagent, the speed that this system generates effective NADH or NADPH concentration is 0.03-365 days, this system generates the absorbancy of effective NADH or NADPH concentration between 0.5-5.0, wavelength 340nm, optical path 10mm.
3, nicotinamide coenzyme reagent according to claim 1 is characterized in that: be made up of by every liter content following component
L～L-Ala 34.0～45.0g
Serum lactic dehydrogenase 2000～5000u
α～ketoisocaproic 0.5～3.5g
EDTA.Na
2.2H
2O 1.0～0.3g
NAD 0.1～0.3g
D～glucose 15.0～21.0g
Glucose～6～phosphate dehydrogenase (leuconos toc) 1000～4000u
Dipotassium hydrogen phosphate 0.3～1.3g
Sodium azide 0.1～1.0g
4, nicotinamide coenzyme reagent according to claim 1 is characterized in that: be made up of by every liter content following component
7.50,37 ℃ of Tris buffer solution phs, 10-15g
L～aspartic acid 34.0-43.0g
Malate dehydrogenase (malic acid dehydrogenase) 100-600u
Lactic dehydrogenase 10 00-4000u
A～ketoisocaproic 0.5-4.5g
EDTA.Na
2.2H
2O 1.0-3.0g
NAD 0.1-0.3g
D～glucose 15.0-21.0g
Glucose～6～phosphate dehydrogenase (leuconos toc) 1000-4500u
Dipotassium hydrogen phosphate 0.3-2.0g
Sodium azide 0.1-1.0g
5, nicotinamide coenzyme reagent according to claim 1 is characterized in that: be made up of by every liter content following component
Tris damping fluid 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum diacid 0.05～5.0
Adenosine diphosphate potassium salt 0～0.8
Glutamate dehydrogenase (microbe-derived) 500～6000u
Urase 5000～15000u
NAD 0.1～0.3g
D～glucose 3～21g
Glucose～6～phosphate dehydrogenase (leuconos toc) 1000～4000m
Dipotassium hydrogen phosphate, 0.3～2.0g
EDTA.Na
2.2H
2O 0.1～0.1g
Sodium azide 0.1～1.0g
6, nicotinamide coenzyme reagent according to claim 1 is characterized in that: be made up of by every liter content following component
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum albumin 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000u
NADP 0.1～0.35g
D～glucose 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 1000～4000u
Potassium primary phosphate 0.5～2.0g
EDTA.Na
2.2H
2O 0.1～1.0g
Sodium azide 0.1～1.0g
7, nicotinamide coenzyme reagent according to claim 1, it is characterized in that: be made up of by every liter content following component, R1 is:
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum albumin 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000u
NADP 0.1～0.5g
D～glucose 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 1000～4000u
Potassium primary phosphate 0.3～2.0g
EDT?A.Nα
2.2H
2O 0.1～1.0g
Sodium azide 7.0MM 0.1～1.0g
R2 is
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
α～ketoisocaproic 0.5～3.5g
Bovine serum albumin 0.05～5.0g
Adenosine diphosphate potassium salt 0～1.5g
Glutamate dehydrogenase (beef liver source) 1000～6000u
EDTA.Na
2.2H
2O 0.1～1.0g
Creatinine imino-desaturase 500～4000u
Mannitol 3～15g
Sodium azide 0.1～1.0g
R1∶R2＝200∶50
8, nicotinamide coenzyme reagent according to claim 1 is characterized in that: be made up of by every liter content following component
8.0,37 ℃ of Tris buffer solution phs, 10.0～15.0g
Phosphoenolpyruvic acid 0.1～0.5g
Phosphoric acid enol pyruvic acid carboxylase 200～1000u
Malate dehydrogenase (malic acid dehydrogenase) 500～3000u
Bovine serum albumin 0.05～5.0g
Sal epsom 0.5～3.0g
Sodium oxamate 0.1～0.4g
NADH 0.1～0.35g
NAD 0.1～0.35g
D～grape 3～21g
Glucose～6 phosphate dehydrogenases (leuconos toc), 2000～10000u
Potassium primary phosphate 0.3～20g
Sodium azide 0.1～1.0g
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