CN1548547A - Enzyme analysis reagent and its prepn - Google Patents
Enzyme analysis reagent and its prepn Download PDFInfo
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- CN1548547A CN1548547A CNA031170935A CN03117093A CN1548547A CN 1548547 A CN1548547 A CN 1548547A CN A031170935 A CNA031170935 A CN A031170935A CN 03117093 A CN03117093 A CN 03117093A CN 1548547 A CN1548547 A CN 1548547A
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Abstract
The present invention is enzyme analysis reagent compounded with enzyme reaction product oxidizing nicotinamide coenzyme and used in measuring body fluid. The present invention features that the enzyme analysis reagent has oxidizing nicotinamide coenzyme or analog as reaction product, rather than reducting nicotinamide coenzyme or analog, added to form enzyme-substrate-NAD or enzyme-substrate-NADP slow reaction system in the reagent. During measurement, the oxidizing nicotinamide coenzyme or analog is reduced into reducting nicotinamide coenzyme or analog. The enzyme analysis reagent includes alanine aminotransferase reagent, aspartate aminotransferase reagent, urea reagent, ammonia reagent, inosine reagent, etc.
Description
Technical field
The present invention relates to enzymatic analysis reagent and method for making thereof
Background technology
Adopt the analyte in reduced form nicotinamide coenzyme or its analogue (as: NADH, NADPH, thioNAD or thioNADPH) the mensuration body fluid sample, it is a kind of widely used method, the degree of oxidation of reduced form nicotinamide coenzyme or its analogue 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, reduced form nicotinamide coenzyme or its analogue are oxidized in the reagent, the absorbancy of reactive system changes, by the absorbancy changing value of assaying reaction system, just can calculate the concentration of analyte in the sample.
The mensuration of ammonia concentration in the plasma sample for example, the ammonia in the blood plasma generate glutaminate with the a-ketoglutaric acid 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 the a-oxoglutarate 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.
But because reduced form nicotinamide coenzyme or its analogue, particularly NADH and NADPH can only adopt powdered reagent or liquid double reagent in this fermentoid reagent.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 NADPH.
The reduced form nicotinamide coenzyme is in the application of liquid single agents, Joseph De Giorgio etc. are (referring to United States Patent (USP): 5804402 and 5705356) invented the stabilized enzyme system that adds NADH or NADPH in measuring reagent, as glucose-6-phosphate dehydrogenase (G6PD) and non-specificity substrate glucose thereof, the NAD or the NADP of NADH or the oxidized generation of NADPH in the reagent preservation process slowly are reduced to NADH or NADPH again, thereby keep measuring the concentration of effective reduced form nicotinamide coenzyme in the reagent.In their invention, do not add reaction product NAD or NADP when measuring reagent production or preparation, still according to traditional reagent production or compound method, add NADH or NADPH, 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.In addition, in this invention, Joseph DeGiorgio etc. do not propose to utilize reaction product efficiency test compositions and methods yet.
RichardA.Kaufman etc. are (referring to United States Patent (USP): 5589348) utilize glucose-6-phosphate dehydrogenase (G6PD) and specificity substrate G-6-P thereof, original position generates reduced coenzyme simultaneously or prior to measuring sample fast in reagent mensuration process, is used for the mensuration of analyte.In their invention, the generating rate of reduced form nicotinamide coenzyme must 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 must be lower than the volumetric molar concentration of the oxidized coenzyme that is used for reduced coenzyme in the reagent, therefore after this class is measured reagent and is mixed with single liquid reagent, generate the same instability in NADH or NADPH and the common mensuration reagent.This method is used for liquid double reagent more.In their invention, 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 books concentration when measuring analyte content in the sample contrasts calculating, thereby obtains the content of wanting analyte in this.Adopt this method to be difficult in actual applications the enzyme content in the body fluid sample is measured.
Summary of the invention
The present invention is the reaction product that adds coenzyme or its analogue when measuring reagent production or preparation, as NAD, NADP, thioNAD or thioNADP, and long response time enzyme and substrate, form systems such as long response time enzyme-substrate-NAD, enzyme-substrate-thioNAD, enzyme-substrate-NADP or enzyme-substrate-thioNADP.Effectively NADH, thio-NADH, NADPH or thio-NADPH content are generated by this system in the mensuration reagent, simultaneously should be owing to constantly generate mensuration required reduced coenzyme or its analogue, thereby improved the stability of reagent greatly, measure reagent and can be mixed with the single agents of liquid stabilising, and reduce the production cost of reagent greatly.
The present invention is to use enzyme reaction product oxidized form nicotinamide coenzyme (NAD, NADP or its analogue are as thio-NAD, thio-NADP etc.) preparation to measure reagent, is used for measuring the enzymic measuring reagent of body fluid sample analyte.Be characterized in production or process for preparation, the enzymic measuring reagent of indication of the present invention does not add the required reduced form nicotinamide coenzyme of assaying reaction or its analogue, but add its reaction product oxidized form nicotinamide coenzyme or its analogue (as: NAD, NADP, thio-NAD or thio-NADP etc.) and generate required enzyme of reduced coenzyme and substrate system accordingly, by in reagent, forming enzyme-substrate-NAD, enzyme-substrate-(thio-NAD), long response time systems such as enzyme-substrate-NADP or enzyme-substrate-(thio-NADP), this class oxidized coenzyme or the slow circulation of its analogue are reduced to reduced form nicotinamide coenzyme or its analogue, when being reduced type nicotinamide coenzyme or its analogue and reaching certain concentration, the enzyme process reagent of indication of the present invention just can reach the purpose of measuring determinand.Because the embedded enzyme and the speed of substrate system reduction-oxidation type nicotinamide coenzyme or its analogue significantly are lower than analyte and mensuration enzyme or substrate system thereof to reduced form nicotinamide coenzyme or its analogue rate of oxidation, therefore this embedded enzyme and substrate system do not influence the assaying reaction of analyte.This class reagent comprises alanine aminotransferase reagent, aspartate amino transferase reagent, urea reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent etc.
The present invention has proposed a kind of production method of utilizing the mensuration reagent of the former reactant of the reverse generation of reaction product simultaneously first.
The present invention discloses a kind of preparation of mensuration reagent and production method of saving and improveing, do not use NADH, NADPH, thio-NADH or thio-NADPH or its analogue 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 alanine aminotransferase reagent, aspartate amino transferase reagent, urea reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent etc.
According to principle of the present invention, the reduced coenzyme that reagent can adopt in measuring or the generation system of its analogue are a lot, comprise Hexose phosphate dehydrogenase-glucose-oxidized form nicotinamidase or its analogue system, serum lactic dehydrogenase-pyruvic acid-oxidized form nicotinamide coenzyme or its analogue system, glycerol dehydrogenase-glycerine-oxidized form nicotinamide coenzyme or its analogue system and (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-oxidized form nicotinamide coenzyme or its analogue system etc.In order to reduce the generating rate of reduced form nicotinamide coenzyme or its analogue effectively, do not influence the mensuration performance of reagent, can adjust the enzyme in the above-mentioned generation system and substrate and concentration thereof, make its speed that generates reduced form nicotinamide coenzyme or its analogue when measuring analyte, not influence the rate of oxidation of reduced form nicotinamide coenzyme or its analogue.For example, Hexose phosphate dehydrogenase-glucose-oxidized form nicotinamide coenzyme or its analogue system can be adjusted to Hexose phosphate dehydrogenase-wood sugar-oxidized form nicotinamide coenzyme or its analogue system, serum lactic dehydrogenase-pyruvic acid-oxidized form nicotinamide coenzyme or its analogue system can be adjusted to serum lactic dehydrogenase-a-ketoglutaric acid-oxidized form nicotinamide coenzyme or its analogue system, and glycerol dehydrogenase-glycerine-oxidized form nicotinamide coenzyme or its analogue system can be adjusted to glycerol dehydrogenase-ethylene glycol-oxidized form nicotinamide coenzyme or its analogue system, and (glucose-6-phosphate dehydrogenase (G6PD))-(G-6-P)-oxidized form nicotinamide coenzyme or its analogue system can be adjusted to (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or its analogue system etc.Consider that the substrate material that may contain in the sample when measuring sample has influence on the generating rate of reduced form nicotinamide coenzyme or its analogue, and then the system that preferentially selects for use in above-mentioned each system of the assaying reaction that influences analyte is glycerol dehydrogenase-ethylene glycol-oxidized form nicotinamide coenzyme or its analogue system and (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or its analogue system, and the system that the most preferentially selects for use is (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or its analogue system.Below be example with (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or its analogue system, further set forth the principle and the method for making of reagent of the present invention.
The reduced coenzyme that reduced coenzyme among the present invention or its analogue generation system generate is after reaching certain concentration, can carry out the concentration determination of analyte with other mensuration composition of reagent, this moment, reduced coenzyme or its analogue generation system can be finished the formation reaction of reduced coenzyme or its analogue fully, also can still continue to generate reduced coenzyme or its analogue.These characteristics have guaranteed to adopt the present invention can prepare or produce the liquid reagent of single stable.The speed that reduced coenzyme among the present invention or its analogue generation system generate reduced coenzyme or its analogue can be the speed of any not interference measurement reaction, the effective reduced form niacinamide of the generation that can adopt is auxilliary or it is like between the speed of the concentration of thing is in 0.001-365 days, the reduced coenzyme that generates or the absorbancy (340nm or 405nm wavelength, optical path 10mm) within the 0.5-5.0 scope of its analogue; Generating rate was less than 0.001 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 reduced coenzyme of generation or the absorbancy of its analogue are 0.8-3.0 (wavelength 340nm or 405nm or 405nm, optical path 10mm); The preferential generating rate that adopts is in 1-7 days, and the absorbancy of the reduced coenzyme of generation is 1.0-2.8, wavelength 340nm or 405nm, optical path 10mm.The concentration range of glucose-6-phosphate dehydrogenase (G6PD) is between 100-100000U/L, and between the more suitable 10000U/L, 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 10-200mM.Oxidized coenzyme or its analogue comparatively proper concentration between 0.05-0.85mM.The scope of preferentially selecting for use is between 0.20-0.50mM.
The mensuration reagent of the present invention's preparation, the reduced coenzyme wherein or the content of its analogue, also can adopt reduced coenzyme or its analogue generation system among the present invention partly to generate, other parts still adopt traditional method to add, to shorten the rise time of reduced coenzyme or its analogue, help the market supply of flexible adaptation reagent.
Reduced coenzyme or its analogue generation system can add damping fluid, sanitas, heavy metal complexing agent, tensio-active agent and enzyme stabilizers etc. in conjunction with measuring reagent.Damping fluid commonly used comprises: three (methylol) aminomethane (Tris) damping fluid, imidazole buffer, diethanolamine buffer, phosphate buffered saline buffer, Good ' s damping fluid, as: 3-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPOS), 3-(N-morpholino) propanesulfonic acid (MOPS), 2-(N-morpholino) ethyl propanesulfonic acid (MES), piperazine-N, N-two (2-ethylsulfonic acid) (PIPES), N-(2-hydroxyethyl) piperazine-N-(2-ethylsulfonic acid) (HEPES), 3-[N, two (hydroxyethyl) ammonia of N-]-2-ammonia hydroxy-propanesulfonic acid (DIPSO), N-(2-hydroxyl second) second piperazine-N-2-hydroxypropyl sulfonic acid (HEPPSO), piperazine-N, N-two (2-hydroxypropyl sulfonic acid) (POPSO), N-three (methylol) methyl]-3-amino-2-hydroxypropyl sulfonic acid (TAPSO) etc., wherein the application of three (methylol) aminomethane buffer solution is comparatively general, preferentially selecting damping fluid among the present invention for use is three (hydroxyl first) methylamino-methane (Tris) damping fluids, the concentration of damping fluid is typically chosen within the 10-20mM scope, and the 50-100mM scope is more suitable.The sanitas that can preferentially adopt is a sodium azide, adopts other biological anticorrosion 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 such as TritonX-100 etc. in the reagent.
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 reduced coenzyme or its analogue, 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 reduced coenzyme or its analogue generation system slowly generate reduced coenzyme or its analogue.When the growing amount of reduced coenzyme or its analogue reaches certain concentration, reagent just can be used for measuring, during the obtaining liq double reagent, the generation system of reduced coenzyme seven its analogues that adopt, as glycerol dehydrogenase-ethylene glycol-oxidized form nicotinamide coenzyme or its analogue system, (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or its analogue system etc., should be in same reagent part.
Specific embodiment
Embodiment 1: preparation: alanine aminotransferase reagent, form by every liter content by following component,
Tris damping fluid 100mM, PH7.15,37 ℃,
L-L-Ala 500mM,
Serum lactic dehydrogenase 2.7ku/L,
A-ketoglutaric acid 15mM,
EDTA.Na
2.2H
2O6mM，
NAD?0.27mM，
D-glucose 50mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM
Sodium azide 1.5mM
The method of the above-mentioned reagent preparation solution of root a tree name, after the preparation of above alanine aminotransferase reagent is finished, put 2-8 ℃ 1-3 days, reagent blank absorbancy 〉=1.10,340nm, 10mm optical path.When measuring sample, adopt rate method, the sample reagent ratio is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Embodiment 2: aspartate amino transferase reagent
Tris damping fluid 80mM, PH7.65,37 ℃,
L-aspartic acid 240mM,
Malate dehydrogenase (malic acid dehydrogenase) 600U/L
Lactic dehydrogenase 10 00U/L,
A-ketoglutaric acid 12mM,
EDTA.Na
2.2H
2O6mM，
NAD?0.28mM，
D-glucose 80mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM
Sodium azide 1.5mM
The method of the above-mentioned reagent preparation solution of root a tree name after above alanine aminotransferase reagent preparation is finished, is put 2-8 ℃, and 1-5 days, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path.When measuring sample, adopt rate method, the sample reagent ratio is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Give an example 2: aspartate amino transferase reagent
Tris damping fluid 80mM, pH7.65,37 ℃
L-aspartic acid 240mM
Malate dehydrogenase (malic acid dehydrogenase) 600U/L
Lactic dehydrogenase 10 00U/L
A-ketoglutaric acid 12mM
EDTA.Na
2.2H
2O?6mM，
NAD?0.28mM，
D-glucose 80mM,
Glucose-6-phosphate dehydrogenase (G6PD) (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 days, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path.When measuring sample, adopt rate method, the sample reagent ratio is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
Give an example 3: urea reagent
Tirs damping fluid 120mM, 7.5,37 ℃ of pH
A-ketoglutaric acid 7.5mM,
Bovine serum albumin 5g/L,
Adenosine diphosphate potassium salt 1.3mM,
Glutamate dehydrogenase (microbe-derived) 550U/L,
Urase 12000U/L,
NAD0.29mM，
D-glucose 100mM,
G6P-DH (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM,
EDTA.Na
2.2H
2O?1mM，
Sodium azide 4.0mM,
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 〉=1.2,340nm, 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: ammonia reagent
Tris damping fluid 100mM, pH8.0,37 ℃,
A-ketoglutaric acid 7.5mM,
Bovine serum albumin 5g/L,
Adenosine diphosphate potassium salt 2.5mM,
L-glutamic acid deamination acid (beef liver source) 4500U/L,
NADP?0.32mM，
D-glucose 40mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 3700U/L,
Potassium primary phosphate 5mM,
EDTA.Na
2.2H
2O?1mM，
Sodium azide 7.0mM,
According to the method for above-mentioned reagent preparation solution, after above ammonia reagent preparation is finished, put 2-8 ℃ of sky, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path.When measuring sample or calibration object, adopt useful two-point method, the sample reagent ratio is 1: 10, and 37 ℃ of temperature, are selected 2 available points at 0 second time of lag in the minute 120 seconds, reading minute.
Give an example 5: the creatinine liquid double reagent
Reagent 1:
Tris damping fluid 100mM, pH8.0,37 ℃,
A-ketoglutaric acid 7.5mM,
Bovine serum albumin 5g/L,
Adenosine diphosphate potassium salt 2mM,
L-glutamic acid desaminase (beef liver source) 3500U/L,
NADP0.40mM，
D-glucose 40mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 3700U/L,
Potassium primary phosphate 5mM,
EDTA.Na
2.2H
2O?1mM，
Sodium azide 7.0mM,
According to the method for above-mentioned reagent preparation solution, after the preparation of above creatinine reagent is finished, put 2-8 ℃ 1-7 days, reagent blank absorbancy 〉=1.20,340nm, 10mm optical path.
Reagent 2:
Tris damping fluid 100mM, pH8.0,37 ℃,
A-ketoglutaric acid 7.5mM,
Bovine serum albumin 5g/L,
Adenosine diphosphate potassium salt 2mM,
Glutamate dehydrogenase (beef liver source) 3500U/L,
EDTA.Na
2.2H
2O?1mM，
Creatinine imino-dehydrogenase 1 100U/L
Sweet acid anhydride sugar alcohol 55mM
Sodium azide 7.0mM,
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 was hatched 300 seconds in the mensuration temperature after adding sample or calibration object, 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 formulation 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: carbonic acid gas reagent
Tris damping fluid 50mM, Ph8.0,37 ℃,
Phosphoenolpyruvic acid 1.8mM,
Phosphoric acid enol pyruvic acid carboxylase 500U/L,
Malate dehydrogenase (malic acid dehydrogenase) 1250U/L
Bovine serum albumin 1g/L
Sal epsom 10mM,
Sodium oxamate 2.5mM,
NADH0.1mM
NAD0.45mM
D-glucose 100mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 7500U/L,
Potassium primary phosphate 5mM
Sodium azide 7.7mM,
According to the method for above-mentioned reagent preparation solution, after above carbonic acid gas reagent preparation is finished, put reagent blank absorbancy 〉=1.20,340nm, 10mm optical path the 2-8 ℃ of airtight 1-15 of leaving standstill days.Measure and read the 1st absorbance at once after sample or calibration object add reagent, read the 2nd absorbance in the time of 300 seconds.Because carbonic acid gas extensively exists in the environment, adopt fresh deionized water to boil the preparation of cooling back during proposed arrangement carbonic acid gas reagent, the complete airtight preservation of preparation back reagent.Carve in the routine reagent that to add part NADH be in order there to be the carbonic acid gas of handing over ground to get rid of in the environment to disturb.
Give an example 7: alanine aminotransferase reagent
Tris damping fluid 100mM, pH7.15,37 ℃,
L-L-Ala 500mM,
Serum lactic dehydrogenase 2.7ku/L,
A-ketoglutaric acid 15mM,
EDTA.Na
2.2H
2O?6mM，
Thio-NAD0.27mM，
D-glucose 50mM,
Glucose-6-phosphate dehydrogenase (G6PD) (leuconos toc) 3700U/L,
Dipotassium hydrogen phosphate 5mM
Folded ammonia sodium 1.5mM
According to the method for above-mentioned reagent preparation solution, after the preparation of above alanine aminotransferase reagent is finished, put 2-8 ℃ 1-10 days, reagent blank absorbancy 〉=1.10,405nm, 10mm optical path.When measuring sample, adopt bundle rate method, the sample reagent ratio is 1: 12,37 ℃ of temperature, 90 seconds time of lag, minute 180 seconds, reading number of times 〉=6 points.
The above-mentioned 1-for example compound method of 7 reagent for example only is used to illustrate principle of the present invention and application thereof, and the present invention never is confined to above-mentioned range of application of giving an example; In addition, the professional and technical personnel in association area of the present invention, can according to principle of the present invention and method make with and so on various mensuration reagent, but do not detach principle range of application of the present invention.
Claims (9)
1, utilize the mensuration reagent of reaction product and long response time enzyme thereof and substrate system preparation, this kind reagent is used for measuring people's body fluid sample by the concentration of thing.
2, utilize the preparation of reaction product and long response time enzyme thereof and substrate system to measure compositions and methods, the reagent of this method preparation can be loaded in all kinds of bottles for using.
3, the reaction product of 1 middle indication of claim the is oxidized form nicotinamide coenzyme or its analogue, as NAD, NADP, thio-NAD or thio-NADP.
4, the long response time enzyme of 1 middle indication of claim the and substrate system are enzyme-substrate-oxidized form nicotinamide coenzyme or its analogue system, as: Hexose phosphate dehydrogenase-wood sugar-oxidized form nicotinamide coenzyme or its analogue system, serum lactic dehydrogenase-a-ketoglutaric acid-oxidized form nicotinamide coenzyme or its analogue system, glycerol dehydrogenase-ethylene glycol-oxidized form nicotinamide coenzyme or its analogue system, (glucose-6-phosphate dehydrogenase (G6PD))-glucose-oxidized form nicotinamide coenzyme or common analogue system etc.
5, embed long response time enzyme-substrate-oxidized form nicotinamide coenzyme or its analogue, slowly generate the enzymatic reaction system of reductibility nicotinamide coenzyme or its analogue, with the reductibility nicotinamide coenzyme or its analogue that utilize generation, as:, NADPH, thio-NADH or thio-NADPH etc., the enzymic measuring reagent that analyte concentration in the body fluid sample is measured.
6, the enzyme-substrate oxidized form nicotinamide coenzyme of 5 middle indications of claim the or its analogue system are the systems of (glucose-6-phosphate dehydrogenase (G6PD))-glucose-(thio-NAD).
The enzyme-substrate of 5 middle indications of claim the-oxidized form nicotinamide coenzyme or its analogue system are the systems of (glucose-6-phosphate dehydrogenase (G6PD))-glucose-(thio-NAD).
The enzyme-substrate oxidized form niacinamide of 5 middle indications of claim the is assisted or its analogue system is (glucose-6-phosphorus enzyme desaturase)-glucose-NADP system.
The enzyme-substrate oxidized form nicotinamide coenzyme of 5 middle indications of claim the or analogue system are the systems of (G-6-P takes off enzyme)-glucose-(thio-NADP).
The speed that the glucose of 5 middle indications of claim the-6-phosphorus enzyme desaturase-glucose-oxidized form nicotinamide coenzyme or its analogue enzymatic reaction system generate effective NADH or its analogue concentration was preferably within 1-7 days within 0.001-365 days.
7, the absorbancy of effective NADH concentration of 6 indications of claim the is between 0.5-5.0, wavelength 340nm, optical path 10mm.Be preferably between the 1.0-2.5 wavelength 340nm, optical path 10mm.
The absorbancy of effective NADPH concentration of 6 middle indications of claim the between 0.5-5.0, wavelength 340nm, optical path 10mm.Be preferably between the 1.0-2.5 wavelength 340nm, optical path 10mm.
The absorbancy of effective thio-NADH concentration of 5 middle indications of claim the between 0.5-5.0, wavelength 405nm, optical path 10mm.Be preferably between the 1.0-2.5 wavelength 405nm.Optical path 10mm.
The absorbancy of effective thio-NADPH concentration of 8 middle indications of claim the between 0.5-5.0, wavelength 405nm, optical path 10mm.Be preferably between the 1.0-2.5 wavelength 405nm, optical path 10mm.
8, (glucose-6-phosphate dehydrogenase (G6PD)) of 5 middle indications of claim the-glucose-oxidized form nicotinamide coenzyme or its analogue enzymatic reaction system generate effective NADPH concentration speed within 0.001-365 days, be preferably within 1-7 days.
The speed that (glucose-6-phosphate dehydrogenase (G6PD)) of 5 middle indications of claim the-glucose-oxidized form nicotinamide coenzyme or its analogue enzymatic reaction system generate effective thio-NADH concentration was preferably within 1-7 days within 0.001-365 days.
The speed that (glucose-6-phosphate dehydrogenase (G6PD)) of 5 middle indications of claim the-glucose-oxidized form nicotinamide coenzyme or its analogue enzymatic reaction system generate effective thio-NADPH concentration was preferably between 1-7 days within 0.001-365 days.
9, the enzymic measuring reagent of 1 and the 2nd middle indication of claim the refers to alaninyl transferring enzyme reagent, aspartate amino transferase reagent, urea reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent, monoamine oxidase reagent and adenosine deaminase reagent etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800852B (en) * | 2005-01-06 | 2012-04-18 | 上海复旦张江生物医药股份有限公司 | Improved clinical diagnosis reagent for detection by rate method |
| CN104140996A (en) * | 2014-08-04 | 2014-11-12 | 宁波美康生物科技股份有限公司 | Liquid stable enzymic method homocysteine kit |
| CN106885905A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of urea detection reagent and detection method with superior detection line and sensitivity for analysis |
| CN109613225A (en) * | 2018-12-28 | 2019-04-12 | 上海高踪医疗器械科技有限公司 | A kind of carbon dioxide detection reagent box |
| CN112226483A (en) * | 2020-11-05 | 2021-01-15 | 洛阳恒恩生物科技有限公司 | High-stability reduced nicotinamide coenzyme determination reagent and preparation method thereof |
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2003
- 2003-05-23 CN CNA031170935A patent/CN1548547A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1800852B (en) * | 2005-01-06 | 2012-04-18 | 上海复旦张江生物医药股份有限公司 | Improved clinical diagnosis reagent for detection by rate method |
| CN104140996A (en) * | 2014-08-04 | 2014-11-12 | 宁波美康生物科技股份有限公司 | Liquid stable enzymic method homocysteine kit |
| CN106885905A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of urea detection reagent and detection method with superior detection line and sensitivity for analysis |
| CN109613225A (en) * | 2018-12-28 | 2019-04-12 | 上海高踪医疗器械科技有限公司 | A kind of carbon dioxide detection reagent box |
| CN112226483A (en) * | 2020-11-05 | 2021-01-15 | 洛阳恒恩生物科技有限公司 | High-stability reduced nicotinamide coenzyme determination reagent and preparation method thereof |
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