CN101750351A - Creatinine diagnosis/determination reagent (box) and creatinine concentration determination method - Google Patents
Creatinine diagnosis/determination reagent (box) and creatinine concentration determination method Download PDFInfo
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- CN101750351A CN101750351A CN200810244200A CN200810244200A CN101750351A CN 101750351 A CN101750351 A CN 101750351A CN 200810244200 A CN200810244200 A CN 200810244200A CN 200810244200 A CN200810244200 A CN 200810244200A CN 101750351 A CN101750351 A CN 101750351A
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- coenzyme
- stabilizing agent
- hydrogen peroxide
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Abstract
The invention relates to a creatinine diagnosis/determination reagent (box) employing enzyme colorimetric method and enzyme coupling method technique; meanwhile, the invention also relates to creatinine concentration determination method, reagent composition and ingredients, and belongs to the medical testing determination technical field. The main ingredients of the reagent (box) include: buffer solution, coenzyme, hydrogen peroxide, 2-ketoglutarate, creatinine deiminase, glutamate oxidase, glutamate synthase and stabilizer; a series of enzymic reactions occur through the mixture of sample and reagent in certain volumetric proportion, and then the reactant is placed under an ultraviolet/visible light analyzer for detecting the ascending degree of absorbance at a main wavelength of 340nm, and the creatinine concentration is determined accordingly.
Description
Technical field
The present invention relates to a kind of creatinine diagnosis/determination reagent (box), the invention still further relates to the method for measuring creatine concentration simultaneously, belong to medical test determination techniques field.
Background technology
Measure creatinine and mainly contain chemical assay (Jaffe method), enzyme process, high performance liquid chromatography (HPLC) and capillary electrophoresis etc.
Chemical assay is with low cost, and is easy and simple to handle, is one of the most frequently used method of present domestic mensuration creatinine.Creatinine in the sample and picrate effect generate the picric acid creatinine compound of yellowish red color.The shortcoming of this method is that specificity is not high, and vitamin C, acetone, acetoacetate, ethyldopa and high concentration glucose, protein and some microbiotic such as benzyl penicillin, Cefoxitin, cephazoline etc. also can generate red with the alkaline picric acid reaction.
High performance liquid chromatography (HPLC) (HPLC), creatinine is positively charged in weak acid environment, can separate with other compositions are fine by the cation-exchange chromatography post, measures its light absorption at 234nm.Precision height, specificity that this method is analyzed are good, but this law is unsuitable for clinical samples analysis in enormous quantities, usually as the reference method of creatinine assay, are used to estimate kit and some scientific research purpose of commercially available creatinine assay.Retrieval is found, the Chinese patent application that application number is 90106198.0, the applying date is 1990.12.18 discloses with high performance liquid chromatography and has directly measured pseudouridine (abbreviation pseudouridine) in people's urine, measure creatinine simultaneously by spectrophotometric method again, use pseudouridine and pseudouridine/creatinine mark respectively as nasopharyngeal carcinoma and lung cancer.(this patent and ours irrelevant)
Capillary electrophoresis, serum specimen is done pre-service with high speed centrifugation, and urine specimen can be used low-speed centrifugal, removes visible component, and supernatant is measured 235nm place absorbance after moving the electrocapillary electrophoretic separation with micella.It is wide that this law is measured the range of linearity, operate comparatively easy, but need with specific installation with carry out the pre-service of serum specimen, routine clinical use difficulty.
The enzymatic determination method mainly contains Creatinine deiminase (Creatinine deiminase) and Creatininase (Creatininase) two big classes.
Retrieval finds, the patented claim that application number is 02139298.6, the applying date is 2002.11.15 discloses the mensuration reagent of using the preparation of enzyme reaction product oxidized form nicotinamide coenzyme.The enzymic measuring reagent of this invention indication does not add the required reduced form nicotinamide coenzyme of assaying reaction or its analog, and add its reaction product oxidized form nicotinamide coenzyme or its analog and generate required enzyme of reduced coenzyme and substrate system accordingly, by in reagent, forming long response time systems such as enzyme-substrate-NAD or enzyme-substrate-NADP, this class oxidized coenzyme or the slow circulation of its analog are reduced to reduced form nicotinamide coenzyme or its analog, when the reduced form nicotinamide coenzyme that is reduced or its analog reached certain concentration, the enzyme process reagent of this invention indication just can reach measured alanine aminotransferase in the sample, aspartate amino transferase, urea, ammonia, the purpose of creatinine and carbon dioxide etc.This characteristic feature of an invention is that the test of supporting reagent, ammonia reagent, creatinine reagent and carbon dioxide reagent etc. for alanine aminotransferase reagent, aspartate amino transferase reagent, urea provides an endogenous synthetic alanine aminotransferase reagent, aspartate amino transferase reagent, urea to support reaction needed substrate-reduced form nicotinamide coenzymes such as reagent, ammonia reagent, creatinine reagent and carbon dioxide reagent.One of its shortcoming is, this system is in the needed reduced form nicotinoyl of reaction of formation ammonia coenzyme, also offset the activity of part alanine aminotransferase, aspartate amino transferase, urea, ammonia, creatinine and carbon dioxide etc., caused the accuracy of reagent test to reduce greatly.Two of its shortcoming is that this reagent must react a period of time before official testing in advance, so that can produce enough reduced form nicotinoyl ammonia coenzyme, has so just caused the result that slow action cannot save a critical situation, brings very big inconvenience to test.
According to application number be 02139298.6, the applying date is the patent introduction of 2002.11.15, this system is owing to constantly generate and measure required NADH or NADPH, thereby improved the stability of reagent greatly, and reduces the production cost of reagent greatly.The stabilizing agent that adds NADH or NADPH in its tangible reagent has not been difficulty or the problem that increases cost, on the contrary than in reagent, increase a reactive system economy many.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, metering reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for creatine concentration, simultaneously, the present invention also will provide in order to realize the creatinine diagnosis/determination reagent (box) of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out creatine concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Creatine concentration determination method of the present invention is as follows:
Creatinine+water
Creatinine deiminaseN-methyl hydantoin+ammonia
Ammonia+hydrogen peroxide+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+coenzyme
Glutamate synthetaseGlutamine+2-oxoglutaric acid+reduced coenzyme
This method is used Creatinine deiminase (creatinine deaminase; EC 3.5.4.21) enzyme (idol) connection dglutamic oxidase (glutamate oxidase; EC 1.4.3.7; EC 1.4.3.11; EC 1.4.3.15), glutamate synthetase (glutamate synthase; EC 1.4.1.13) enzymatic reaction colorimetric end-point method.The reaction of Creatinine deiminase enzymolysis creatinine produces ammonia, the effect of uniting dglutamic oxidase, glutamate synthetase again by (idol), coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured the degree that reduced coenzyme rises in 340nm place absorbance, by measuring the degree that 340nm place absorbance rises, can calculate the concentration of creatinine.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the creatinine diagnosis/determination reagent of the present invention (box) of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Creatinine deiminase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 15mmol/L
Creatinine diagnosis/determination reagent of the present invention (box) can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid.
Reagent (box) can be dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, Creatinine deiminase, dglutamic oxidase, glutamate synthetase.
Coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.Reagent (box) can be dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, dglutamic oxidase, glutamate synthetase.
Reagent 3
Damping fluid, stabilizing agent, Creatinine deiminase.
Coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for creatine concentration, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The creatinine diagnosis/determination reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Creatinine deiminase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 15mmol/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of creatinine.
Embodiment two
The creatinine diagnosis/determination reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 15mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Creatinine deiminase 10000U/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of creatinine.
Embodiment three
The creatinine diagnosis/determination reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Hydrogen peroxide 12mmol/L
2-oxoglutaric acid 15mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Dglutamic oxidase 12000U/L
Glutamate synthetase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Creatinine deiminase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring creatine concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), and about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects the degree that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of creatinine.
The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0008; Absorbance time response curve should be the rising curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 1.5mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 4%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.2 ± 0.1 Δ A/mmol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. the method for measurement of concentration of the creatinine of enzymic colorimetric and enzyme-linked method, its method is as follows:
Creatinine+water
Creatinine deiminaseN-methyl hydantoin+ammonia
Ammonia+hydrogen peroxide+2-oxoglutaric acid
Dglutamic oxidaseGlutamic acid+oxygen+water
Glutamic acid+coenzyme
Glutamate synthetaseGlutamine+2-oxoglutaric acid+reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance rises, calculate the concentration measurement result of creatinine.
2. a creatinine diagnosis/determination reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
DPN diphosphopyridine nucleotide---6mmol/L
Creatinine deiminase 1000---80000U/L
Dglutamic oxidase 1000---80000U/L
Glutamate synthetase 1000---80000U/L
Hydrogen peroxide 1---50mmol/L
2-oxoglutaric acid 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
It is characterized in that: reagent (box) can be dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described creatinine diagnosis/determination reagent of claim 2 (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid.
4. according to the described creatinine diagnosis/determination reagent of claim 2 (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, Creatinine deiminase, dglutamic oxidase, glutamate synthetase.Coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.
5. according to the described creatinine diagnosis/determination reagent of claim 2 (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, hydrogen peroxide, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, dglutamic oxidase, glutamate synthetase; Reagent 3 is made up of damping fluid, stabilizing agent, Creatinine deiminase.Coenzyme, Creatinine deiminase, dglutamic oxidase, glutamate synthetase, hydrogen peroxide, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described creatinine diagnosis/determination reagent of claim 2 (box), it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244200A CN101750351A (en) | 2008-12-10 | 2008-12-10 | Creatinine diagnosis/determination reagent (box) and creatinine concentration determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244200A CN101750351A (en) | 2008-12-10 | 2008-12-10 | Creatinine diagnosis/determination reagent (box) and creatinine concentration determination method |
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|---|---|
| CN101750351A true CN101750351A (en) | 2010-06-23 |
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| CN200810244200A Pending CN101750351A (en) | 2008-12-10 | 2008-12-10 | Creatinine diagnosis/determination reagent (box) and creatinine concentration determination method |
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- 2008-12-10 CN CN200810244200A patent/CN101750351A/en active Pending
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Open date: 20100623 |