CN1766640A - Creatinine content determination method and creatinine diagnosis kit - Google Patents
Creatinine content determination method and creatinine diagnosis kit Download PDFInfo
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- CN1766640A CN1766640A CN 200410065167 CN200410065167A CN1766640A CN 1766640 A CN1766640 A CN 1766640A CN 200410065167 CN200410065167 CN 200410065167 CN 200410065167 A CN200410065167 A CN 200410065167A CN 1766640 A CN1766640 A CN 1766640A
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- creatinine
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- coenzyme
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Abstract
The invention relates to a method for measuring the creatinine content and a creatinine diagnosis agent box in the field of medical testing technology. The agent box comprises: buffer solution, coenzyme, creatinine, antinase, tryptophan oxygenase, aldehyde dehydrogenase and stabilizer. It mixes the sample and the agent with a certain volume ratio to do enzyme coupling reaction; then it dispositions the end resting material on the chemical analyzer to detect the speed of the main wavelength light absorption to measure the content of the creatinine.
Description
Technical field
The present invention relates to a kind of method of measuring creatinine content, the invention still further relates to the creatine diagnosis reagent kit that is used to realize this method 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---with low cost, easy and simple to handle, be 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, because 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 only as the reference method of creatinine assay, are used to estimate kit and some scientific research purpose of commercially available creatinine assay.
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 contain 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 the purpose of measuring alanine aminotransferase, aspartate amino transferase, urea, ammonia, creatinine and carbon dioxide etc. in the sample.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.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of assay method that can overcome the creatinine content of above prior art shortcoming, provide the creatine diagnosis reagent kit of this method of realization simultaneously, adopt the reagent in this kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out creatinine content determination, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
It is as follows that the present invention measures the method step of creatinine content:
1), with sample and the reagent mix mainly formed by coenzyme (being oxidized coenzyme), Creatininase, kreatinase, sarcosine oxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbance descends, calculate the size of creatinine content.
Common step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the speed that predominant wavelength 340nm absorbance descends, calculate the size of creatinine content.
The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction time was controlled at conventional 2-30 minute.
The present invention uses Creatininase coupling kreatinase, sarcosine oxidase, aldehyde dehydrogenase reaction continuous monitoring method or colourimetry.Creatininase becomes creatinine into creatine, effect by coupling kreatinase, sarcosine oxidase, aldehyde dehydrogenase again, with oxidized coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, thereby measured the amplitude that reduced coenzyme rises in 340nm place absorbance, by measuring the amplitude that 340nm place absorbance rises, can calculate the content of creatinine.
Creatine diagnosis reagent kit of the present invention can be single agent, comprising:
Damping fluid 40--200mmol/l
Coenzyme 0.5--10mmol/l
Creatininase 1000--50000U/l
Kreatinase 1000--50000U/l
Sarcosine oxidase 1000--20000U/l
Aldehyde dehydrogenase 500--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Also above single agent can be made into following pair of agent:
Reagent I
Damping fluid 40--200mmol/l
Coenzyme 0.5--10mmol/l
Kreatinase 1000--50000U/l
Sarcosine oxidase 1000--20000U/l
Aldehyde dehydrogenase 500--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Creatininase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, and coenzyme, kreatinase, sarcosine oxidase, aldehyde dehydrogenase etc. can be placed on reagent II, reagent II composition wherein, Creatininase also can be placed on reagent I, so can form multiple formulations, does not describe in detail one by one at this.
Reagent can also be made into following three reagent, more help the stable of reagent:
Reagent I
Damping fluid 40--200mmol/l
Coenzyme 0.5--10mmol/l
Stabilizing agent 10--80% (cumulative volume)
Reagent II
Damping fluid 40--200mmol/l
Kreatinase 1000--50000U/l
Sarcosine oxidase 1000--20000U/l
Aldehyde dehydrogenase 500--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Reagent III
Damping fluid 40--200mmol/l
Creatininase 1000--50000U/l
Stabilizing agent 10--80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription, and the composition of reagent I wherein, coenzyme etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, kreatinase, sarcosine oxidase, aldehyde dehydrogenase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, Creatininase also can be placed among reagent I or the reagent II, so can form multiple formulations, do not describe in detail one by one.
The pH scope of buffering agent can be 6.0～11.0.Buffering agent can be three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphate (Phosphate) damping fluid, triethanolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
Above oxidation coenzyme can be NADP
+, NAD
+Or thio-NAD
+Deng oxidized form nicotinamide coenzyme or derivatives thereof.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent 10～80% or 10～50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stabilizing agent can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine or the salt etc.
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 creatine diagnosis reagent kit of the present invention of following formula components relation is comparatively desirable:
Damping fluid 80--120mmol/l
DPN diphosphopyridine nucleotide--5mmol/l
Creatininase 8000--20000U/l
Kreatinase 8000--20000U/l
Sarcosine oxidase 6000--10000U/l
Aldehyde dehydrogenase 8000--20000U/l
Stabilizing agent 20--50% (cumulative volume)
Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy operating of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The creatine diagnosis reagent kit of present embodiment comprises:
Damping fluid 80mmol/l
DPN diphosphopyridine nucleotide mmol/l
Creatininase 8O00U/l
Kreatinase 8000U/l
Sarcosine oxidase 6000U/l
Aldehyde dehydrogenase 8000U/l
Stabilizing agent 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 340nm absorbance rises, thereby calculate the content of creatinine.
Present embodiment the present invention uses Creatininase coupling kreatinase, sarcosine oxidase, aldehyde dehydrogenase reaction continuous monitoring method or colourimetry.Creatininase becomes creatinine into creatine, effect by coupling kreatinase, sarcosine oxidase, aldehyde dehydrogenase again, with oxidized coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, thereby measured the amplitude that reduced coenzyme rises in 340nm place absorbance, by measuring the amplitude that 340nm place absorbance rises, can calculate the content of creatinine.
Embodiment two (two agent)
The creatine diagnosis reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Coenzyme 3mmol/l
Kreatinase 14000U/l
Sarcosine oxidase 8000U/l
Aldehyde dehydrogenase 14000U/l
Stabilizing agent 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Creatininase 14000U/l
Stabilizing agent 50% (cumulative volume)
When measuring creatinine content, temperature is controlled at 30 ℃, 15 minutes reaction time, and test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is positive reaction.
Concrete determination step is:
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 340nm absorbance rises, thereby calculate the content of creatinine.
The reaction time of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The creatine diagnosis reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
Coenzyme 5mmol/l
Stabilizing agent 20mmol/l
Reagent II
Damping fluid 120mmol/l
Kreatinase 20000U/l
Sarcosine oxidase 10000U/l
Aldehyde dehydrogenase 20000U/l
Stabilizing agent 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Creatininase 20000U/l
Stabilizing agent 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction time, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is positive reaction.
Concrete determination step is:
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 340nm absorbance rises, thereby calculate the content of creatinine.
The reaction time of each reactions steps was controlled at 20 minutes.
Embodiment four
The creatine diagnosis reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Coenzyme 2mmol/l
Stabilizing agent 20mmol/l
Reagent II
Damping fluid 100mmol/l
Creatininase 10000U/l
Kreatinase 10000U/l
Sarcosine oxidase 8000U/l
Aldehyde dehydrogenase 10000U/l
Stabilizing agent 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is positive reaction.
After adding sample and reagent, make it to mix, following reaction take place:
The end reaction thing is placed under the biochemical analyzer, detect the amplitude that predominant wavelength 340nm absorbance rises, thereby calculate the content of creatinine.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, degree of accuracy good, is not subjected to the pollution of inside and outside source material.
Claims (10)
1. creatinine content determination method, step is as follows:
1), with sample and the reagent mix of mainly forming by coenzyme, Creatininase, kreatinase, sarcosine oxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Creatinine+water
CreatininaseCreatine
Creatine+water
KreatinaseMethyl amimoacetic acid+urea
Methyl amimoacetic acid+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycocoll
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbon dioxide+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbance descends, calculate the size of creatinine content.
2. according to the described creatinine content determination method of claim 1, it is characterized in that: described step 2) be: the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbance descends, calculate the content of creatinine.
3, according to claim 1 or 2 described creatinine content determination methods, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction time was controlled at 2-30 minute.
4, according to claim 1 or 2 described creatinine content determination methods, it is characterized in that: the proportional control of tested creatinine sample and reagent is 1/10 to 1/500.
5. creatine diagnosis reagent kit is grouped into by following one-tenth:
Damping fluid 40--200mmol/l
Oxidized coenzyme 0.5--10mmol/l
Creatininase 1000--50000U/l
Kreatinase 1000--50000U/l
Sarcosine oxidase 1000--20000U/l
Aldehyde dehydrogenase 500--50000U/l
Stabilizing agent 10--80% (cumulative volume)
6. according to the described creatine diagnosis reagent kit of claim 5, it is characterized in that: described reagent is made into single agent, two agent or three doses.
7. according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: described stabilizing agent is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, ammonium sulfate or the salt.
8. according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: the pH scope of described buffering agent is 6.0-11.0.
9. described according to Claim 8 creatine diagnosis reagent kit is characterized in that: described buffering agent is a kind of in three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, triethanolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
10, according to the described creatine diagnosis reagent kit of claim 9, it is characterized in that: described oxidized coenzyme is NADP
+, NAD
+Or thio-NAD
+A kind of in the oxidized form nicotinamide coenzyme or derivatives thereof.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410065167 CN1766640A (en) | 2004-10-28 | 2004-10-28 | Creatinine content determination method and creatinine diagnosis kit |
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|---|---|---|---|
| CN 200410065167 CN1766640A (en) | 2004-10-28 | 2004-10-28 | Creatinine content determination method and creatinine diagnosis kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375066A (en) * | 2011-09-20 | 2012-03-14 | 天津希恩思生化科技有限公司 | Creatinine content detecting reagent and kit, and manufacturing and using methods of kit |
| CN107505470A (en) * | 2017-08-10 | 2017-12-22 | 威特曼生物科技（南京）有限公司 | Stable creatinine detection reagent box and its application method |
| CN112577940A (en) * | 2019-09-30 | 2021-03-30 | 厦门大学 | Method for rapidly and quantitatively detecting concentration of creatinine in urine at low cost |
| CN116730877A (en) * | 2023-06-12 | 2023-09-12 | 宁夏恒康科技有限公司 | Preparation method of creatine monohydrate |
-
2004
- 2004-10-28 CN CN 200410065167 patent/CN1766640A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375066A (en) * | 2011-09-20 | 2012-03-14 | 天津希恩思生化科技有限公司 | Creatinine content detecting reagent and kit, and manufacturing and using methods of kit |
| CN102375066B (en) * | 2011-09-20 | 2013-10-16 | 天津希恩思生化科技有限公司 | Creatinine content detecting reagent and kit, and manufacturing and using methods of kit |
| CN107505470A (en) * | 2017-08-10 | 2017-12-22 | 威特曼生物科技（南京）有限公司 | Stable creatinine detection reagent box and its application method |
| CN112577940A (en) * | 2019-09-30 | 2021-03-30 | 厦门大学 | Method for rapidly and quantitatively detecting concentration of creatinine in urine at low cost |
| CN116730877A (en) * | 2023-06-12 | 2023-09-12 | 宁夏恒康科技有限公司 | Preparation method of creatine monohydrate |
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Open date: 20060503 |