CN1757750A - Method of determining creatnine content and reagent box for diagnosing creatnine - Google Patents
Method of determining creatnine content and reagent box for diagnosing creatnine Download PDFInfo
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- CN1757750A CN1757750A CN 200410064885 CN200410064885A CN1757750A CN 1757750 A CN1757750 A CN 1757750A CN 200410064885 CN200410064885 CN 200410064885 CN 200410064885 A CN200410064885 A CN 200410064885A CN 1757750 A CN1757750 A CN 1757750A
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- coenzyme
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Abstract
A method for measuring the content of creatinine by use of the creatinine testing reagent kit includes such steps as proportionally mixing said reagents with specimen, enzyme coupling reaction, taking resultant, detecting the variation of primary wave in optical absorbancy by biochemical analyzer and calculating the content of creatinine. Said reagent kit is composed of 9 reagents including buffering liquid, adenosin triphosphate, coenzyme, stabilizer, etc.
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 mixture of yellowish red color.The shortcoming of this method is that specificity is not high, because vitamins C, acetone, etheric acid, methyldopa and high concentration glucose, protein and some microbiotic such as penicillin G, cefoxitin, Kefzol 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 photoabsorption 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 test kit and some scientific research purpose of commercially available creatinine assay.
Capillary electrophoresis---serum specimen is done pre-treatment with high speed centrifugation, and urine specimen can be used low-speed centrifugal, removes formed elements, and supernatant liquor is measured 235nm place absorbancy after moving the electrocapillary electrophoretic separation with micella.It is wide that this law is measured linearity range, operate comparatively easy, but need with specific installation with carry out the pre-treatment 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 patent application 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 analogue, and add its reaction product oxidized form nicotinamide coenzyme or its analogue 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 analogue are reduced to reduced form nicotinamide coenzyme or its analogue.When the reduced form nicotinamide coenzyme that is reduced or its analogue 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 carbonic acid gas etc. in the sample.This characteristic feature of an invention is that the test of supporting reagent, ammonia reagent, creatinine reagent and carbonic acid gas 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 carbonic acid gas reagent.One of its shortcoming is, this system is in the needed reduced form nicotinoyl of formation reaction ammonia coenzyme, also offset the activity of part alanine aminotransferase, aspartate amino transferase, urea, ammonia, creatinine and carbonic acid gas etc., caused the accuracy of reagent test to reduce greatly.Two of its shortcoming is that this reagent must react for some 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 measuring 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 test 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.
The step that the present invention measures creatinine content is as follows:
1), with sample and the reagent mix of mainly forming by adenosine triphosphate, coenzyme, Creatinine deiminase, N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Carboxamide sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbancy 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 absorbancy 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 times was controlled at conventional 2-30 minute.
The present invention uses Creatinine deiminase coupling N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase reaction colorimetric or continuous monitoring method.Creatinine deiminase hydrolysis creatinine produces the N-methyl hydantoin, N-methyl hydantoin enzyme acts on N-methyl hydantoin and adenosine triphosphate generation carboxamide sarkosine then, produce sarkosine by carboxamide sarkosine hydroamidase again, produce formaldehyde by the effect of coupling sarcosine oxidase again, effect by the coupling aldehyde dehydrogenase again is with coenzyme (NAD
+, NADP
+Or other analogue---do not have absorption peak at the 340nm place) be reduced into reducibility coenzyme (NADH, NADPH or other analogue---absorption peak is arranged) at the 340nm place, thereby measured reduced coenzyme in 340nm place absorbancy ascensional range, by measuring the ascensional range of 340nm place absorbancy, can calculate the content of creatinine.
Creatine diagnosis reagent kit in order to realization the inventive method can be single agent, comprising:
Damping fluid 40---200mmol/l
Adenosine triphosphate 0.2---20mmol/l
Coenzyme 0.5---20mmol/l
Creatinine deiminase 1000---20000U/l
N-methyl hydantoin enzyme 1000---20000U/l
Carboxamide sarkosine hydroamidase 1000---10000U/l
Sarcosine oxidase 1000---20000U/l
Aldehyde dehydrogenase 500---50000U/l
Stablizer 10---80% (cumulative volume)
Also above single agent can be made into following pair of agent:
Reagent I
Damping fluid 40---200mmol/l
Adenosine triphosphate 0.2---20mmol/l
Coenzyme 0.5---20mmol/l
N-methyl hydantoin enzyme 1000---20000U/l
Carboxamide sarkosine hydroamidase 1000---10000U/l
Sarcosine oxidase 1000---20000U/l
Aldehyde dehydrogenase 500---50000U/l
Stablizer 10---80% (cumulative volume)
Reagent II
Damping fluid 40---200mmol/l
Creatinine deiminase 1000---20000U/l
Stablizer 10---80% (cumulative volume)
The prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, adenosine triphosphate, N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase etc. can be placed on reagent II, reagent II composition wherein, Creatinine deiminase also can be placed on reagent I, so can form multiple formulations, not describe in detail one by one at this.
Reagent can also be made into following three reagent:
Reagent I
Damping fluid 40---200mmol/l
Adenosine triphosphate 0.2---20mmol/l
Coenzyme 0.5---20mmol/l
Stablizer 10---80mmol/l
Reagent II
Damping fluid 40---200mmol/l
N-methyl hydantoin enzyme 1000---20000U/l
Carboxamide sarkosine hydroamidase 1000---10000U/l
Sarcosine oxidase 1000---20000U/l
Aldehyde dehydrogenase 500---50000U/l
Stablizer 10---80% (cumulative volume)
Reagent III
Damping fluid 40---200mmol/l
Creatinine deiminase 1000---20000U/l
Stablizer 10---80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription; The composition of reagent I wherein, adenosine triphosphate, coenzyme etc. can be placed among reagent II or the reagent III.Reagent II composition wherein, N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase etc. also can be placed among reagent I or the reagent III.Reagent III composition wherein, Creatinine deiminase also can be placed among reagent I or the reagent II.So can form multiple formulations, not describe in detail one by one.
The pH scope of buffer reagent can be 6.0～11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-l-propyl alcohol (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 oxidized 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 stablizer 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, stablizer 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 system component relation is comparatively desirable:
Damping fluid 80---120mmol/l
Adenosine triphosphate 2---10mmol/l
Coenzyme 2---8mmol/l
Creatinine deiminase 6000---12000U/l
N-methyl hydantoin enzyme 6000---12000U/l
Carboxamide sarkosine hydroamidase 4000---8000U/l
Sarcosine oxidase 8000---16000U/l
Aldehyde dehydrogenase 5000---20000U/l
Stablizer 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 handling 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
Adenosine triphosphate 2mmol/l
Coenzyme 2mmol/l
Creatinine deiminase 6000U/l
N-methyl hydantoin enzyme 6000---12000U/l
Carboxamide sarkosine hydroamidase 4000U/l
Sarcosine oxidase 8000U/l
Aldehyde dehydrogenase 5000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, 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:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Carboxamide sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy rises, calculate the content of creatinine.
Present embodiment is used Creatinine deiminase coupling N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase reaction colorimetric or continuous monitoring method.Creatinine deiminase hydrolysis creatinine produces the N-methyl hydantoin, N-methyl hydantoin enzyme acts on N-methyl hydantoin and adenosine triphosphate generation carboxamide sarkosine then, produce sarkosine by carboxamide sarkosine hydroamidase again, produce formaldehyde by the effect of coupling sarcosine oxidase again, effect by the coupling aldehyde dehydrogenase again is with coenzyme (NAD
+, NADP
+Or other analogue---do not have absorption peak at the 340nm place) be reduced into reducibility coenzyme (NADH, NADPH or other analogue---absorption peak is arranged) at the 340nm place, thereby measured reduced coenzyme in 340nm place absorbancy ascensional range, by measuring the ascensional range of 340nm place absorbancy, can calculate the content of creatinine.
Embodiment two (two agent)
The creatine diagnosis reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
Adenosine triphosphate 6mmol/l
Coenzyme 5mmol/l
N-methyl hydantoin enzyme 9000U/l
Carboxamide sarkosine hydroamidase 6000U/l
Sarcosine oxidase 12000U/l
Aldehyde dehydrogenase 13000U/l
Stablizer 50% (cumulative volume)
Reagent II
Damping fluid 100mmol/l
Creatinine deiminase 9000U/l
Stablizer 50% (cumulative volume)
When measuring creatinine content, temperature is controlled at 30 ℃, 15 minutes reaction times, 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:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Oxygen formyl sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde takes off the helium enzymeCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy rises, calculate the content of creatinine.
The reaction times 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
Adenosine triphosphate 10mmol/l
Coenzyme 8mmol/l
Stablizer 20mmol/l
Reagent II
Damping fluid 120mmol/l
N-methyl hydantoin enzyme 12000U/l
Carboxamide sarkosine hydroamidase 8000U/l
Sarcosine oxidase 16000U/l
Aldehyde dehydrogenase 20000U/l
Stablizer 50% (cumulative volume)
Reagent III
Damping fluid 120mmol/l
Creatinine deiminase 12000U/l
Stablizer 50% (cumulative volume)
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, 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:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Carboxamide sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy rises, calculate the content of creatinine.
The reaction times 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
Adenosine triphosphate 4mmol/l
Coenzyme 2mmol/l
Stablizer 30mmol/l
Reagent II
Damping fluid 100mmol/l
Creatinine deiminase 8000U/l
N-methyl hydantoin enzyme 6000U/l
Carboxamide sarkosine hydroamidase 8000U/l
Sarcosine oxidase 16000U/l
Aldehyde dehydrogenase 10000U/l
Stablizer 50% (cumulative volume)
On Biochemical Analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, 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:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Carboxamide sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy rises, calculate the content of creatinine.
In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.
Claims (9)
1. creatinine content determination method, step is as follows:
1), with sample and the reagent mix of mainly forming by adenosine triphosphate, coenzyme, Creatinine deiminase, N-methyl hydantoin enzyme, carboxamide sarkosine hydroamidase, sarcosine oxidase, aldehyde dehydrogenase, make it to take place the reaction of following principle:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
N-methyl hydantoin+water+adenosine triphosphate
N-methyl hydantoin enzyme
Carboxamide sarkosine+adenosine diphosphate (ADP)+phosphate radical
Carboxamide sarkosine+water
Carboxamide sarkosine hydroamidase
Sarkosine+ammonium ion+bicarbonate radical
Sarkosine+oxygen+water
Sarcosine oxidaseHydrogen peroxide+formaldehyde
+ glycine
Formaldehyde+coenzyme+water
Aldehyde dehydrogenaseCarbonic acid gas+hydrogen peroxide
+ reducibility coenzyme+hydrogen ion
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbancy 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 absorbancy 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 times was controlled at 2-30 minute.
4, according to claim 1 or 2 described creatinine content determination methods, it is characterized in that: the ratio 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
Adenosine triphosphate 0.2---20mmol/l
Coenzyme 0.5---20mmol/l
Creatinine deiminase 1000--20000U/l
N-methyl hydantoin enzyme 1000---20000U/l
Carboxamide sarkosine hydroamidase 1000---10000U/l
Sarcosine oxidase 1000---20000U/l
Aldehyde dehydrogenase 500---50000U/l
Stablizer 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 stablizer is one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, sulfuric acid amine 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 buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, phosphate buffered saline buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or glycylglycine (Glycylglycine) damping fluid.
9, according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: described coenzyme is NADP
+, NAD
+Or thio-NAD
+A kind of in the oxidized form nicotinamide coenzyme or derivatives thereof.
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|---|---|---|---|
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|---|---|---|---|
| CN 200410064885 CN1757750A (en) | 2004-10-10 | 2004-10-10 | Method of determining creatnine content and reagent box for diagnosing creatnine |
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| Publication Number | Publication Date |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773833A (en) * | 2013-12-19 | 2014-05-07 | 深圳市雷诺华科技实业有限公司 | Creatinine measurement reagent |
-
2004
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773833A (en) * | 2013-12-19 | 2014-05-07 | 深圳市雷诺华科技实业有限公司 | Creatinine measurement reagent |
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