CN1757753A - Determination method of creatnine content and reagent box for diagnosing creatnine - Google Patents

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 buffer liquid, adenosine, pyruvic acid, stabilizer, etc.

Description

Creatinine content determination method and creatine diagnosis reagent kit

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.

It is as follows that the present invention measures the method steps of creatinine content:

1), with sample and the reagent mix of mainly forming by adenosine triphosphate, pyruvic acid, Creatinine deiminase, N-methyl hydantoin enzyme, pyruvic oxidase, catalase, reduced form chromogen, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

2), detect the end reaction thing in the speed that predominant wavelength 400--600nm absorbancy rises, 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 400--600nm absorbancy rises, 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 enzyme linked reaction and reduced form chromogen (Chromogen) combined systems such as creatinine coupling Creatinine deiminase, N-methyl hydantoin enzyme, pyruvic oxidase, peroxidase, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen (Quioneimine) or indoleamine chromogen (Indamine) dyestuff, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect the size of creatinine content.The advantage of this enzyme linked reaction system has: colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen in (1) its utilization or indoleamine chromogen dyestuff reflects creatinine content, and the good advantage of tolerance range is arranged.(2) reacted constituent of this enzyme linked reaction system composition all adds, and is not subjected to the pollution of inside and outside source material, and test result is accurate.

Be used to realize that the creatine diagnosis reagent kit of the inventive method can be single agent, comprise:

Damping fluid 40--200mmol/l

Adenosine triphosphate 0.2--20mmol/l

Pyruvic acid 1--20mmol/l

Creatinine deiminase 1000--20000U/l

N-methyl hydantoin enzyme 1000--20000U/l

Pyruvic oxidase 1000--60000U/l

Catalase 1000--50000U/l

Reduced form chromogen combination 0.1--20mmol/l

Stablizer 10--80% (cumulative volume)

3-methyl-2-Proxel hydrazone (3-methyl-2-benzothiazolinone-hydrszone is called for short MBTH) that above reduced form chromogen combination can be 0.1-20mmol/l or the amino anti-arsenic of 4-(4-aminoantipyrine) of 0.1-20mmol/l combine with one of following 14 kinds of compositions of 0.1--20mmol/l:

PHENOL 99.8 MIN ((CARBOLIC ACID)) (phenol)

N-ethyl-N-(3-thiopropyl)-m-thialdine amine (N-ethyl-N-(3-sulfopropyl)-m-anisidine is called for short ESPAS)

N, and the two ethyls of N--m-Tolylamine (N, N-Diethyl-m-toluidine)

2, and the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-(2,4-Dichloriphenol)

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid (2,4,6-Tribromo-3-hydroxy-benzenesulfonic acid is called for short TBHB)

3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid (3,5-Dichlorophenol sulfonic acid) of 5-

3, the two chloro-2-hydroxyl-Phenylsulfonic acids (3,5-Dichloro-2-hydroxy-benzenesulfonicacid is called for short DHBS) of 5-

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium is called for short TOOS)

Three bromine hydroxy-benzoic acid (Tribromohydroxybenzoic acid)

Two monomethylanilines (Dimethylaniline is called for short DMA)

N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine is called for short EHSPT)

2,2 '-AZINO-two (3-ethylbenzene thiazoles-6-sulfonic acid) (2,2 '-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) is called for short ABTS

Vanillic acid (Vanillic acid (4-hydroxy-3-methoxybenzoic acid) is called for short HMB)

3-methyl-ethyl-hydroxyanilines (3-methyl-ethyl-hydroxyaniline is called for short MEHA)

Also above single agent can be made into following pair of agent, more help eliminating inside and outside source phosphate radical and pollute:

Reagent I

Damping fluid 40--200mmol/l

Adenosine triphosphate 0.2--20mmol/l

Pyruvic acid 1--20mmol/l

N-methyl hydantoin enzyme 1000--20000U/l

Pyruvic oxidase 1000--60000U/l

Catalase 1000--50000U/l

Reduced form chromogen combination (one) 0.1--20mmol/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.

Reagent II

Damping fluid 40--200mmol/l

Creatinine deiminase 1000--20000U/l

Reduced form chromogen combination (two) 0.1--20mmol/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination (two) can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

The prescription of two agent is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can transpositions.The composition of reagent I wherein, adenosine triphosphate, N-methyl hydantoin enzyme etc. can be placed on reagent II.The composition of reagent II wherein, Creatinine deiminase 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, not only more help eliminating inside and outside source phosphate radical and pollute, also more help the stable of reagent:

Reagent I

Damping fluid 40--200mmol/l

Adenosine triphosphate 0.2--20mmol/l

Pyruvic acid 1--20mmol/l

Reduced form chromogen combination (one) 0.1--20mmol/l

Stablizer 10--80mmol/l

Reduced form chromogen combination (one) can be that the amino anti-arsenic of 4-of the 3-methyl-2-Proxel hydrazone of 0.1-20mmol/l or 0.1-20mmol/l is one of in the two.

Reagent II

Damping fluid 40--200mmol/l

N-methyl hydantoin enzyme 1000--20000U/l

Pyruvic oxidase 1000--60000U/l

Catalase 1000--50000U/l

Stablizer 10--80% (cumulative volume)

Reagent III

Damping fluid 40--200mmol/l

Creatinine deiminase 1000--20000U/l

Reduced form chromogen combination (two) 0.1--20mmol/l

Stablizer 10--80% (cumulative volume)

Reduced form chromogen combination (two) can be one of following 14 kinds of compositions of 0.1--20mmol/l: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

Three doses prescription is not limited only to above-mentioned prescription, and reduced form chromogen combination () and (two) can divide any position that is opened in reagent I, II or III.The composition of reagent I wherein, adenosine triphosphate, pyruvic acid can be placed among the reagent II.The composition of reagent II, N-methyl hydantoin enzyme, pyruvic oxidase, catalase etc. also can be placed among the reagent I, and peroxidase can also be placed on reagent III.The composition of reagent III, Creatinine deiminase can divide any position that is opened in reagent I or 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, trolamine (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.

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 of the present invention of following system component relation is comparatively desirable:

Damping fluid 80--120mmol/l

Adenosine triphosphate 2--10mmol/l

Pyruvic acid 5--10mmol/l

Creatinine deiminase 6000--10000U/l

N-methyl hydantoin enzyme 6000--10000U/l

Pyruvic oxidase 8000--12000U/l

Catalase 8000--20000U/l

Reduced form chromogen combination 0.5--10mmol/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.

In addition, one of striking features of the present invention is the pollution that can eliminate inside and outside source phosphate radical, the effect of eliminating inside and outside source phosphate radical occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source phosphate radical, and all be the effect that results from creatinine at the needed phosphate radical of second half section time test creatinine content.

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

Pyruvic acid 5mmol/l

Creatinine deiminase 6000U/l

N-methyl hydantoin enzyme 6000U/l

Pyruvic oxidase 8000U/l

Catalase 8000U/l

The amino anti-arsenic 2mmol/l of 4-

PHENOL 99.8 MIN ((CARBOLIC ACID)) 10mmol/l

Stablizer 50% (cumulative volume)

On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 495～505nm, more than the test commplementary wave length 600nm, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 495～505nm absorbancy rises, thereby calculate the content size of creatinine.

Present embodiment is used enzyme linked reaction and reduced form chromogen combined systems such as creatinine coupling Creatinine deiminase, N-methyl hydantoin enzyme, pyruvic oxidase, peroxidase, colourless reduced form chromogen combination is oxidized to coloured quinone-imine chromogen or indoleamine chromogen, therefore the height (different dyes absorbing wavelength difference is between 400--600nm) of measuring dyestuff content can directly reflect the size of creatinine content.

Embodiment two (two agent)

The creatine diagnosis reagent of present embodiment has:

Reagent I

Damping fluid 100mmol/l

Adenosine triphosphate 6mmol/l

Pyruvic acid 8mmol/l

N-methyl hydantoin enzyme 8000U/l

Pyruvic oxidase 10000U/l

Catalase 10000U/l

The amino anti-arsenic 1mmol/l of 4-

Stablizer 50% (cumulative volume)

Reagent II

Damping fluid 100mmol/l

Creatinine deiminase 8000U/l

2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid 1mmol/l

Stablizer 50% (cumulative volume)

When measuring creatinine content, temperature is controlled at 30 ℃, 15 minutes reaction times, and test predominant wavelength 546nm, more than the test commplementary wave length 630nm, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 546nm absorbancy rises, thereby calculate the content size 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

Pyruvic acid 10mmol/l

3-methyl-2-Proxel hydrazone 2mmol/l

Stablizer 20mmol/l

Reagent II

Damping fluid 120mmol/l

N-methyl hydantoin enzyme 10000U/l

Pyruvic oxidase 12000U/l

Catalase 20000U/l

Stablizer 50% (cumulative volume)

Reagent III

Damping fluid 120mmol/l

Creatinine deiminase 10000U/l

Two monomethylaniline 2mmol/l

Stablizer 20% (cumulative volume)

On automatic clinical chemistry analyzer, set: temperature 25,20 minutes reaction times, test predominant wavelength 578nm, more than the test commplementary wave length 700nm, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate the content size of creatinine.

The reaction times of each reactions steps was controlled at 10 minutes.

Embodiment four

The creatine diagnosis reagent of present embodiment has:

Reagent I

Damping fluid 100mmol/l

Adenosine triphosphate 5mmol/l

N-methyl hydantoin enzyme 8000U/l

Pyruvic oxidase 10000U/l

Hydrogen peroxidase 12 000U/l

The amino anti-arsenic 1mmol/l of 4-

Stablizer 50% (cumulative volume)

Reagent II

Damping fluid 100mmol/l

Pyruvic acid 7mmol/l

Creatinine deiminase 10000U/l

Two monomethylaniline 2mmol/l

Stablizer 50% (cumulative volume)

Measure 5 '-during activity of 5 '-nucleotidase, temperature is controlled at 30 ℃, 10 minutes reaction times, and test predominant wavelength 578nm, more than the test commplementary wave length 700nm, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

The end reaction thing is placed under the biochemical analyzer, detect the speed that predominant wavelength 578nm absorbancy rises, thereby calculate the content size of creatinine.

The reaction times of each reactions steps was controlled at 10 minutes.

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 (10)

1. creatinine content determination method, step is as follows:

1), with sample and the reagent mix of mainly forming by adenosine triphosphate, pyruvic acid, Creatinine deiminase, N-methyl hydantoin enzyme, pyruvic oxidase, catalase, reduced form chromogen, 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

Phosphate radical+pyruvic acid+oxygen+water Pyruvic oxidaseAcetylphosphate+

Carbonic acid gas+hydrogen peroxide

Hydrogen peroxide+reduced form chromogen combination PeroxidaseThe indoleamine chromogen or

Quinone-imine chromogen+water

2), detect the end reaction thing in predominant wavelength 400---the speed that the 600nm absorbancy rises, 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, detecting predominant wavelength 400---the speed (journey) that the 600nm absorbancy rises is spent, and calculates 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

Pyruvic acid 1---20mmol/l

Creatinine deiminase 1000---20000U/l

N-methyl hydantoin enzyme 1000---20000U/l

Pyruvic oxidase 1000---60000U/l

Catalase 1000---50000U/l

Reduced form chromogen combination 0.1---20mmol/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 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 buffer reagent is 6.0～11.0, and described buffer reagent is a kind of in three (carboxymethyl) aminomethane-hydrochloride buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.

9, according to claim 5 or 6 described creatine diagnosis reagents, it is characterized in that: one of following 14 kinds of compositions of 3-methyl-2-Proxel hydrazone that described reduced form chromogen is 0.1-20mmol/l and 0.1-20mmol/l combine: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.

10, according to claim 5 or 6 described creatine diagnosis reagents, it is characterized in that: described reduced form chromogen is combined as by the amino anti-arsenic of the 4-of 0.1-20mmol/l and one of 14 kinds of compositions below the 0.1-20mmol/l and combines: PHENOL 99.8 MIN ((CARBOLIC ACID)), N-ethyl-N-(3-thiopropyl)-m-thialdine amine, N, the two ethyls of N--m-Tolylamine, 2, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) of 4-, 2,4,6-three bromo-3-hydroxyl-Phenylsulfonic acid, 3, the two chlorine PHENOL 99.8 MIN ((CARBOLIC ACID)) sulfonic acid of 5-, 3, the two chloro-2-hydroxyl-Phenylsulfonic acids of 5-, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine sodium salt, three bromine hydroxy-benzoic acid, two monomethylanilines, N-ethyl-N-(2-hydroxyl-3-thiopropyl)-m-Tolylamine, 2,2 '-AZINO-two (3-ethylbenzene thiazole-6-sulfonic acid), vanillic acid, 3-methyl-ethyl-hydroxyanilines.