CN101498662A - Reagent kit for monoamine oxidase MAO single-reagent measurement - Google Patents

Abstract

The invention discloses a kit of single reagent used for diagnosing monoamine oxidase (MAO) in serum. MAO hydrolyzed benzylamine, butyl amine, amyl amine, Beta-phenylethyl amine and other amine compounds are utilized to produce oxygen; the oxygen reacts with glutamate dehydrogenase in a coupling way; oxidized coenzyme is formed by the reaction of reduced coenzyme; and the activity of sample MAO formed by quantitative reaction is calculated through measuring the descending speed of the absorbance of light with a wavelength of 340 nm; in addition, enzyme and substrate enzyme which can slowly generate reduced nicotinoyl coenzyme are added in the reaction process, so that an enzyme-substrate enzyme-nicotinoyl coenzyme slow reaction system is formed in single reagent and the nicotinoyl coenzyme can be compensated slowly and circularly and further the single reagent can be stabilized when the concentration of the single reagent achieves a certain balance. The kit is convenient for use and simple is structure, can be used on a common ultraviolet/visible light analyzer or a semi-automatic/full-automatic biochemical analyzer for rapid measurement without using a special or additional apparatus and has low cost.

Description

Reagent kit for monoamine oxidase MAO single-reagent measurement

Technical field

The application relates to the enzyme process single reagent and measures the method for activity of monoamine oxidase and the method for making of kit.

Background technology

Monoamine oxidase (MAO) can reflect Fibrotic biochemical process, is the important indicator of reflection liver fibrosis and hepatocellular damage, more and more comes into one's own in the diagnosis of cirrhosis.

MAO is the protein of cupric, extensively be present in the connective tissue of organs such as liver, kidney, brain and in the cell mitochondrial, its distribution liver〉heart〉kidney〉brain〉lung〉skeletal muscle, also contain MAO in blood platelet and the placenta, its isodynamic enzyme has three kinds, be respectively MAO-I, MAO-II and MAO-III, two MAO-A of subunit and MAO-B are arranged.By the homologous sequence analysis, find that monoamine oxidase has the zone of four high conservatives.In MAO-B, these conservative regions comprise: 1) ADP binding site; 2) substrate binding structural domain; 3) flavin adenine dinucleotide (FAD) (FAD) covalent bond site; 4) C end regions is mainly striden the relevant α spiral of film with one of formation.

MAO is the water-soluble enzyme that can act on different monoamine compounds, and the bridge formation that participates in the collagen maturation final stage forms.After fiber formed, MAO broke away from, thereby caused the active rising of MAO in the serum.The hepatopath finally causes liver fibrosis because of chronic inflammation sexual stimulus in the liver produces the fibrous connective tissue hyperplasia.Increase because collagen is synthetic in this process, increase and degree of hepatic fibrosis positive correlation so MAO is active in the serum.

In the existing determination techniques, the main method of its mensuration has: fluorescence method, immunodepression and chemical spectrophotometric method.Fluorescence method is to be that substrate detects monoamine oxidase with β-phenethyl amine.Immunological method is to utilize monoamine oxidase antibody and monoamine oxidase to take place to answer, and detects the isodynamic enzyme of the monoamine oxidase that generates.Spectrophotometric method is to measure by discharge hydrogen peroxide deoxidation 10-N-methylamino formoxyl-3,7-dimethylamino-10-hydrogen-phenothiazine colour formers such as (MCDP) with monoamine oxidase catalysis amine.At present, mainly with benzylamine and P-Toluidine-β-azo naphthols as substrate.

The technology of the main mensuration MAO that uses is to use the method that adopts the double reagent kit in the market, and this is because the kit of double reagent by the control potential of hydrogen, can increase the stability of reagent.But because the double reagent that adopted takies more reagent freight space, and on automanual instrument complex operation, length consuming time is so limited their practicality.

Therefore, also exist at present exploitation quick, simple to operate, have the good detection effect and needs of the kit that can in multiple detecting instrument, use.Present patent application provides the kit of a kind of single reagent diagnosis MAO, and the method for preparing this kit.The kit that the application provides can be widely used in the various detecting instruments activity of monoamine oxidase being detected, by reprocessing cycle enzyme system of coupling in reaction system, slowly replenish the amount of coenzyme, thereby guaranteed the stability of reagent and the accuracy of clinical measured value.In addition, the freight space that the single reagent that the application provides takies is few, and is easy and simple to handle, easy to use, highly sensitive, and antijamming capability is strong, and it is fast to have a finding speed, accuracy height, effects such as stable reagent.

Summary of the invention

Therefore, in order to overcome the aforesaid problems that in detecting the MAO field, exist at present, please provide the kit of the single reagent of activity of monoamine oxidase in a kind of stable mensuration serum, the blood plasma in this.

The invention provides a kind of 50-250mM that comprises, the single reagent kit of the mensuration activity of monoamine oxidase of the glutamte dehydrogenase of α-ketoglutaric acid of the damping fluid of pH7.0-10, the aminated compounds of 5-30mM, 5-20mM, the reduced coenzyme of 0.2-0.4mM, 0.2-5Ku, it also comprises the regeneration enzyme circulation system of being made up of regenerative system enzyme, substrate and coenzyme.This regeneration enzyme circulation system comprises that concentration is the substrate of 5-100mM, the system enzyme of 10-1000u.

The reaction rate of the described regeneration enzyme circulation system of the application is not disturbed main reaction less than the speed of main reaction.The preferred regeneration enzyme circulation system of the application comprises glucose dehydrogenase-glucose-reduced coenzyme or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-reduced coenzyme or its analog system, glycerol dehydrogenase-glycerine-reduced coenzyme or its analog system, lactic dehydrogenase-pyruvic acid-reduced coenzyme or its analog system; Perhaps glucose dehydrogenase-glucose-oxidized coenzyme or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-oxidized coenzyme or its analog system, glycerol dehydrogenase-glycerine-oxidized coenzyme or its analog system, lactic dehydrogenase-pyruvic acid-oxidized coenzyme or its analog system.

The preferred oxidized coenzyme of the application is oxidized form nicotinoyl coenzyme or its analog, and preferred reduced coenzyme is oxidized form nicotinoyl coenzyme or its analog corresponding reduced form nicotinoyl coenzyme or its analog.Preferred oxidized form nicotinoyl coenzyme or its analog are NAD, NADP, thio-NAD or thio-NADP, and preferred reduced form nicotinoyl coenzyme or its analog are NADH, NADPH, thio-NADH or thio-NADPH.

Especially, the aminated compounds described in the kit that provides of the application is selected from benzylamine, butylamine, amylamine, β-phenethyl amine, tyrasamine or derivatives thereof.Described damping fluid is selected from trishydroxymethylaminomethane-hydrochloride buffer, triethanolamine damping fluid, imidazoles-hydrochloride buffer, glycylglycine damping fluid, sodium carbonate-sodium bicarbonate buffer liquid, boric acid-sodium borate buffer liquid, glycine buffer, citric acid-sodium citrate buffer solution, phosphate buffer or phosphate buffer.

The kit that the application provides preferably also comprises the sequestrant of 0.1-3g/L, the antiseptic of 0.1-3g/L, the stabilizing agent of 1-30%, the surfactant of 0.1-5g/L, the reaction accelerator of 0.1-5mM; Described stabilizing agent is one or more in ethylene glycol, propylene glycol, glycerine, bovine serum albumin(BSA), ethylenediamine tetraacetic acid, thioglycol, sweet mellow wine, lactose, flavin adenine dinucleotide (FAD) and the flavin mononucleotide (FMN); Described surfactant is cationic surfactant, anionic surfactant or amphoteric surfactant.

On the other hand, the regeneration enzyme circulation system that provides of the application also is used for preparing the single reagent kit that detects carbon dioxide, adenosine deaminase, ammonia, creatinine, urea, alanine aminotransferase and aspartate amino transferase.

The application's beneficial effect mainly shows: 1) this method institute reagent preparation is a single agents, so easy to use, simple to operate; 2) this reagent can be on automatic clinical chemistry analyzer fast detecting, also can on semi-automatic or manual instrument, use, so easy to utilize; 3) composition of participation coupling reaction all adds, and can not introduce the pollution of extra interior allogenic material; 4) the regeneration enzyme circulation system speed of the application's coupling is less than the speed of main reaction, does not disturb main reaction; 5) the single reagent kit of the application's preparation, good stability can use for a long time, 37 degree stability experiment proofs blank absorbency A0 〉=0.8 after a year and a half.

The kit of the detection MAO of Cun Zaiing is the kit of double reagent in the market.The application then provides a kind of kit of single reagent that is.

The several problems that need consider when design the application are:

1) optimum reaction conditions of measuring according to the selected monoamine oxidase of the substrate of selecting, as the concentration of damping fluid, ionic strength and pH or the like;

2) the suitableeest stable pH of reduced coenzyme and the long response time pH of the regenerating coenzyme circulation system;

3) removal that ammonia disturbs in the sample;

4) stability problem of whole system;

5) take into account reaction rate problem under the above-mentioned all conditions.

On this basis, the applicant discloses a kind of kit that is used to detect the single reagent of MAO.In the application's a specific embodiments, described kit is mainly taked following method and step:

At first,, make it to take place following reaction with needs sample of measuring and the various material mixings that contain aminated compounds, α-ketoglutaric acid, glutamte dehydrogenase, reduced coenzyme and the regeneration enzyme circulation system,

Monoamine oxidase

Aminated compounds+2 water+oxygen------→ corresponding aldehyde product+ammonium ion+hydrogen peroxide+2OH

Glutamte dehydrogenase

Ammonium ion+α-ketoglutaric acid+reduced coenzyme------→ glutamic acid+oxidized coenzyme+water

Corresponding glucose dehydrogenase

Oxidized coenzyme+grape carbohydrate substrate------→ reduced coenzyme+corresponding aldehyde+water

Then, post reaction mixture on automatic clinical chemistry analyzer, is detected the decline rate of 340nm place absorbance, thereby calculate the active size of monoamine oxidase.

The application's kit also comprises a kind of regeneration enzyme circulation system in the single reagent preparation, replenish system by the generation that in detectable, adds indicative coenzyme, utilize the reversibility of reaction, by the former reactant of the reverse generation of product, thus the amount of replenishing former reactant.This kit by coupling in reaction system one step regeneration enzyme circulation system, thereby guaranteed stable in preparation, storage and application process of each component in the single reagent technically.This regeneration enzyme circulation system can also be applied in the development of following single reagent for example: carbon dioxide reagent, adenosine deaminase reagent, ammonia reagent, creatinine reagent, urea reagent, alanine aminotransferase reagent and aspartate amino transferase reagent.

Concrete, the regeneration enzyme circulation system that comprises in the kit of the disclosed single reagent of the application includes but not limited to glucose dehydrogenase-glucose-reduced form nicotinamidase or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-reduced form nicotinamidase or its analog system, glycerol dehydrogenase-glycerine-reduced form nicotinamidase or its analog system, lactic dehydrogenase-pyruvic acid-reduced form nicotinamidase or its analog system; Perhaps glucose dehydrogenase-glucose-oxidized form nicotinamidase or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-oxidized form nicotinamidase or its analog system, glycerol dehydrogenase-glycerine-oxidized form nicotinamidase or its analog system, lactic dehydrogenase-pyruvic acid-oxidized form nicotinamidase or its analog system.

According to the application's disclosed method, the kit principal ingredient of the single reagent that is mixed with is as follows, but the composition that it will be appreciated by those skilled in the art that this kit is not limited to following compositions, it can comprise other of equal value reagent identical with described composition function, also comprises other compositions or material that reagent preparation box well-known to those skilled in the art is used always:

Damping fluid 50-250mM (pH7.0-11.0)

Aminated compounds 5-30mM

α-ketoglutaric acid 5-20mM

Reduced coenzyme 0.2-0.4mM

Glutamte dehydrogenase 0.2-5ku

D-glucose 5-100mM

Glucose dehydrogenase 10-1000U

Stabilizing agent 1-30%

Antiseptic 0.1-3g/L

Sequestrant 0.1-3g/L

Surfactant 0.1-5g/L

Reaction accelerator 0.1-5mM

The pH scope of preparation the application's the selected damping fluid of kit is between 7.0-11.0.Described damping fluid can be any one of following buffer system: Tris-HCL damping fluid, triethanolamine damping fluid, imidazole hydrochloride damping fluid, glycylglycine damping fluid, citrate buffer solution, carbonic acid buffer, phosphate buffer or the like.

The above-mentioned stabilizing agent of mentioning can be to be selected from one or more of following compositions: ethylene glycol, propylene glycol, glycerine, ammonium sulfate, thioglycol, bovine serum albumin(BSA), lactose or the like.

The above-mentioned sequestrant of mentioning can be selected from following compositions a kind of: EDTA, NTA, CyDTA, DTPA, EDTA-OH, TTHA, HIDA acid or slaine.Its application target mainly is the bivalent metal ion in the chelating sample, reduces the interference of bivalent metal ion to measuring.Its preferred concentration range is: 0.1-3.0g/L.

Above-mentionedly mention that surfactant is can be to be selected from one or more of following compositions: cationic surfactant refers to: cetyl trimethyl ammonium bromide, hexadecylpyridinium chloride etc.Anionic surfactant is meant: sodium dodecylsulphonate, neopelex, sodium taurocholate etc.Amphoteric surfactant is meant: Triton X series, alkyl, betaine series etc.Its preferred concentration range is: 0.1-5g/L.

In the specific embodiments of wood application, its method that detects MAO is operating as: 1) sample (calibration object is made sample) is joined in the reagent, make that the ratio of sample and reagent is 1:9～1:11, mix, 37 ℃ of reactions are after 5 minutes, read the absorbance of (for example 2 minutes, 3 minutes, 5 minutes) in the Fixed Time Interval, and calculate the rate of change of absorbance; Calculate the activity of MAO according to following formula:

ε: mM extinction coefficient

Tv: total reaction volume (mL)

Sv: sample volume (mL)

L: cuvette optical path (cm)

The present invention is described in detail below with reference to embodiment; wherein embodiment illustrates and non-limiting effect; those skilled in the art can make improvements at the technical of specific embodiment of following disclosure fully, all can fall into protection scope of the present invention but be no more than the improvement of having done within the scope of claim of the present invention or the spirit of the present invention.

Specific embodiments

Embodiment 1: the preparation of the single reagent of kit

The preparation that the application is exemplary the kit of 3 kinds of single reagents, the composition of 3 kinds of single reagents of its preparation raw material, preparation steps and acquisition is respectively:

1) preparation of reagent:

Needed raw material

Title	Molecular weight	Purity	Manufacturer
				Tris-base	121.1	99.9％	Beijing is glad through biotech company of section
Nacl	58.44	Analyze pure	Beijing Yili Fine Chemicals Co., Ltd.
				NaN3	65.01	99％	Merck
EDTA-2Na	372.24
				BSA		98.0％	Amresco
ADP—K	501.3
				TritonX—100	647	99.0％	Beijing is glad through biotech company of section
α-ketoglutaric acid	146.1	99.5％	Beijing is glad through biotech company of section
				Benzylamine	107.15	99.5％	acros
D-glucose	180.16	99.8％	Beijing is glad through biotech company of section
				GLDI		237u/mg	Toyobo
NADH	709.9		roche
				GDH		72.8u/mg

2) collocation method

Various materials are at room temperature dissolved in small quantity of deionized water, and mixing transfers that pH is 7.0 ~ 11.0, constant volume in damping fluid then, places for 4-8 ℃ and can use in 4-7 days.

3) composition of the kit of 3 kinds of single reagents of Huo Deing (called after kit I, kit II and kit III respectively) is as follows:

Kit I:

Tris-Hcl damping fluid 50mM

Benzylamine 5mM

α-ketoglutaric acid 5mM

Reduced coenzyme 0.2mM

Glutamte dehydrogenase 0.2ku

Mannitol 0.1%

D-glucose 5mM

Glucose dehydrogenase 10U

EDTA 1g/L

NaN3 1g/L

ADP—K 0.1mM

Triton x-100 1g/L

Kit II box:

Phosphate buffer 100mM

Benzylamine 20mM

α-ketoglutaric acid 10mM

Reduced coenzyme 0.25mM

Glutamte dehydrogenase 275u

Mannitol 0.1%

Pyruvic acid 20mM

Lactic dehydrogenase 70U

EDTA 2g/L

NaN3 2g/L

ADP—K 1.5mM

Triton x-100 5g/L

Reagent III:

Glycylglycine damping fluid 250mM

Benzylamine 30mM

α-ketoglutaric acid 20mM

Reduced coenzyme 0.4mM

Glutamte dehydrogenase 5ku

Mannitol 0.5%

Glucose 6 phosphatase 11 00mM

Glucose 6 phosphate dehydrogenase 10 00U

EDTA 3g/L

NaN3 3g/L

ADP—K 5mM

Triton x-100 5g/L

Embodiment 2: with method and the step of MAO in the kit test sample

1) detected parameters is:

37 ℃ of temperature

Wavelength 340nm (main) ﹠amp; 405nm (pair)

Cuvette optical path 1.0cm

The method of testing rate method

The Direction of Reaction negative reaction

2) detecting step is:

At first, calibrate with the water zeroing; The sample that adds 0.018ml then; Add 0.18ml reagent afterwards, 37 ℃ hatch 4 minutes after, tested 2 minutes, calculate the variation (Δ A/min) of test duration section per minute mean light absorbency.Theoretical k value is-1740.

3) normal reference value of MAO: 0-12U/L in the serum

Embodiment 3: the antijamming capability of detection kit I, II, III

To 3 kinds of single reagent boxes of embodiment 1 configuration, promptly the performance of kit I, II, III is carried out following detection simultaneously:

1, the anti-interference experiment of kit

The testing sample that contains ascorbic acid, cholerythrin, Hb variable concentrations respectively according to ordinary skill in the art configuration, adopt kit I, II, the III activity of MAO in the test sample in detecting instrument, thereby detect the antijamming capability (each sample triplicate, get its mean value) of three kinds of kits to various chaff interferences.Testing result (mean value) is as shown in table 1:

Table 1

The result of table 1 shows, when ascorbic acid≤50mg/dL, cholerythrin≤40mg/dL or Hb≤200mg/dL, it does not influence the detection of MAO in kit I, II, the III sample, and promptly described chaff interference does not produce the detection of kit and disturbs.

The anti-ammonia interference test of 2 kits

The testing sample that contains the ammonia of variable concentrations respectively according to ordinary skill in the art configuration, adopt kit I, II, the III activity of MAO in the test sample in detecting instrument, thereby detect the antijamming capability (each sample triplicate, get its mean value) of three kinds of kits to ammonia.Experimental result is as shown in table 2:

Table 2:

Therefore the result of table 2 shows when the interference of ammonia reaches 50uM, can not influence the detection of the application's kit to MAO, illustrate that the disclosed kit of the application can resist≤interference of the ammonia of 50uM, and the not anti-ammonia of contrast agents box disturbs.

Embodiment 4: the accuracy of detection kit I, II, III and precision

Adopt the detection method of this area routine respectively, kit I, the II of embodiment 1 preparation, accuracy and the precision of III are detected.

1. accuracy

To the target value be 8 control liquid under the same conditions, adopt kit I, II, III to the active continuous detecting of the MAO of same control liquid 6 times, the mean value and the target value scope (4-12) of testing result compared, to detect the accuracy of described kit.Testing result to the accuracy of reagent basin I, II, III is as shown in table 3:

Table 3

The result of table 3 shows: the mean value of 6 testing results of kit I, II, III is in target value scope (4-12), and inaccuracy relative deviation (CV (%)) is no more than ± 5%.Illustrate that the accuracy of the disclosed three kinds of kits of the application meets the technical indicator of diagnostic kit.

1. precision

Under the same conditions, adopt kit I, II, III to the active continuous detecting of the MAO of a serum 20 times, the coefficient of variation of each kit relatively is to detect the precision of described kit.Testing result to the precision of kit I, II, III is as shown in table 4:

Table 4

The result of table 4 shows: the coefficient of variation of the MAO activity of the same sample that kit I, II, III detect is no more than ± and 10%, illustrate that the precision of the disclosed three kinds of kits of the application meets the technical indicator of diagnostic kit.

Embodiment 5: the sensitivity of kit I, II, III detects

Adopt method known to those skilled in the art, with blank solution with a sample that carries out 10 gradients of proportional diluted with physiological saline, on detecting instrument, adopt kit I, II, III continuously to absorbance measurement 14 times, read the original absorbance of each mensuration, calculate the value of the absorbance of each sample of deduction behind the blank absorbency then, calculate its average, standard deviation.Here adopt 99.7% possibility to calculate the detection lower bound.The average of each sample deducted 3 times standard deviation separately, 3 times standard deviation with blank solution compares then, if the former is higher than the latter, we just assert has minimum absorbance that 99.7% possibility occurs necessarily greater than the absorbance of blank, can quantitative reporting the result.

1) data (table 5) of the original absorbance of the measured value of kit I

Table 5

2) absorbance data (table 6) after the deduction blank

Table 6

Table 5 and 6 result show that the biological detection limit of kit I is: 0.00054 Δ A/u/L

2) data (table 7) of the original absorbance of the measured value of kit II

Table 7

2) absorbance data (table 8) after the deduction blank

Table 8

Table 7 and 8 result show that the biological detection limit of kit II is: 0.00044 Δ A/u/L

3) data (table 9) of the original absorbance of the measured value of kit III

Table 9

2) absorbance data (table 10) after the deduction blank

Table 10

Table 9 and 10 data show that the biological detection limit of kit III is: 0.0010 Δ A/u/L

Embodiment 6 kit I, the clinical liver cancer of II, III, patients with liver fibrosis measured value

Collect hepatopath's serum samples such as being diagnosed as liver cancer, liver fibrosis from hospital, on Olympus400, with the MAO activity of the kit for preparing according to the conventional method test sample, its result is as shown in table 11:

Table 11

The presentation of results of table 11 conforms to clinical disease with the testing result of the disclosed 3 kinds of kits of the application to the MAO activity of the clinical sample made a definite diagnosis, illustrates that the disclosed 3 kinds of kits of the application go for clinical detection to the MAO activity.

In a word, the application's advantage is, the single reagent box is simple to operate, easy to use, can be applied on the various biochemical analyzers such as full-automatic and semi-automatic; By the interference of external source ammonia in the lengthening response delay time accelerated reaction elimination, the indirect regeneration of coupled cofactor, stability, accuracy, the anti-interference of enhancing reagent, long storage time still can accurately be measured the activity of the monoamine oxidase in the sample afterwards.

It will be understood by those skilled in the art that the application's disclosed method and the single reagent box of producing thus can be widely used in Hitachi, Olympus and the various automatic clinical chemistry analyzers of Synchron.The condition determination of each instrument is as follows: the ratio of 37 degrees centigrade of temperature, 4 minutes reaction time, test predominant wavelength 340nm, commplementary wave length 405nm, mensuration sample and kit is 1:10, the reflection direction is negative reaction, be 4 minutes time delay, and 2 minutes detection times, theoretical k value is-1746.According to the experience of prior art and clinical staff, carry out under the prerequisite of creative work not needing, those skilled in the art can be optimized the optimum detection parameter of various detecting instruments.Parameter Optimization to various detecting instruments does not exceed outside the claimed scope of the application.

Claims (13)

1, a kind of single reagent kit of measuring activity of monoamine oxidase, comprise 50-250mM, α-ketoglutaric acid of the damping fluid of pH7.0-10, the aminated compounds of 5-30mM, 5-20mM, the reduced coenzyme of 0.2-0.4mM, the glutamte dehydrogenase of 0.2-5Ku is characterized in that also comprising the regeneration enzyme circulation system of being made up of regenerative system enzyme, substrate and coenzyme.

2, according to the kit of claim 1, the wherein said regeneration enzyme circulation system comprises that concentration is the substrate of 5-100mM, the system enzyme of 10-1000u.

3, according to the kit of claim 1 or 2, it is characterized in that the speed of the reaction rate of the described regeneration enzyme circulation system less than main reaction, do not disturb main reaction.

4, according to the kit of claim 1 or 2, the wherein said regeneration enzyme circulation system comprises glucose dehydrogenase-glucose-reduced coenzyme or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-reduced coenzyme or its analog system, glycerol dehydrogenase-glycerine-reduced coenzyme or its analog system, lactic dehydrogenase-pyruvic acid-reduced coenzyme or its analog system; Perhaps glucose dehydrogenase-glucose-oxidized coenzyme or its analog system, glucose 6 phosphate dehydrogenases-glucose 6 phosphoric acid-oxidized coenzyme or its analog system, glycerol dehydrogenase-glycerine-oxidized coenzyme or its analog system, lactic dehydrogenase-pyruvic acid-oxidized coenzyme or its analog system.

5, according to the kit of claim 4, it is characterized in that described oxidized coenzyme is oxidized form nicotinoyl coenzyme or its analog, reduced coenzyme is oxidized form nicotinoyl coenzyme or its analog corresponding reduced form nicotinoyl coenzyme or its analog.

6, according to the kit of claim 5, it is characterized in that oxidized form nicotinoyl coenzyme or its analog are NAD, NADP, thio-NAD or thio-NADP, reduced form nicotinoyl coenzyme or its analog are NADH, NADPH, thio-NADH or thio-NADPH.

7. according to the kit of claim 6, the absorbance that it is characterized in that described reduced coenzyme NADH between 0.6-2.0, wavelength 340nm, optical path 10mm.

8,, it is characterized in that also comprising the sequestrant of 0.1-3g/L, the antiseptic of 0.1-3g/L, the stabilizing agent of 1-30%, the surfactant of 0.1-5g/L, the reaction accelerator of 0.1-5mM according to the kit of claim 1 or 2.

9,, it is characterized in that described aminated compounds is selected from benzylamine, butylamine, amylamine, β-phenethyl amine, tyrasamine or derivatives thereof according to the kit of claim 1 or 2.

10,, it is characterized in that described damping fluid is selected from trishydroxymethylaminomethane-hydrochloride buffer, triethanolamine damping fluid, imidazoles-hydrochloride buffer, glycylglycine damping fluid, sodium carbonate-sodium bicarbonate buffer liquid, boric acid-sodium borate buffer liquid, glycine buffer, citric acid-sodium citrate buffer solution, phosphate buffer or phosphate buffer according to the kit of claim 1 or 2.

11, kit according to Claim 8, it is characterized in that be stabilizing agent be in ethylene glycol, propylene glycol, glycerine, bovine serum albumin(BSA), ethylenediamine tetraacetic acid, thioglycol, sweet mellow wine, lactose, flavin adenine dinucleotide (FAD) and the flavin mononucleotide (FMN) one or more.

12, kit according to Claim 8 is characterized in that described surfactant is cationic surfactant, anionic surfactant or amphoteric surfactant.

13, a kind of regeneration enzyme circulation system can be expanded the purposes that is used for preparing the single reagent kit that detects carbon dioxide, adenosine deaminase, ammonia, creatinine, urea, alanine aminotransferase and aspartate amino transferase.