CN102154444B

CN102154444B - Method for measuring concentration of asymmetric dimethylarginine and diagnostic reagent kit

Info

Publication number: CN102154444B
Application number: CN 201010615214
Authority: CN
Inventors: 宋占科; 刘希; 刘瑶
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2010-12-30
Filing date: 2010-12-30
Publication date: 2013-09-25
Anticipated expiration: 2030-12-30
Also published as: CN102154444A

Abstract

The invention relates to a method for measuring the concentration of asymmetric dimethylarginine and a diagnostic reagent kit. The method comprises the following steps of: degrading asymmetric dimethylarginine (ADMA) by adopting dimethylarginine dimethylamino hydrolase to generate dimethylamine and citrulline; reacting the citrulline with adenosine triphosphate (ATP) and aspartate under the action of arginine succinate synthetase to generate adenosine monophosphate (AMP); generating inosine monophosphate (IMP) and ammonia by the AMP under the action of AMP deaminase; generating oxidization nicotinamide adenine dinucleotide (NAD) by the ammonia and deamination NAD under the action of NAD synzyme; and calculating the concentration of the ADMA by detecting the concentration of the NAD. The method and the reagent kit are accurate in results, and high in speed, repeatability and interference resistance, and are convenient to popularize and use.

Description

Measure method and the diagnostic kit of asymmetric diethylarginine concentration

Technical field

The present invention relates to measure method and the diagnostic kit of asymmetric diethylarginine concentration.Especially, the present invention relates to utilize diethylarginine dimethylamino lytic enzyme, ATP, aspartic acid, argininosuccinate synthetase, AMP desaminase, NAD synthetic enzyme and deaminizating oxidized coenzyme NAD to generate oxidized coenzyme, by detecting method and the test kit that coenzyme concentration records asymmetric diethylarginine concentration.

Background technology

Asymmetric diethylarginine (ADMA) be a kind of can be in human blood and people urine detected endogenous molecule, form in the hydrolytic process of the albumen that methylates in vivo.ADMA can be by suppressing the synthetic biological action of bringing into play of nitrogen protoxide.Constantly have to have statistics significantly between newly-increased clinical study proof ADMA and important bad cardiovascular event or the dead incidence and cognation independently, and then infer that ADMA is as a kind of important new cardiovascular risk factor.

Traditional relatively cardiovascular risk factor, ADMA can also be applied in traditional risks and assumptions can't explain (for example hemodialysis patient) in the unusual high Incidence of CHD case.Rising that there are some researches show ADMA at present may play the important pathological physiological role in diseases such as atherosclerosis, congestive heart failure, hypercholesterolemia, hypertension, diabetes, chronic kidney hypofunction, preeclampsia, hyperhomocysteinemiainjury, liver failure, erective dysfunction.

The method of measuring asymmetric diethylarginine concentration in serum or the blood plasma has a variety of, comprises high performance liquid chromatography (HPLC), euzymelinked immunosorbent assay (ELISA) (ELISA), high performance liquid chromatography-mass spectrometry method and enzyme process etc.

High performance liquid chromatography and high performance liquid chromatography-mass spectrometry combination method detects, complicated operation, and need specific apparatus.CN 101438148A (WO2007103124-A2) utilizes tandem mass spectrometry, based on the specific charge after the ADMA ionization, measures the content of ADMA in the sample on the internal standard substance basis of using known quantity.WO2006128419-A1 points out after using particular matter to remove sample albumen, utilize silicate positive liquid chromatography tandem mass spectrometry, adding 13C6-Arginine and D6-ADMA simultaneously is interior mark, can measure arginine, ADMA, Symmetric dimethylarginine (SDMA) in the sample simultaneously.

Euzymelinked immunosorbent assay (ELISA) (ELISA): be mainly used in experimental study at present.Liquid phase chromatography is convenient a little relatively, and its shortcoming is that sample pre-treatments is more loaded down with trivial details, and most of operation also needs manual operations, and time-consuming is long, is difficult to realize fully automated.Business-like ELISA test kit (DLD Diagnostika GmbH at present; Germany) analysis principle: use acylting agent with the ADMA acylations in the sample earlier; add enzyme plate then; ADMA competitiveness on being combined in solid phase is combined the ADMA polyclonal antibody; adopt standard substance and the enzymic-labelled antibody competition combination of different levels; absorbancy, production standard curve are read in colour developing then.After the same step operation of sample and standard substance, obtain corresponding A DMA concentration at typical curve; CN 1656123A (WO2003102031-A) utilizes the diethylarginine dimethylamino lytic enzyme (DDAH) of sudden change, namely guaranteeing the sudden change substrate A DMA affinity of DDAH and be not enough to be hydrolyzed into dimethylamine and citrulline, and proposed to utilize this mutant enzyme specificity in conjunction with enzymoimmunoassay (ELISA), fluorescence polarization immunoassay (FPIA), radioimmunoassay (RIA), the equiprobable detection method of clone's enzyme donor immunoassay (CEDIA) of ADMA character thus, but existing market is not found the report that its commercialization is used; US20090053741A points out, based on the characteristics that ADMA is combined with the polypeptide fragment of nitric oxide synthetase, has proposed a kind of detection method of using competitive ELISA.

Enzyme process: based on the specific detection of enzyme, general good reproducibility easily is automated, but does not also have matured product in the market.WO200046395-A with the testing sample Deproteinization after, using arginase to remove arginine disturbs, behind the ion exchange column roughing out ADMA, use diethylarginine dimethylamino lytic enzyme (DDAH) hydrolysis ADMA to produce dimethylamine and citrulline, and then use colorimetric method for determining citrulline content, measure ADMA content, but this method operation is quite loaded down with trivial details, and chronic, be difficult to promote in clinical labororatory; WO2006030620-A1 (JP2006081481, A) utilize diethylarginine dimethylamino lytic enzyme (DDAH) hydrolysis ADMA to produce dimethylamine and citrulline, measure according to following several method: 1) dimethylamine desaturase coupling dimethylamine and mPMS generate reduced form mPMS, and reduced form mPMS and the first moon generate for (formazan) reagent (NTB or WST-8) reaction back and replace by the first moon; 2) dimethylamine coupling NADPH under the effect of dimethylamine monooxygenase, the decline speed of reaction of mensuration NADPH; 3) citrulline generates AMP under the argininosuccinate synthetase effect, and uses luciferase (Luciferase) to come the coupling fluorescein to measure the content of residue ATP; 4) citrulline generates AMP under the argininosuccinate synthetase effect, and the coupling phosphoenolpyruvic acid generates pyruvic acid, and then detects the pyruvic acid content that generates under the pyruvic oxidase effect.The disclosure patent that need illustrate is not mentioned by the AMP desaminase and is detected AMP content.Owing to also do not have ripe enzyme process to detect method and the test kit of ADMA concentration at present in the prior art, therefore, exist measuring the method for ADMA concentration and the demand of test kit convenient, sensitive, exactly.

Summary of the invention

Therefore, goal of the invention of the present invention is to seek a kind of method and the test kit that can measure ADMA concentration convenient, sensitive, exactly.

Therefore, a first aspect of the present invention relates to a kind of method of measuring asymmetric diethylarginine concentration, and it comprises the steps:

1) in sample, add diethylarginine dimethylamino lytic enzyme, make the reaction of asymmetric diethylarginine and water produce dimethylamine and citrulline,

2) in the mixture of step 1), add ATP, aspartic acid and argininosuccinate synthetase, citrulline reacted produce AMP,

3) to step 2) mixture in add the AMP desaminase, make the reaction of AMP and water produce IMP and ammonia,

4) in the mixture of step 3), add ATP, NAD synthetic enzyme and deaminizating oxidized coenzyme NAD, make the ammonia generation oxidized coenzyme that reacts,

5) record the concentration of asymmetric diethylarginine by the concentration of the oxidized coenzyme NAD that measure to produce,

Alternatively, above-mentioned diethylarginine dimethylamino lytic enzyme and ATP, aspartic acid, argininosuccinate synthetase, AMP desaminase, NAD synthetic enzyme and deaminizating oxidized coenzyme NAD are added in the sample in two batches.

Preferably, the consumption of described diethylarginine dimethylamino lytic enzyme is 0.5-100kU/L, more preferably 1-50kU/L, more preferably 2-20kU/L, most preferably 3-10kU/L; Preferably, the consumption of described ATP is 1-10mM, more preferably 2-6mM, most preferably 5mM; Preferably, the consumption of described aspartic acid is 2-15mM, most preferably 10mM; Preferably, the consumption of described argininosuccinate synthetase is 0.2-50kU/L, more preferably 0.3-20kU/L, most preferably 5kU/L; Preferably, the consumption of described NAD synthetic enzyme is 1-100kU/L, more preferably 5-50kU/L, most preferably 15-30kU/L; Preferably, the consumption of described AMP desaminase is 1-100kU/L, more preferably 5-50kU/L, most preferably 15-30kU/L; Preferably, the consumption of described deaminizating oxidized coenzyme NAD is 1-10mM.

Preferably, described water derives from sample or extra the adding, and preferably, sample is blood or urine.

Preferably, adopt electrochemistry during the concentration of the oxidized coenzyme NAD that measure to produce, chemistry is given out light or the method for desaturase linked reaction is carried out, preferably, employing catalyzed oxidation type coenzyme NAD is that the desaturase of NADH is converted into the concentration that NADH detects oxidized coenzyme NAD with oxidized coenzyme NAD, preferably, the desaturase that described catalyzed oxidation type coenzyme NAD is NADH is Hexose phosphate dehydrogenase or D-3 HBD, the corresponding hydrogen donor substrate corresponding with saccharase, i.e. glucose or the D-3 hydroxybutyric acid of adding.

Preferably, when the desaturase that by catalyzed oxidation type coenzyme NAD is NADH is converted into the concentration of NADH detection oxidized coenzyme NAD with oxidized coenzyme NAD, comprise that also step 6) adds Lipoyl dehydrogenase and tetrazolium salts chromogen, by detecting the first moon that generates records oxidized coenzyme NAD for concentration concentration, preferably, the tetrazolium salts chromogen is selected from WST-1, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10, WST-11, WST-20, WST-21, XTT, INT, TB, NTB, MOPMS or PMS.

Preferably, the content that described catalyzed oxidation type coenzyme NAD is the desaturase of NADH is 0.1-500kU/L, more preferably 1-200kU/L, more preferably 5-100kU/L, most preferably 20-60kU/L; Preferably, the content of described hydrogen donor substrate is 0.1-100mM, more preferably 0.5-50mM, more preferably 5-50mM, most preferably 25mM; Preferably, the content of described Lipoyl dehydrogenase is 0.1-500kU/L, more preferably 1-100kU/L, more preferably 5-20kU/L, most preferably 10kU/L; Preferably, the content of tetrazolium salts chromogen is 0.1-50mM, more preferably 0.5-10mM, more preferably 1-5mM, most preferably 2mM.

Preferably, described method also comprises removes the step 7) that endogenous citrulline and ammonia disturb in the sample, and this step was carried out before step 1),

Step 7) makes citrulline be converted into ammonia by adding argininosuccinate synthetase and AMP desaminase in sample,

The ammonia of Chan Shenging and endogenic ammonia are removed by following step 8) or step 9) then,

Step 8) adds L-glutamic acid, ATP, PK (pyruvate kinase, Pyruvate kinase), PEP (phosphoenolpyruvic acid, Phosphoenolpyruvic acid) and glutamine synthetase, make ammonia be converted into glutamine and ADP, ADP and PEP generate ATP and pyruvic acid under PK catalysis, at last by adding the glutamine synthetase inhibitor termination reaction, preferably, glutamine synthetase inhibitor is the yellow imide of L-methionine(Met), or

Step 9) adds glutamate dehydrogenase, 2-oxoglutaric acid and NADPH, make ammonia be converted into L-glutamic acid, at last by adding glutamate dehydrogenase inhibitor termination reaction, preferably, the glutamate dehydrogenase inhibitor is selected from a kind of or its combination in p-chloromercuri-benzoate pyridine, 4-4 '-dipyridyl disulfide or 2, the 2 '-dipyridyl disulfide.

Preferably, the content of described L-glutamic acid is 0.1-100mM, more preferably 1-20mM, more preferably 2-10mM, most preferably 5mM; Preferably, the content of described PK is 0.1-100kU/L, more preferably 1-20kU/L, more preferably 2-10kU/L, most preferably 5kU/L; Preferably, the content of described PEP is 0.1-100mM, more preferably 1-20mM, more preferably 2-10mM, most preferably 5mM; Preferably, the content of described glutamine synthetase is 0.1-100kU/L, more preferably 1-20kU/L, more preferably 2-10kU/L, most preferably 5kU/L; Preferably, the content of described glutamine synthetase inhibitor is 0.1-200mM, more preferably 1-100mM, more preferably 5-50mM, most preferably 20mM; Preferably, the content of described glutamate dehydrogenase is 0.1-100kU/L, more preferably 1-20kU/L, more preferably 2-10kU/L, most preferably 5kU/L; Preferably, the content of described 2-oxoglutaric acid is 0.1-20mM, more preferably 0.5-5mM, most preferably 1mM; Preferably, the content of described NADPH is 0.1-20mM, more preferably 0.5-5mM, most preferably 1mM; Preferably, the content of described glutamate dehydrogenase inhibitor is 0.1-200mM, more preferably 1-100mM, more preferably 5-50mM, most preferably 20mM.

A second aspect of the present invention relates to a kind of asymmetric diethylarginine concentration diagnostic kit, and it comprises following compositions:

Diethylarginine dimethylamino lytic enzyme 0.1-500kU/L,

ATP 0.5-20mM，

Aspartic acid 0.5-20mM,

Argininosuccinate synthetase 0.1-500kU/L,

NAD synthetic enzyme 0.1-500kU/L,

AMP desaminase 0.1-500kU/L and

Deaminizating oxidized coenzyme NAD 0.5-20mM,

Described test kit is preferably three reagent test kits or two reagent test kits, wherein diethylarginine dimethylamino lytic enzyme is arranged in different reagent with other compositions, preferably, argininosuccinate synthetase also is arranged in different reagent with the AMP desaminase with other compositions, wherein said test kit is powdered reagent box or liquid reagent box, preferably, when being the liquid reagent box, described test kit also comprises:

Damping fluid 10-500mM, and

Stablizer 0.01-50% weightmeasurement ratio,

Preferably, described damping fluid is Good ' s damping fluid, phosphoric acid buffer, glycine buffer, acetate buffer, and described stablizer is BSA, EDTA, tensio-active agent, glycerine or N.F,USP MANNITOL.

Preferably, the content of described diethylarginine dimethylamino lytic enzyme is 0.5-100kU/L, more preferably 1-50kU/L, more preferably 2-20kU/L, most preferably 3-10kU/L; Preferably, the content of described ATP is 1-10mM, more preferably 2-6mM, most preferably 5mM; Preferably, the content of described aspartic acid is 2-15mM, most preferably 10mM; Preferably, the content of described argininosuccinate synthetase is 0.2-50kU/L, more preferably 0.3-20kU/L, most preferably 5kU/L; Preferably, the content of described NAD synthetic enzyme is 1-100kU/L, more preferably 5-50kU/L, most preferably 15-30kU/L; Preferably, the content of described AMP desaminase is 1-100kU/L, more preferably 5-50kU/L, most preferably 15-30kU/L; Preferably, the content of described deaminizating oxidized coenzyme NAD is 1-10mM, most preferably 2mM; Preferably, the concentration of described damping fluid is 20-200mM, most preferably 50mM; Preferably, the concentration of described stablizer is the 0.1-10% weightmeasurement ratio, more preferably 0.2%-3%, most preferably 0.3% weightmeasurement ratio.

Preferably, described test kit comprises that also catalyzed oxidation type coenzyme NAD is desaturase and the corresponding substrate of NADH, preferably, described test kit also comprises Hexose phosphate dehydrogenase, when comprising Hexose phosphate dehydrogenase but when not containing glucose in the testing sample, described test kit also comprises glucose, preferably, described test kit also comprises Lipoyl dehydrogenase and tetrazolium salts chromogen, preferably, Lipoyl dehydrogenase is positioned at different reagent with the tetrazolium salts chromogen, preferably, Lipoyl dehydrogenase and diethylarginine dimethylamino lytic enzyme are positioned at same reagent, and preferably, the tetrazolium salts chromogen is selected from WST-1, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10, WST-11, WST-20, WST-21, XTT, INT, TB, NTB, MOPMS or PMS.

Preferably, the content that described catalyzed oxidation type coenzyme NAD is the desaturase of NADH is 0.1-500kU/L, more preferably 1-200kU/L, more preferably 5-100kU/L, most preferably 20-60kU/L; Preferably, the content of described substrate is 0.1-50mM, more preferably 0.5-10mM, more preferably 1-5mM, most preferably 2mM; Preferably, the content of described Lipoyl dehydrogenase is 0.1-500kU/L, more preferably 1-100kU/L, more preferably 5-20kU/L, most preferably 10kU/L; Preferably, the content of tetrazolium salts chromogen is 0.1-50mM, more preferably 0.5-10mM, more preferably 1-5mM, most preferably 2mM.

Preferably, when argininosuccinate synthetase is arranged in different reagent with the AMP desaminase with other compositions, the reagent at argininosuccinate synthetase and AMP desaminase place also comprises L-glutamic acid, ATP, PK, PEP and glutamine synthetase, and test kit comprises the glutamine synthetase inhibitor of the reagent outside the reagent that is arranged in argininosuccinate synthetase and AMP desaminase place simultaneously, preferably, glutamine synthetase inhibitor is the yellow imide of L-methionine(Met); Or, when argininosuccinate synthetase is arranged in different reagent with the AMP desaminase with other compositions, the reagent at argininosuccinate synthetase and AMP desaminase place also comprises glutamate dehydrogenase, 2-oxoglutaric acid and NADPH, and test kit comprises the glutamate dehydrogenase inhibitor of the reagent outside the reagent that is arranged in argininosuccinate synthetase and AMP desaminase place simultaneously, preferably, the glutamate dehydrogenase inhibitor is selected from a kind of or its combination in p-chloromercuri-benzoate pyridine, 4-4 '-dipyridyl disulfide or 2, the 2 '-dipyridyl disulfide.

Preferably, described test kit does not contain diethylarginine dimethylamino lytic enzyme and argininosuccinate synthetase moves in the reagent at former diethylarginine dimethylamino lytic enzyme place, and this test kit is for detection of citrulline concentration.

Preferably, described test kit does not contain diethylarginine dimethylamino lytic enzyme, argininosuccinate synthetase and aspartic acid and the AMP desaminase moves in the reagent at former diethylarginine dimethylamino lytic enzyme place, and this test kit is for detection of AMP concentration.

In other words, the purpose of this invention is to provide a kind of method of the convenient and practical asymmetric diethylarginine concentration of mensuration, and the asymmetric diethylarginine diagnostic kit of using this method.

Analysis principle of the present invention is mainly based on the asymmetric diethylarginine concentration of circulation enzymatic assays.The major technology route is: adopt the asymmetric diethylarginine of diethylarginine dimethylamino lytic enzyme (DDAH) (EC 3.5.3.18) degraded to produce dimethylamine and citrulline, citrulline and ATP, aspartic acid produces AMP under argininosuccinate synthetase (ASS) (EC 6.3.4.5) effect, AMP produces IMP and ammonia under AMP desaminase (EC 3.5.4.6) effect, ammonia and deaminizating oxidized coenzyme generate oxidized coenzyme under the effect of NAD synthetic enzyme (EC 6.3.1.5), calculate the concentration of oxidized coenzyme, and then calculate the concentration of asymmetric diethylarginine.

Reaction can be carried out according to following steps:

With sample with contain diethylarginine dimethylamino lytic enzyme (DDAH), water, Triphosaden (ATP), aspartic acid (L-Aspartate), argininosuccinate synthetase (ASS), monophosphate adenosine deaminase (AMP deaminase, the AMP desaminase), the reagent react of deaminizating oxidized form NAD, NAD synthetic enzyme (NAD synthase), detect the content of the oxidized coenzyme NAD that generates, specifically as shown in Figure 1.

Owing to may there be the interference of endogenous citrulline and ammonia in the sample, therefore, method of the present invention can additionally comprise the step of the interference of removing endogenous citrulline and ammonia, and the principle of this step is as follows;

At first, citrulline transforms ammonification under argininosuccinate synthetase and the effect of AMP desaminase;

Secondly, ammonia can be removed by several different methods, method one: ammonia changes into glutamine under the glutamine synthetase effect, and glutamine synthetase is at inhibitor (for example, a kind of or its combination of the yellow imide of L-methionine(Met) and analogue thereof) effect lose activity down (specifically principle is seen Fig. 2);

Method two: ammonia is under the glutamate dehydrogenase effect, be converted to L-glutamic acid, and glutamate dehydrogenase at inhibitor (for example, p-chloromercuri-benzoate pyridine, 4-4 '-dipyridyl disulfide, 2,2 '-a kind of or combination in the dipyridyl disulfide) effect is suppressed activity (specifically reaction principle is seen Fig. 3) down.

The detection of the oxidized coenzyme NAD that produces when utilizing method of the present invention to detect ADMA can be used certain methods well known in the art, can be by electrochemistry, chemistry method such as give out light, and also can be with multiple desaturase in interior linked reaction.

Be example with the Hexose phosphate dehydrogenase, oxidized coenzyme NAD and glucose generate NADH under the Hexose phosphate dehydrogenase effect, directly detect the NADH that generates, and can measure the concentration of oxidized coenzyme, and its principle as shown in Figure 4.

For the coenzyme NAD H that changes into by oxidized coenzyme NAD and then the method that detects its concentration, also can under the Lipoyl dehydrogenase effect, tetrazolium type chromogen be changed into the first moon for (wavelength is 450nm), measure the pigment first moon for producing advancing the speed of absorbancy, calculate the concentration of asymmetric diethylarginine, by increasing a circulating reaction system, improve detection sensitivity, its principle as shown in Figure 5.

This test kit also can be applied to the detection of citrulline in the sample, does not add DDAH in the reagent, ASS is put into the reagent at the original place of DDAH.

This test kit also can be applied to the detection of AMP in the sample, does not add DDAH, ASS and L-aspartic acid in the reagent, simultaneously the AMP desaminase is put into the reagent at original DDAH place.

Using the test kit of present method can be three reagent, also can be double reagent.

Test kit can be powdered reagent, is dissolved in water before namely using, and also can be liquid reagent, directly use.

For the character such as stable and anti-interference, freeze proof of guaranteed reagent,G.R., generally need in reagent, add complementary materials such as tensio-active agent, salt ion, sanitas, concentration is 0.01 to 50%.

The asymmetric diethylarginine detection kit of this method of use that the present invention provides can be used at automatic clinical chemistry analyzer and semi-automatic biochemical analyzer, does not need other instrument, measurement result is accurate, and speed is fast, good reproducibility, immunity from interference is strong, is convenient to promote the use of.

Description of drawings

Fig. 1: the principle of asymmetric diethylarginine circulation enzymatic assays.

Fig. 2: the principle of the first method of the interference of removal endogenous citrulline and ammonia.

Fig. 3: the principle of the second method of the interference of removal endogenous citrulline and ammonia.

Fig. 4: utilize Hexose phosphate dehydrogenase to detect the principle of oxidized coenzyme NAD.

Fig. 5: utilize Lipoyl dehydrogenase to improve the principle of ADMA detection sensitivity.

Fig. 6: the correlation data of test kit of the present invention and LC-MS/MS method.

Fig. 7: the sample concentration linearity range during test kit measure sample of the present invention.

Embodiment

To further specify the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.

Following experimental technique is ordinary method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.

Embodiment 1: double reagent test kit prescription 1 reaches the comparative experiments with liquid chromatography-mass spectrography

First prescription of test kit of the present invention is the double reagent test kit, and its composition is as follows respectively:

Reagent 1 composition and concentration:

Good ' s damping fluid: 50mM,

ATP： 5mM，

ASS： 5kU/L，

L-aspartic acid: 10mM,

AMP desaminase: 20kU/L,

Deaminizating oxidized coenzyme NAD:2mM,

NAD synthetic enzyme: 20kU/L,

Glucose: 50mM,

Hexose phosphate dehydrogenase: 50kU/L,

L-glutamic acid 5mM,

Glutamine synthetase: 5kU/L,

PK： 5KU/L，

PEP： 5mM，

WST-3： 3mM，

BSA： 0.3％，

Reagent 2 compositions and concentration:

Good ' s damping fluid: 50mM,

DDAH： 5KU/L，

Lipoyl dehydrogenase: 10kU/L,

Yellow imide: the 20mM of L-methionine(Met),

BSA： 0.3％。

This detection method is rate method, and 180 μ L reagent 1 are hatched 5min for 37 ℃ with after 20 μ L samples mix, add reagent 2, hatch 5min again, add reagent 2 simultaneously after, the speed that absorbancy raises under the 450nm when detecting 2-5min, thus calculate the concentration of asymmetric diethylarginine in the sample.Simultaneously, liquid chromatography-mass spectrography-mass spectrometry method (LC-MS/MS) (Vishwanathan according to maturation, K., R.L.Tackett, et al. (2000). " Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry. " Journal of Chromatography B:Biomedical Sciences and Applications 748 (1): the 157-166) concentration of asymmetric diethylarginine in the mensuration same sample.Experimental result and the correlation data of the two see Table 1.

Table 1: the result of test kit of the present invention and LC-MS method relatively

Sequence number	Enzyme process μ M	LC-MS-MSμM
			1	0.43	0.46
2	0.58	0.56
			3	0.92	0.89
4	0.67	0.7
			5	1.23	1.32
6	0.56	0.61
			7	0.85	0.83
8	0.67	0.65
			9	2.35	2.56
10	0.75	0.8
			11	2.85	2.9
12	3.12	3.21
			13	0.64	0.66
14	0.61	0.59
			15	0.51	0.58
16	0.39	0.43

17	0.42	0.43
			18	0.56	0.59

See Fig. 6 by the dependency curve that above-mentioned data obtain, as seen the two match is good.

Embodiment 2: double reagent test kit prescription 2 and measuring repeatability and linearity range

Second prescription of test kit of the present invention also is the double reagent test kit, and its composition is as follows respectively:

Reagent 1 composition and concentration:

Phosphoric acid buffer: 50mM,

ATP： 5mM，

ASS： 5kU/L，

L-aspartic acid: 10mM,

AMP desaminase: 20kU/L,

Deaminizating oxidized coenzyme NAD:2mM,

NAD synthetic enzyme: 20kU/L,

NADPH： 1mM，

2-oxoglutaric acid: 1mM,

Glucose: 5mM,

Hexose phosphate dehydrogenase: 50kU/L,

Glutamate dehydrogenase: 5kU/L,

WST-3： 2mM，

BSA： 0.3％，

Reagent 2 compositions and concentration:

Phosphoric acid buffer: 50mM,

DDAH： 5KU/L，

Lipoyl dehydrogenase: 10kU/L,

P-chloromercuri-benzoate pyridine: 20mM,

BSA： 0.3％。

Use the method for the asymmetric diethylarginine concentration of this kit measurement identical with the method for embodiment 1.Use a plurality of samples of this test kit horizontal survey simultaneously, the repeatability during the detection kit measure sample the results are shown in Table 2.Sample by behind the different dilutions, is detected with test kit of the present invention, and the sample concentration linearity range when verifying test kit measure sample of the present invention the results are shown in Figure 7.As seen, test kit measuring repeatability height of the present invention, the measure linear scope is wide

Table 2: the repeated result during kit measurement serum sample of the present invention:

Embodiment 3: double reagent test kit prescription 3

The 3rd prescription of test kit of the present invention is the double reagent test kit, and its composition is as follows respectively:

Reagent 1 composition and concentration:

Good ' s damping fluid: 50mM,

ATP： 6mM，

ASS： 0.3kU/L，

L-aspartic acid: 2mM,

AMP desaminase: 15kU/L,

Deaminizating oxidized coenzyme NAD:10mM,

NAD synthetic enzyme: 15kU/L,

Glucose: 5mM,

Hexose phosphate dehydrogenase: 20kU/L,

L-glutamic acid: 2mM

Glutamine synthetase: 2kU/L,

PK： 2kU/L，

PEP： 2mM，

WST-3： 1mM，

BSA： 0.05％，

Reagent 2 compositions and concentration:

Good ' s damping fluid: 50mM,

DDAH： 2kU/L，

Lipoyl dehydrogenase: 5kU/L,

Yellow imide: the 5mM of L-methionine(Met),

BSA： 0.05％。

Embodiment 4: double reagent test kit prescription 4

The 4th prescription of test kit of the present invention is the double reagent test kit, and its composition is as follows respectively:

Reagent 1 composition and concentration:

Good ' s damping fluid: 50mM,

ATP： 2mM，

ASS： 20kU/L，

L-aspartic acid: 15mM,

AMP desaminase: 30kU/L,

Deaminizating oxidized coenzyme NAD:1mM,

NAD synthetic enzyme: 5kU/L,

Glucose: 50mM,

Hexose phosphate dehydrogenase: 60kU/L,

L-glutamic acid: 10mM

Glutamine synthetase: 10kU/L,

PK： 10KU/L，

PEP： 10mM，

WST-3： 5mM，

BSA： 0.5％，

Reagent 2 compositions and concentration:

Good ' s damping fluid: 50mM,

DDAH： 20kU/L，

Lipoyl dehydrogenase: 20kU/L,

Yellow imide: the 50mM of L-methionine(Met),

BSA： 0.5％。

Embodiment 5:

embodiment

3 and 4 prescriptions compare with the dependency of embodiment 1 prescription

Embodiment

3 and 4 prescriptions are done dependency relatively with embodiment 1 prescription, and three's match as a result is good.The result is as shown in table 3.

The dependency comparative result of table 3:

embodiment

3 and 4 prescriptions and embodiment 1 prescription.

Sequence number	1 μ M fills a prescription	3 μ M fill a prescription	4 μ M fill a prescription
				1	0.39	0.41	0.4
2	0.45	0.47	0.44
				3	0.49	0.48	0.47
4	0.57	0.56	0.59
				5	1.13	1.15	1.12
6	1.45	1.47	1.4
				7	2.67	2.7	2.65
8	0.65	0.65	0.64
				9	0.71	0.75	0.76
10	0.56	0.54	0.58
				11	0.43	0.48	0.45
12	0.52	0.52	0.52
				13	0.65	0.65	0.63
14	0.52	0.53	0.54
				15	2.98	2.91	2.87
16	1.52	1.51	1.47

Claims

1. asymmetric diethylarginine concentration diagnostic kit, it comprises following compositions:

2. asymmetric diethylarginine concentration diagnostic kit according to claim 1, described test kit is two reagent test kits, wherein said diethylarginine dimethylamino lytic enzyme is arranged in different reagent with other compositions.

3. asymmetric diethylarginine concentration diagnostic kit according to claim 1, described test kit is three reagent test kits, wherein said diethylarginine dimethylamino lytic enzyme is arranged in different reagent with other compositions.

4. asymmetric diethylarginine concentration diagnostic kit according to claim 3, wherein said argininosuccinate synthetase also is arranged in different reagent with the AMP desaminase with other compositions.

5. asymmetric diethylarginine concentration diagnostic kit according to claim 1, described test kit is the powdered reagent box.

6. asymmetric diethylarginine concentration diagnostic kit according to claim 1, described test kit is the liquid reagent box.

7. asymmetric diethylarginine concentration diagnostic kit according to claim 6, described test kit also comprises:

Damping fluid 10-500mM, and

Stablizer 0.01-50% weightmeasurement ratio.

8. asymmetric diethylarginine concentration diagnostic kit according to claim 7, wherein said damping fluid is Good's damping fluid, phosphoric acid buffer, glycine buffer or acetate buffer; Described stablizer is BSA, EDTA, tensio-active agent, glycerine or N.F,USP MANNITOL.

9. asymmetric diethylarginine concentration diagnostic kit according to claim 1, described test kit comprises that also catalyzed oxidation type coenzyme NAD is desaturase and the corresponding substrate of NADH.

10. asymmetric diethylarginine concentration diagnostic kit according to claim 9, the desaturase that wherein said catalyzed oxidation type coenzyme NAD is NADH is Hexose phosphate dehydrogenase.

11. asymmetric diethylarginine concentration diagnostic kit according to claim 10 is when comprising Hexose phosphate dehydrogenase but when not containing glucose in the testing sample, described test kit also comprises glucose.

12. asymmetric diethylarginine concentration diagnostic kit according to claim 9, described test kit also comprises Lipoyl dehydrogenase and tetrazolium salts chromogen.

13. asymmetric diethylarginine concentration diagnostic kit according to claim 12, wherein Lipoyl dehydrogenase is positioned at different reagent with the tetrazolium salts chromogen.

14. asymmetric diethylarginine concentration diagnostic kit according to claim 12, Lipoyl dehydrogenase and diethylarginine dimethylamino lytic enzyme are positioned at same reagent.

15. asymmetric diethylarginine concentration diagnostic kit according to claim 12, wherein said tetrazolium salts chromogen is selected from WST-1, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10, WST-11, WST-20, WST-21, XTT, INT, TB, NTB, MOPMS or PMS.

16. according to claim 1 or 9 described asymmetric diethylarginine concentration diagnostic kits, it is characterized in that, when argininosuccinate synthetase is arranged in different reagent with the AMP desaminase with other compositions, the reagent at argininosuccinate synthetase and AMP desaminase place also comprises L-glutamic acid, ATP, PK, PEP and glutamine synthetase, and test kit comprises the glutamine synthetase inhibitor of the reagent outside the reagent that is arranged in argininosuccinate synthetase and AMP desaminase place simultaneously.

17. asymmetric diethylarginine concentration diagnostic kit according to claim 16, wherein said glutamine synthetase inhibitor are L-methionine(Met) Huang imide.

18. according to claim 1 or 9 described asymmetric diethylarginine concentration diagnostic kits, it is characterized in that, when argininosuccinate synthetase is arranged in different reagent with the AMP desaminase with other compositions, the reagent at argininosuccinate synthetase and AMP desaminase place also comprises glutamate dehydrogenase, 2-oxoglutaric acid and NADPH, and test kit comprises the glutamate dehydrogenase inhibitor of the reagent outside the reagent that is arranged in argininosuccinate synthetase and AMP desaminase place simultaneously.

19. asymmetric diethylarginine concentration diagnostic kit according to claim 18, wherein said glutamate dehydrogenase inhibitor is selected from a kind of or its combination in p-chloromercuri-benzoate pyridine, 4-4'-dipyridyl disulfide or 2, the 2'-dipyridyl disulfide.