CN102863495A - Stable composition containing NAD+ or NADH - Google Patents
Stable composition containing NAD+ or NADH Download PDFInfo
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- CN102863495A CN102863495A CN2011101880289A CN201110188028A CN102863495A CN 102863495 A CN102863495 A CN 102863495A CN 2011101880289 A CN2011101880289 A CN 2011101880289A CN 201110188028 A CN201110188028 A CN 201110188028A CN 102863495 A CN102863495 A CN 102863495A
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Abstract
The invention provides a stable composition containing NAD+ or NADH. Bioactivity of NAD+ or NADH can be stably maintained for a long time. By the use of a stabilizer which contains trehalose, glycerol, a buffer solution and a surfactant, stability of NAD+ and NADH dissolved state is raised. The invention also provides a method for stabilizing NAD+ or NADH, and also provides an application of a composition composed of trehalose, glycerol, the buffer solution, the surfactant and a pH regulator in increasing stability of NAD+ or NADH. The composition can be used for a lactate dehydrogenase reagent, an alanine aminotransferase reagent, an aspartate aminotransferase reagent, a urea kid and the like, and is widely applied.
Description
Technical field
The invention belongs to biological technical field, related to a kind of NAD of containing
+Or the stable composition of NADH, and the method that increases NAD+ or NADH stability.
Background technology
Reduced nicotinamide-adenine dinucleotide (nicotinamide adenine dinucleotide is referred to as NAD) is a kind of coenzyme of passing on electronics, and it appears in a lot of metabolism reactions of cell.NADH is its reduction form.NAD is the coenzyme of desaturase, such as ethanol dehydrogenase (ADH), is used for Oxidation of Alcohol.It is bringing into play irreplaceable effect in glycolysis-, glyconeogenesis, tricarboxylic acid cycle and respiratory chain.Intermediate product can be passed NAD with the hydrogen of taking off, and makes it to become NADH.NADH then can as the carrier of hydrogen, by the mode of chemiosmotic coupling, synthesize ATP in respiratory chain.
Aspect extinction, NADH respectively has an absorption peak at 260nm and 340nm place, and NAD then only has 260nm one place's absorption peak, and this is both important attribute of difference.This also is in a lot of metabolic tests simultaneously, measures the physical basis of metabolic rate.NAD is 1.78 * 10L/(molcm) in the specific absorbance of 260nm, and NADH is 6.2 * 10L/ (molcm) in the specific absorbance of 340nm.
NAD has a lot of application at the external diagnosis reagent prescription: such as serum lactic dehydrogenase reagent, alanine transaminase reagent, aspartic transaminase reagent, urea kit, AHB etc.Therefore this protective material has more widely application.
NAD and NADH coenzyme are unsettled.NAD is alkali labile molecule, and the NADH of reduction form is unstable to acid, is known degradation pathway such as epimerization.In the prior art, the means of stablizing NAD and NADH have: (1) is such as CN200680027359.1 disclosed (open day 2008-07-30), make it stable by the method for preparing derivative, or to the particular requirement of storage condition, increase its stability such as cooling or stored dry.(2) such as CN200680027359.1 disclosed (open day 2008-07-30), adopt stablizer, increase the stability of NAD or NADH such as trehalose, polyvinylpyrrolidone and serum albumin formation polymer network.(3) such as CN87100939 disclosed (open day 1987-11-04), increase its stability with alkaline aqueous solution environment, polyhydroxy alkyl solvent (20-90%V/V) such as propylene glycol, boric acid.(4) such as CN200410043900.0 disclosed (open day 2005-05-18), adopt tensio-active agent to increase its stability such as 0.1～2ml tween 20.
The contriver notices, even under inviolent condition (in aqueous solution), coenzyme NAD and NADH only is hydrolyzed by ambient moisture also can cause the measuring result out of true.Therefore, the contriver recognizes that raising NAD+ and the stability of NADH when the dissolution in vitro state will have vital role to the accuracy that improves measuring result.
Summary of the invention
The object of the present invention is to provide a kind of NAD of containing
+Or the stable composition of NADH, the present composition can improve NAD+ and the stability of NADH when the dissolution in vitro state.
Another object of the present invention is to provide a kind of stable NAD
+Or the method for NADH.
Another object of the present invention is to provide a kind of composition and use thereof that comprises trehalose, glycerine, damping fluid and tensio-active agent, namely for increasing NAD
+Or the stability of NADH.
Purpose of the present invention can be achieved through the following technical solutions: by using trehalose, glycerine, damping fluid, tensio-active agent are mixed the certain solution of composition, and then with NAD
+And/or the NADH dissolving wherein.The above-mentioned substance combination is called stablizer in the present invention, finds to significantly improve the NAD of dissolved state by research
+And/or the stability of NADH.
Specifically, the present invention contains NAD
+Or the stable composition of NADH comprises NAD
+Or NADH and described stablizer, described stablizer comprises trehalose, glycerine, damping fluid and tensio-active agent.NAD
+Or the content of NADH in composition is 3-40g/l.
The present invention is by using stablizer to improve NAD
+And/or NADH is in the stability of dissolved state, and stablizer can be made up according to different ratios by following material:
(1) trehalose, content is: 10g/l～60 g/l, more preferably 30g/l～40 g/l.
(2) glycerol content is: 5g/l～30g/l, more preferably 15g/l～20g/l.
(3) damping fluid can be the combination that the slow salt of tris salt buffer, goods salt buffer, phosphoric acid salt rushes liquid, carbonate buffer solution or these damping fluids.In the described Tris damping fluid, the content of Tris is 50mmol/l～100mmol/l.The content of phosphoric is 50mmol/l～1mol/l in the described phosphate buffered saline buffer.
(4) tensio-active agent can be the mixture of polysorbas20, tween 80, polyoxyethylene laurel ether, polyoxyethylene glycol, SDS or these several materials, wherein, described polysorbas20 content is 11g/l～10g/l, and tween 80 content is 1g/l～10g/l, and polyoxyethylene laurel ether content is 1g/l～10g/l.Polyethyleneglycol content is 10mmol/l～500mmol/l, and SDS content is 1g/l～10g/l.
Aforesaid stablizer is for increasing NAD
+Or the purposes of NADH stability.Described stablizer can be used for the preparation of serum lactic dehydrogenase reagent, alanine transaminase reagent, aspartic transaminase reagent, urea kit.
The present invention further provides a kind of stable NAD
+Or the method for NADH, comprise NAD
+Or NADH is dissolved in the solution that contains trehalose, glycerine, damping fluid, tensio-active agent.In the described solution, content of trehalose is: 10g/l～60 g/l, glycerol content is: 5g/l～30g/l, damping fluid can be the combination that the slow salt of tris salt buffer, goods salt buffer, phosphoric acid salt rushes liquid, carbonate buffer solution or these damping fluids, in the described Tris damping fluid, the content of Tris is 50mmol/l～100mmol/l, and the content of phosphoric is 50mmol/l～1mol/l in the described phosphate buffered saline buffer; Tensio-active agent can be the mixture of polysorbas20, tween 80, polyoxyethylene laurel ether, polyoxyethylene glycol, SDS or these several materials, wherein, described polysorbas20 content is 11g/l～10g/l, and tween 80 content is 1g/l～10g/l, and polyoxyethylene laurel ether content is 1g/l～10g/l.Polyethyleneglycol content is 10mmol/l～500mmol/l, and SDS content is 1g/l～10g/l.
The present invention is by being applied to improve NAD with trehalose, glycerine, damping fluid, tensio-active agent
+Or the stability of NADH, having significantly improved NAD+ and the NADH stability when the dissolution in vitro state, the maintenance phase can reach 18 months under the condition of storage of routine.The contriver finds by the contrast experiment, and when not adopting this stablizer, the maintenance phase is generally 12 months under the same stored condition.
The present invention can be applied to the process for preparation of the test kits such as serum lactic dehydrogenase reagent, alanine transaminase reagent, aspartic transaminase reagent, urea kit, AHB, therefore has wider using value.
Further specify the present invention below in conjunction with specific embodiment, should be understood that embodiment only is used for explanation the present invention, and unrestricted the present invention.
Embodiment
Embodiment 1
The application of the present invention in the lactic dehydrogenase enzyme reagent kit:
Reagent preparation box reagent 1 at first: lactic acid 40mmol/l, 100mmol/l Tris solution uses 1mol/l hydrochloric acid or sodium hydroxide to regulate pH to 9.2.
Reagent preparation box reagent 2 again: contain trehalose 10g/l, glycerine 1g/l, Tris 0.1mol/l, then the solution of polysorbas20 0.1g/l use 1mol/l hydrochloric acid or sodium hydroxide that its pH is transferred to 7.0, takes by weighing some NAD again
+Be dissolved in the above-mentioned protective material of 10ml, make its concentration reach 20g/l, stir.
Mentioned reagent 1 and reagent 2 form the lactic dehydrogenase enzyme reagent kit, and this test kit can reach 18 months 2～8 ℃ of maintenance phases.By simultaneous test, the lactic dehydrogenase enzyme reagent kit that does not add stablizer only can keep 12 months under the same conditions.
Embodiment 2
The present invention is in the application at the AHB test kit:
Reagent preparation box reagent 1 at first: contain trehalose 5g/l, glycerine 10g/l, Tris 0.1mol/l, then the solution of polysorbas20 0.5g/l transfer to 7.0 with its pH, takes by weighing 10mg NAD again and be dissolved in the above-mentioned protective material of 10ml, stirs.
Reagent preparation box reagent 2 again: nitric acid 50mmol/l solution, α-oxo butyric acid 3mmol/l.Regulate pH to 7.5.
Mentioned reagent 1 and reagent 2 form the AHB test kit, and this test kit can reach 18 months 2～8 ℃ of maintenance phases.By simultaneous test, the lactic dehydrogenase enzyme reagent kit that does not add stablizer only can keep 12 months under the same conditions.
Claims (13)
1. contain NAD
+Or the stable composition of NADH, it is characterized in that, contain NAD
+Or NADH and stablizer, described stablizer comprises trehalose, glycerine, damping fluid, tensio-active agent.
2. the NAD that contains as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described content of trehalose is 10g/l～60 g/l.
3. NAD as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described glycerol content is 5g/l～30g/l.
4. NAD as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described damping fluid can be the combination that the slow salt of Tris damping fluid, goods salt buffer, phosphoric acid salt rushes liquid, carbonate buffer solution or these damping fluids.
5. NAD as claimed in claim 4
+Or the stable composition of NADH, it is characterized in that, in the described Tris damping fluid, the content of Tris is 50mmol/l～100mmol/l.
6. NAD as claimed in claim 4
+Or the stable composition of NADH, it is characterized in that, the content of phosphoric is 50mmol/l～1mol/l in the described phosphate buffered saline buffer.
7. NAD as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described tensio-active agent can be the mixture of polysorbas20, tween 80, polyoxyethylene laurel ether, polyoxyethylene glycol, SDS or these several materials, wherein, described polysorbas20 content is 11g/l～10g/l, and tween 80 content is 1g/l～10g/l, and polyoxyethylene laurel ether content is 1g/l～10g/l, polyethyleneglycol content is 10mmol/l～500mmol/l, and SDS content is 1g/l～10g/l.
8. NAD as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described pH value is 5.0-7.0.
9. NAD as claimed in claim 1
+Or the stable composition of NADH, it is characterized in that, described stablizer comprises: trehalose, content is: 10g/l～60 g/l; Glycerine, content is: 5g/l～30g/l; The Tris salt buffer with the content meter of Tris, is 50mmol/l～100mmol/l; Polysorbas20, content are 11g/l～10g/l.
10. such as the purposes of the arbitrary described stablizer of claim 1 to 9, it is characterized in that, for increasing NAD
+Or the purposes of NADH stability.
11. the purposes such as the arbitrary described stablizer of claim 1 to 9 is characterized in that, described stablizer is used for the preparation of serum lactic dehydrogenase reagent, alanine transaminase reagent, aspartic transaminase reagent, urea kit.
12. stable NAD
+Or the method for NADH, it is characterized in that, with NAD
+Or NADH is dissolved in the mixing solutions that contains trehalose, glycerine, damping fluid and tensio-active agent, and the pH of mixing solutions is 5.0-7.0.
13. method as claimed in claim 12, it is characterized in that, in the described solution, content of trehalose is: 10g/l～60 g/l, glycerol content is: 5g/l～30g/l, damping fluid can be the combination that the slow salt of Tris salt buffer, goods salt buffer, phosphoric acid salt rushes liquid, carbonate buffer solution or these damping fluids, in the described Tris damping fluid, the content of Tris is 50mmol/l～100mmol/l, and the content of phosphoric is 50mmol/l～1mol/l in the described phosphate buffered saline buffer; Tensio-active agent can be the mixture of polysorbas20, tween 80, polyoxyethylene laurel ether, polyoxyethylene glycol, SDS or these several materials, wherein, described polysorbas20 content is 11g/l～10g/l, tween 80 content is 1g/l～10g/l, polyoxyethylene laurel ether content is 1g/l～10g/l, polyethyleneglycol content is 10mmol/l～500mmol/l, and SDS content is 1g/l～10g/l.
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|---|---|---|---|
| CN2011101880289A CN102863495A (en) | 2011-07-06 | 2011-07-06 | Stable composition containing NAD+ or NADH |
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|---|---|---|---|
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| CN103217525A (en) * | 2013-03-21 | 2013-07-24 | 上海执诚生物科技股份有限公司 | Composition for improving cystatin C latex coated antibody stability, stabilizer containing the same, preparation method and application thereof |
| CN103698500A (en) * | 2013-12-20 | 2014-04-02 | 上海执诚生物科技股份有限公司 | Method for improving stability of carbon dioxide detection reagent |
| CN104152535A (en) * | 2014-08-26 | 2014-11-19 | 北京瑞正善达生物工程技术有限公司 | In-vitro enzymatic method potassium determining reagent, preparation method and use method thereof |
| CN104198686A (en) * | 2014-08-14 | 2014-12-10 | 苏州康铭诚业医用科技有限公司 | Compound stabilizer for uric acid assay kit |
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| CN103698500B (en) * | 2013-12-20 | 2016-01-27 | 上海执诚生物科技有限公司 | A kind of method strengthening stability of carbon dioxide detection reagent |
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| CN105651763A (en) * | 2016-03-08 | 2016-06-08 | 山东博科生物产业有限公司 | Alpha-hydroxybutyrate dehydrogenase detection reagent with good stability and high accuracy |
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| JP2020033288A (en) * | 2018-08-29 | 2020-03-05 | 株式会社シノテスト | Method for stabilizing NADH and NADPH |
| JP7349117B2 (en) | 2018-08-29 | 2023-09-22 | 株式会社シノテスト | Method for stabilizing NADH and NADPH |
| CN109596522A (en) * | 2018-12-17 | 2019-04-09 | 蓝怡科技集团股份有限公司 | It is a kind of to stablize the solution and kit for saving NADH |
| CN110057640A (en) * | 2019-04-30 | 2019-07-26 | 山东博科生物产业有限公司 | Anti-interference, stable compound calibration object and preparation method containing 33 indexs |
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| CN113456527A (en) * | 2021-07-30 | 2021-10-01 | 北京清大赛尔生物科技有限公司 | NADH microcapsule preparation and preparation method and application thereof |
| CN113456527B (en) * | 2021-07-30 | 2023-04-07 | 北京清大赛尔生物科技有限公司 | NADH microcapsule preparation and preparation method and application thereof |
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Address after: 201204, Lane 3399, Lane 6, Kang Xin Road, Shanghai, Pudong New Area Applicant after: Shanghai Zhicheng Biological Technology Co.,Ltd. Address before: 201204 Shanghai city Pudong New Area Lianxi Road No. 1210 Building 2 Applicant before: Shanghai Zhicheng Biological Technology Co.,Ltd. |
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Address after: 201204, Lane 3399, Lane 6, Kang Xin Road, Shanghai, Pudong New Area Applicant after: SHANGHAI ZHICHENG BIOTECHNOLOGY CO., LTD. Address before: 201204, Lane 3399, Lane 6, Kang Xin Road, Shanghai, Pudong New Area Applicant before: Shanghai Zhicheng Biological Technology Co.,Ltd. |
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Application publication date: 20130109 |