Cyclophorase detection method for quantitative detection of thio-nicotinamide-adenine dinucleotide (Thio-NAD) and kit

An oxidized coenzyme, quantitative detection technology, applied in the measurement of color/spectral characteristics, etc., can solve problems such as combustion or explosion

Active Publication Date: 2012-06-20
浙江夸克生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method is relatively simple to operate, hydrazine hydrate is a highly toxic chemical with an acute toxicity of LD50129 mg / kg (by mouth in rats). It is combustible when exposed to open flames or high heat, and can react strongly with oxidants, causing combustion or explosion.

Method used

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  • Cyclophorase detection method for quantitative detection of thio-nicotinamide-adenine dinucleotide (Thio-NAD) and kit

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Experimental program
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Effect test

Embodiment 1

[0019] Prepare 200mmol / L pH 9.25 Tris-HCl buffer containing NADH 4g / L, sodium deoxycholate 75μmol / L, 3α-HSD 3KU / L, and 5% trehalose as the measurement reagent, which is used for the measurement of the sample solution When, the instrument used is Hitachi 7080 automatic biochemical analyzer, the type of measurement method is the end point method, the temperature is 37℃, sample 4μl, V Reagent It is 300μl, and the main / sub-wavelength of the measurement is 405 / 800nm. After the reagent is added to the sample, the absorbance is read after 300 seconds of reaction at the measurement temperature. The calculation formula of Thio-NAD in the sample solution is: Thio-NAD content (μmol / L)=(A-A 0 )×V 1 ×10 6 / (11900×V 2 ), where A is the absorbance value of the sample tube reaction, A 0 Is the same volume of deionized water instead of the measured absorbance value of the sample, V 1 Is the total reaction volume, V 2 Is the sample volume, and 11900 is the molar absorbance of Thio-NAD at 405 nm. ...

Embodiment 2

[0024] Prepare 2500mmol / L pH9.25 Tris-HCl buffer containing NADH 6g / L, sodium deoxycholate 100μmol / L, 3α-HSD 5KU / L, and 10% trehalose as the measurement reagent, which is used for the measurement of the sample solution At the time, the instrument used is Beckman LX20 automatic biochemical analyzer, the type of measurement method is the end point method, the temperature is 37 ℃, V sample 3μl, V Reagent It is 225μl, and the main / sub-wavelength of the measurement is 410 / 600nm. After the reagent is added to the sample, the absorbance is read after 180 seconds at the measurement temperature and the Thio-NAD content in the sample solution is calculated. The calculation formula of Thio-NAD in the sample solution is: Thio-NAD content (μmol / L)=(A-A 0 )×V 1 ×10 6 / (10850×V 2 ), where A is the absorbance value of the sample tube reaction, A 0 Is the same volume of deionized water instead of the measured absorbance value of the sample, V 1 Is the total reaction volume, V 2 Is the sample vol...

Embodiment 3

[0026] Prepare a 20mmol / L pH 9.25 Tris-HCl buffer containing NADH 2g / L, sodium deoxycholate 25μmol / L, 3α-HSD 0.5KU / L, and 2% trehalose as the measurement reagent, which is used to determine the sample When liquid, the instrument used is Shimadzu UV-2550 ultraviolet-visible spectrophotometer, temperature is 37℃, V sample 40μl, V Reagent It is 3000μl and the measuring wavelength is 420nm. After reagents are added to the sample, the absorbance is read after 20 minutes of reaction at the measuring temperature and the Thio-NAD content in the sample solution is calculated. The calculation formula of Thio-NAD in the sample solution is: Thio-NAD content (μmol / L)=(A-A 0 )×V 1 ×10 6 / (9760×V 2 ), where A is the absorbance value of the sample tube reaction, A 0 Is the same volume of deionized water instead of the measured absorbance value of the sample, V 1 Is the total reaction volume, V 2 Is the sample volume, 9760 is the molar absorbance of Thio-NAD at 420nm.

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Abstract

The invention discloses a cyclophorase detection method for quantitative detection of thio-nicotinamide-adenine dinucleotide (Thio-NAD). The cyclophorase detection method comprises the following steps of: preparing Tris-HCl buffer solution with pH of 8.5 to 10.50, wherein the Tris-HCl buffer solution includes a certain amount of coenzyme I (NADH), a sodium deoxycholate solution, 3Alpha-hydroxysteroiddehydrogenase (3Alpha-HSD) and trehalose, and adding a Thio-NAD-containing sample to be tested; dehydrogenizing and oxidizing sodium cholate or sodium deoxycholate by 3Alpha-HSD to form 3Alpha-ketosteroid; meanwhile, reducing Thio-NAD into reduced-type thio-nicotinamide-adenine dinucleotide (Thio-NADH); re-reducing 3Alpha-ketosteroid into sodium cholate or derivatives thereof by 3Alpha-HSD in the presence of NADH in the solution; meanwhile, dehydrogenating and oxidizing the NADH into NAD<+>, wherein the Thio NADH generated in the reaction has maximum absorption at a 405 nm place; and computing content of THE Thio-NADH of the sample to be tested by measuring optical density of the solution under a specific wavelength after reacting at 37 DEG C for some time. The cyclophorase detection method, provided by the invention, has the advantages of environmental friendliness, and stable test reagent, and is particularly suitable for bulk detection of Thio-NAD.

Description

Technical field [0001] The invention relates to a circulating enzyme detection method for quantitatively detecting a thio-oxidized coenzyme (Thio-NAD) and a kit for the method. Background technique [0002] Thio-oxidized coenzyme I (Thio-NAD) is a derivative of coenzyme I, which is reduced to form thio-reduced coenzyme I (Thio-NADH). As a chemical raw material, thiooxidized coenzyme has an important application in the determination of bile acid by the cyclic enzymatic method. Its content significantly affects the sensitivity of the reagent for the determination of bile acid, and it is important to control the batch difference of the reagent for bile acid determination. One of the experimental factors. [0003] High performance liquid chromatography is a conventional method for the determination of Thio-NAD. This method requires standard control, and the steps are cumbersome and difficult to promote in practice. When using direct spectrophotometry to determine the content of Thio-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 商纯尔陈青松
Owner 浙江夸克生物科技有限公司
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