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Method for measuring relative content of hypoxanthine riboside and adenosine in samples to be tested

A technology of relative content and inosine, which is applied in the field of determining the relative content of inosine and adenosine in adenosine samples, can solve the problems of time-consuming detection and environmental pollution, and achieve the effects of good precision, high sensitivity and accurate results

Inactive Publication Date: 2015-06-24
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, laboratories use high-performance liquid chromatography (HPLC) to determine the relative content of inosine and adenosine in adenosine samples. This method requires special large-scale instruments and standard products for comparison. The detection is time-consuming and requires the use of toxic Solvent methanol, easy to cause environmental pollution

Method used

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  • Method for measuring relative content of hypoxanthine riboside and adenosine in samples to be tested

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Accurately weigh about 0.1 g of the sample, and dilute it with deionized water to a sample solution with a final concentration of 0.05 g / L; prepare a sample solution containing MgCl 2 1mmol / L, EDTA·2Na concentration is 0.5mmol / L, Na 2 HPO 4 Concentration of 5mmol / L, PNP concentration of 0.1KU / L, XTO concentration of 0.2KU / L, POD concentration of 0.5KU / L, 4-AAP concentration of 0.2mmol / L, TOOS concentration of 0.5mmol / L, ethylene glycol 20mmol / L pH7.0 Tris-HCL buffer solution with a concentration of 10ml / L and a sucrose concentration of 5g / L was used as reagent 1; the preparation contained ADA concentration of 10KU / L, ethylene glycol concentration of 10ml / L, and sucrose concentration of 5g / L The 20mmol / L pH 7.0 Tris-HCL buffer is reagent 2. When reagent 1 and reagent 2 are used to measure the sample liquid, the instrument used is the Olympus 2700 automatic biochemical analyzer, the determination method type is the end point method, the temperature is 37°C, V 样品 5μl, V...

Embodiment 2

[0026] Accurately weigh about 0.1 g of the sample, and dilute it with deionized water to a sample solution with a final concentration of 1.25 g / L; prepare a sample solution containing MgCl 2 12.5mmol / L, EDTA·2Na concentration is 10mmol / L, Na 2 HPO 4 Concentration is 25mmol / L, PNP concentration is 5KU / L, XTO concentration is 10KU / L, POD concentration is 25KU / L, 4-AAP concentration is 2.5mmol / L, TOOS concentration is 5mmol / L, ethylene glycol concentration is 50ml / L, the 100mmol / L pH 7.25 Tris-HCL buffer solution with a sucrose concentration of 25g / L is reagent 1; the 100mmol / L buffer solution containing ADA concentration of 50KU / L, ethylene glycol concentration of 50ml / L and sucrose concentration of 25g / L is prepared pH 7.25 Tris-HCL buffer is reagent 2. When reagent 1 and reagent 2 are used to measure sample liquid, the instrument used is Hitachi 7080 automatic biochemical analyzer, the determination method type is endpoint method, the temperature is 37 °C, V 样品 5μl, V 1 3...

Embodiment 3

[0028] Accurately weigh about 0.1 g of the sample, and dilute it with deionized water to a sample solution with a final concentration of 2.5 g / L; prepare a sample solution containing MgCl 2 25mmol / L, EDTA·2Na concentration is 20mmol / L, Na 2 HPO 4 The concentration is 50mmol / L, the concentration of PNP is 10KU / L, the concentration of XTO is 20KU / L, the concentration of POD is 50KU / L, the concentration of 4-AAP is 5mmol / L, the concentration of TOOS is 10mmol / L, and the concentration of ethylene glycol is 100ml / L , 200mmol / L pH7.5 Tris-HCL buffer solution with a sucrose concentration of 50g / L is reagent 1; a 200mmol / L buffer solution containing ADA concentration of 100KU / L, ethylene glycol concentration of 100ml / L and sucrose concentration of 50g / L is prepared. pH 7.5 Tris-HCL buffer is reagent 2. When reagent 1 and reagent 2 are used to measure sample liquid, the instrument used is UV2201 ultraviolet-visible spectrophotometer, the type of determination method is end-point meth...

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Abstract

The invention discloses a method for measuring relative content of hypoxanthine riboside and adenosine in samples to be tested. The method includes converting hypoxanthine riboside in an adenosine sample into purple red quinone derivatives by an enzyme-coupled system composed of PNP (purine nucleoside phosphorylase), XTO (xanthine oxidase) and POD (peroxidase), and measuring solution absorbance value A1 under specific wavelength; measuring absorbance value A2 after adding buffer solution containing adenosine deaminase and keeping reaction for some time, and calculating relative content of hypoxanthine riboside and adenosine in adenosine substrate by the values A1 and A2. The method is green and environment friendly, relative content of hypoxanthine riboside and adenosine can be calculated without reference of calibrators, and detection can be realized by a common visible spectrophotometer. The method is high in flexibility, good in linearity and precision, accurate in results and convenient and quick to operate, and can be used for various types of analyzers to meet requirements of testing the samples in large scale.

Description

technical field [0001] The invention belongs to the technical field of biochemical detection, and in particular relates to a method for determining the relative content of inosine and adenosine in an adenosine sample. Background technique [0002] Adenosine is the substrate for the determination of adenosine deaminase (ADA) by enzymatic chemical method. The liquid ADA determination reagent with adenosine as one of the main raw materials has the characteristics of stability, good anti-interference ability, and automatic analysis. be widely used. Adenosine has good stability when stored at -20°C, and it is easy to decompose to produce inosine after being exposed to moisture and heat. The relative content of inosine and adenosine in adenosine significantly affects the sensitivity of the ADA assay reagent and the blank absorbance value. It is one of the experimental factors that cannot be ignored to control the difference between batches of ADA assay reagents. [0003] At pres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N1/28
Inventor 连国军
Owner WENZHOU MEDICAL UNIV
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