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8-Oxo-2'-deoxyguanosine detection kit and application thereof

A technology of hydroxydeoxyguanosine and detection kit, which is applied in the detection field of 8-hydroxydeoxyguanosine, can solve the problems of higher detection value than real value, incomplete enzymatic hydrolysis, and the specificity of labeling method needs to be improved.

Active Publication Date: 2018-11-02
KEAISE MEDICINE WUHAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned methods all have certain defects, and cannot be used in the clinical determination of the content of 8-OHdG in human tissues or body fluids.
For example: GC-MS method needs derivatization reaction first, and the derivatization process is easy to produce by-products, resulting in false positive results
32 The specificity of P post-labeling method needs to be improved, and it is easy to cause radioactive contamination
The specificity of the immunochemical method is poor, and the urea in the urine will cross-react with 8-OHdG, resulting in a higher detection value than the true value
At present, high-performance liquid chromatography-electrochemical detection (HPLC-ECD) is mainly used to determine 8-OHdG, but this method has the problems of incomplete enzymatic hydrolysis and simultaneous washing out of interfering substances, which can lead to the detection value of 8-OHdG It is higher than the real value; in addition, the cost of the instrument is high, and it is difficult to promote it in clinical practice

Method used

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  • 8-Oxo-2'-deoxyguanosine detection kit and application thereof
  • 8-Oxo-2'-deoxyguanosine detection kit and application thereof
  • 8-Oxo-2'-deoxyguanosine detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 with NaH 2 PO 4 and KH 2 PO 4 Comparative studies for mobile phase detection

[0094] Mobile phase 1: 50mmol / L NaH at pH 3.5 2 PO 4 Solution: methanol = 90%: 10%.

[0095] Mobile phase 2: 50mmol / L KH at pH 3.5 2 PO 4 Solution: methanol = 90%: 10%.

[0096] The injection volume was 50 μL, the flow rate was 1.0 mL / min, the column temperature was 30° C., and the detection wavelength was 300 nm.

[0097] Prepare 8-hydroxydeoxyguanosine standard reference solution with a concentration of 10 μg / mL.

[0098] The results detected by mobile phase 2 are as follows: figure 2 mentioned. The results detected by mobile phase 1 are as follows: image 3 shown.

[0099] It can be seen from the figure that in KH 2 PO 4 When it is the mobile phase, the 8-OHdG peak at 13-16 minutes has a shoulder, and the chromatographic resolution does not meet the detection requirements. and NaH 2 PO 4 When it is the mobile phase, the peak shape of 8-OHdG at 14-16 minutes is ...

Embodiment 2

[0100] Embodiment 2 The selection of mobile phase solvent

[0101] Mobile phase A is NaH 2 PO 4 solution, mobile phase B is methanol or acetonitrile.

[0102] Use 50mmol / L NaH at pH 3.5 2 PO 4 Solution: acetonitrile=90%:10% chromatographic conditions measure 8-hydroxydeoxyguanosine reference substance, acetonitrile sees for the resolution of 8-OHdG target sample peak and solvent peak Figure 4 .

[0103] Use 50mmol / L NaH at pH 3.5 2 PO 4 Solution: methyl alcohol=90%:10% chromatographic conditions measure 8-hydroxydeoxyguanosine reference substance, methyl alcohol sees for the separation degree of 8-OHdG target sample peak and solvent peak Figure 5 .

[0104] Depend on Figure 4 and Figure 5 Visible, the degree of separation of acetonitrile and methanol is consistent, can be used as mobile phase solvent of the present invention.

[0105] Considering that the cost of methanol is lower than that of acetonitrile, and it is more conducive to the application and promoti...

Embodiment 3

[0106] The selection of embodiment 3 column temperature

[0107] Mobile phase A is 50mmol / L NaH of pH3.5 2 PO 4 solution, mobile phase B is methanol, mobile phase A: mobile phase B ratio is 9:1, the injection volume is 50 μL, and the flow rate is 1.0 mL / min.

[0108] Add 8-hydroxydeoxyguanosine reference substance to the urine of healthy people to make the concentration 20 μg / mL, and compare the chromatographic performance of the reference substance under different column temperature conditions of 10°C, 20°C, and 30°C.

[0109] Table 2 Retention time of reference substances under different column temperature conditions

[0110]

[0111] It can be seen from the above table that when the column temperature is 20-30°C, the peak shape of the 8-hydroxydeoxyguanosine component is good, and it can be separated from other impurity peaks. In view of the fact that at 30°C, the peak of 8-hydroxydeoxyguanosine is relatively faster, which is beneficial to improve the efficiency of cl...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an 8-oxo-2'-deoxyguanosine detection kit and a detecting method for 8-oxo-2'-deoxyguanosine. The detection kit comprises a reagent A and a reagent B, wherein the reagent A is a NaH2PO4 solution, and the reagent B is a moving phase solvent. The detecting method for the 8-oxo-2'-deoxyguanosine comprisesthe following steps: 1) pre-treating a to-be-detected sample; 2) setting detecting parameters; and 3) detecting the sample. The detection kit overcomes the problem that column pressure of a chromatographic column is great when monopotassium phosphate is taken as a mobile phase in the prior art, the problem that the chromatographic column and a quaternary pump of a chromatographic instrument are easily blocked, and the problem that easy interference and inaccuracy are caused. Furthermore, combined use of the sample and an efficient liquid-phase chromatogram can be realized, so that automatic detection is realized. The detecting method is accurate, is simple, is strong in specificity, is good in precision and is good in recovery rate. The invention further provides application of the 8-OHdGdetection kit in preparing a kit for lung cancer diagnosis or lung cancer clinic treatment effect monitoring.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection method, a detection kit and an application of 8-hydroxydeoxyguanosine. Background technique [0002] The metabolism of exogenous chemical substances in the body or the normal metabolic process of cells can produce a large number of reactive oxygen species (ROS) free radicals. ROS mainly include superoxide anion groups, hydroxyl radicals, hydrogen peroxide radicals, etc. Active oxygen has unpaired single electrons and has strong chemical reactivity. Excessive ROS levels can cause oxidative damage to cell membrane lipids, proteins, and DNA. However, active oxygen has a very short lifespan and cannot be directly detected in the human body. Therefore, it is necessary to select a product of a non-enzymatic reaction between a biomolecule and active oxygen as a biomarker to replace active oxygen for detection to indirectly reflect the level of acti...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 曾慧慧
Owner KEAISE MEDICINE WUHAN
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