8-Hydroxydeoxyguanosine detection kit and its application

A technology of hydroxydeoxyguanosine and a kit, which is applied in the detection field of 8-hydroxydeoxyguanosine, can solve the problems of higher detection value than the real value, poor specificity of immunochemical method, and simultaneous washing out of interfering substances, etc. Cleaning liquid path and column, good day-to-day precision, and good recovery of standard addition

Active Publication Date: 2021-06-04
KEAISE MEDICINE WUHAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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-Hydroxydeoxyguanosine detection kit and its application
  • 8-Hydroxydeoxyguanosine detection kit and its application
  • 8-Hydroxydeoxyguanosine detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Example 1 Take NaH 2 PO 4 KH 2 PO 4 Comparative study for mobile phase detection

[0094] Mobile phase 1: 50mmol / l Ph3.5 NAH 2 PO 4 Solution: methanol = 90%: 10%.

[0095] Mobile phase 2: 50mmol / L pH 3.5 kH 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 wavelength was 300 nm.

[0097] The 8-hydroxy deoxycoside standard control solution was prepared with a concentration of 10 μg / ml.

[0098] The results detected by mobile phase 2 figure 2 . The results detected by mobile phase 1 image 3 Indicated.

[0099] As can be seen from the figure, with kH 2 PO 4 When the flow of the phase, the peak peak of 8-OHDG at 13-16 minutes has a shoulder, and the chromatographic separation does not meet the test requirements. Nah 2 PO 4 In the case of flowing a phase, the peak peak shape of 8-OHDG at 14-16 minutes is good, in line with the test requirements.

Embodiment 2

[0100] Example 2 Selection of flow phase solvent

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

[0102] Use 50 mmol / l pH 3.5 NAH 2 PO 4 Solution: Acetonitrile = 90%: 10% chromatographic conditions measurable 8-hydroxy deoxymide control, acetonitrile for the separation of the 8-OHDG target sample peak and solvent peak Figure 4 .

[0103] Use 50 mmol / l pH 3.5 NAH 2 PO 4 Solution: Methanol = 90%: 10% chromatographic conditions measured 8-hydroxy deoxymide control, methanol to see the separation of the 8-OHDG target sample peak and solvent peak Figure 5 .

[0104] by Figure 4 with Figure 5 It can be seen that the separation of acetonitrile and methanol can be used as the flow phase solvent of the present invention.

[0105] Considering that methanol is lower than the use of acetonitrile, it is more conducive to the application of clinical large-area applications, so that methanol is preferably selected as organic phases.

Embodiment 3

[0106] Example 3 Selection of column temperature

[0107] Mobile phase A is 50 mmol / l pH 3.5 Nah 2 PO 4 The solution, the mobile phase B is methanol, and the mobile phase A: the mobile phase B is 9: 1, the injection volume is 50 μl, and the flow rate is 1.0 mL / min.

[0108] The 8-hydroxy deoxycine control was added to a healthy human urine to give a concentration of 20 μg / ml, comparison 10 ° C, 20 ° C, 30 ° C different column temperature conditions under different column temperature conditions.

[0109] Table 2 Reserved time for reference under different column temperature conditions

[0110]

[0111] From the above table, the column temperature is 20-30 ° C, and the 8-hydroxyl deoxycoside component peak is good, and can be separated from other impurities peaks. In view of 30 ° C, the peak peak peak peak peak peak is relatively faster, which is advantageous for increasing the efficiency of clinical mass sample detection, and thus the preferred column temperature is 30 ° C....

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Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a detection kit for 8-hydroxydeoxyguanosine and a detection method for 8-hydroxydeoxyguanosine. The detection kit of the present invention comprises: Reagent A: NaH 2 PO 4 Solution; Reagent B: mobile phase solvent. The detection method for 8-hydroxydeoxyguanosine includes: 1) preprocessing the sample to be tested; 2) setting detection parameters; 3) detecting the sample. The detection kit of the present invention improves the problems of high chromatographic column pressure, easy blockage of the chromatographic column and the quaternary pump of the chromatograph, and the problems of easy interference and inaccuracy in the prior art when potassium dihydrogen phosphate is used as the mobile phase. Further, the combination of sample and high-performance liquid chromatography can be realized, so as to realize automatic detection. The detection method of the invention is accurate, simple, strong in specificity, good in precision and good in recovery rate. The present invention also provides the application of the 8-OHdG detection kit in the preparation of a kit for lung cancer diagnosis or clinical treatment effect monitoring of lung cancer.

Description

Technical field [0001] The present invention belongs to the field of biological detection, and more particularly to the detection method of 8-hydroxy deoxycin, detecting the kit and its application thereof. Background technique [0002] The normal metabolic metabolic process of foreign chemicals or cells can produce a large number of active oxygen (ROS) free radicals. ROS mainly includes superoxide anionic group, hydroxyl free radical, hydrogen peroxide free radical, and the like. Active oxygen has unpublished single electron with strong chemical reactivity. Excessive active oxygen levels can cause oxidative damage to cell membrane lipids, proteins and DNA. However, the active oxygen life is extremely short, and it cannot be directly detected in the human body. Therefore, it is necessary to select a product of a biomolecule to carry out the non-enzyme-reacted product as a biomarker, instead of active oxygen for detection, and indirectly reflect the active oxygen in the body. . ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 曾慧慧
Owner KEAISE MEDICINE WUHAN
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