Method for authenticating liver cancer biomarker and detection kit thereof

A technology of biomarkers and kits, applied in the field of medicine, can solve the problems of unsuitability for routine purposes, expensive instruments, large volume of serum samples, etc., and achieve the effects of simplified operation, reasonable price of instruments, and short analysis time.

Inactive Publication Date: 2019-01-11
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, when using enzymatic method, gas-liquid chromatography and high-resolution liquid chromatography, in order to avoid the influence of high-concentration glucose, it needs to be removed, resulting in cumbersome pretreatment process; gas-liquid chromatography-mass spectrometry instruments are expensive and not Suitable for routine purposes; large serum sample volume required
There is currently no literature reporting the simultaneous detection of free mannose and glucose in the serum of patients with liver cancer by a pre-column 1-phenyl-5-methylpyrazolone (PMP)-derived HPLC method

Method used

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  • Method for authenticating liver cancer biomarker and detection kit thereof
  • Method for authenticating liver cancer biomarker and detection kit thereof
  • Method for authenticating liver cancer biomarker and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Precisely weigh mannose (Man), glucosamine (GlcN), galactosamine (GalN), glucuronic acid (GlcUA), glucose (Glc), galactose (Gal), xylose (Xyl), rock Add appropriate amount of algalose (Fuc), add deionized water to prepare two mixed standard solutions containing the above monosaccharide 0.1mg / mL, and prepare immediately for use;

[0053] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5mL EP tube, add 40 μL of 0.3mol / L sodium hydroxide, and vortex to mix;

[0054] (3) PMP derivatization: Add 60 μL 0.5mol / L 1-phenyl-5-methylpyrazolone (PMP) to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0055] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0056] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloro...

Embodiment 2

[0081] (1) Accurately weigh the appropriate amount of mannose (Man), rhamnose (Rha) and glucose (Glc), add deionized water to prepare 5 parts of the same mixed standard solution containing the above monosaccharide 0.1mg / mL, and use it immediately match;

[0082] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5mL EP tube, add 40 μL of 0.3mol / L sodium hydroxide, and vortex to mix;

[0083] (3) PMP derivatization: Add 60 μL 0.5mol / L PMP to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0084] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0085] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0086] (6) Centrifuge the sample at 13000r / min for 10min, take 80μL su...

Embodiment 3

[0107] (1) Accurately weigh the appropriate amount of mannose and glucose, add deionized water to prepare the above monosaccharides containing 0.5mg / mL, 0.25mg / mL, 0.1mg / mL, 0.05mg / mL, 0.01mg / mL, 0.005mg / mL, 0.0025mg / mL, 0.001mg / mL, 0.0005mg / mL mixed standard solution, ready-to-use;

[0108] (2) Create an alkaline environment: Take 40 μL of the mixed monosaccharide standard in a 1.5mL EP tube, add 40 μL of 0.3mol / L sodium hydroxide, and vortex to mix;

[0109] (3) PMP derivatization: Add 60 μL 0.5mol / L PMP to each sample, vortex and mix well, and react in an oven at 70°C for 1 h after centrifugation;

[0110] (4) Neutralization reaction by adding acid: Take out the samples in the oven, let them cool down, add 40 μL of 0.3mol / L hydrochloric acid to each sample, and vortex to mix;

[0111] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0112] (6) Centrifuge the sample at 130...

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Abstract

The invention provides a method for authenticating a liver cancer biomarker and a detection kit thereof. The biomarker comprises free mannose and glucose, obtained by high performance liquid chromatography through derivation of pre-column PMP, in patient serum. The detection method is high performance liquid chromatography derived by pre-column 1-phenyl-5-methyl pyrazolone (PMP). Through the technical scheme, the method has the advantages of simple pretreatment, short analysis time and reasonable instrument cost, conventional use is met, the operating steps are simple and easy to learn, the detection result is high in accuracy, normal persons and liver caner patients can be distinguished just by needing blood sampling, the quantity of required serum is extremely small, and the blood sampling quantity is smaller than 1 mL. The obtained result indicates that the analysis method can quickly quantify free mannose and glucose in the serum of a liver caner patient, and the biomarker is significant for researching the relation between the free mannose and glucose in the serum and liver caner and searching novel markers for liver cancer clinical detection.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying liver cancer biomarkers and a detection kit thereof. Background technique [0002] Liver cancer, also known as malignant liver tumors, is divided into two categories: primary and secondary: the former originates from the liver epithelium or mesenchymal tissue; the latter is metastasized to the liver by other cancers. Liver cancer is a malignant tumor with great harm, and its five-year survival rate is only 10%. my country is the main country with liver cancer. Statistics show that in recent years, new cases of liver cancer in my country account for 59% of the world's total, which is three times the global average incidence rate. On average, one person is diagnosed with liver cancer every 67 seconds. The pathogenesis of liver cancer is very complicated: family history, environment, diet, viral infection, etc. An important reason for the high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽娟胡明慧田清武韩燕
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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