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Method and detection kit for identifying biomarker of cerebral hemorrhage

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

Inactive Publication Date: 2019-02-15
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 intracerebral hemorrhage by pre-column 1-phenyl-5-methylpyrazolone (PMP)-derived HPLC

Method used

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  • Method and detection kit for identifying biomarker of cerebral hemorrhage
  • Method and detection kit for identifying biomarker of cerebral hemorrhage
  • Method and detection kit for identifying biomarker of cerebral hemorrhage

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 1.5 mL centrifuge tube, add 40 μL of 0.3 mol / 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...

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 1.5 mL centrifuge tube, add 40 μL of 0.3 mol / 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, ta...

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 1.5 mL centrifuge tube, add 40 μL of 0.3 mol / 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 sam...

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Abstract

The invention provides a method and a detection kit for identifying a biomarker of cerebral hemorrhage. The biomarker is free mannose and glucose obtained by high performance liquid chromatography based on pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization in serum. The detection method is the high performance liquid chromatography method based on the pre-column PMP derivatization. Thetechnical scheme has the advantages that the pretreatment is simple, the analysis time is short, the instrument price is reasonable, the requirements of conventional use are met, the operation stepsare simple and easy to learn, and the accuracy of a detection result is high, a normal person and a cerebral hemorrhage patient can be distinguished only by blood collection, the amount of the neededserum is extremely small, the blood collection amount is smaller than 1 mL, and the like. The obtained result shows that the analysis method can be used for rapidly determining the content of the freemannose and glucose in the serum of the cerebral hemorrhage patient, so that the method has very important significance for researching a relation between the free mannose and glucose in the serum and the cerebral hemorrhage, and looking for a novel cerebral hemorrhage clinical detection marker.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying biomarkers of cerebral hemorrhage and a detection kit thereof. Background technique [0002] Cerebral hemorrhage refers to hemorrhage caused by non-traumatic vascular rupture in the brain parenchyma, accounting for 20% to 30% of all strokes, and the fatality rate in the acute phase is 30% to 40%. The cause of the occurrence is mainly related to cerebrovascular lesions, that is, closely related to hyperlipidemia, diabetes, high blood pressure, aging of blood vessels, and smoking. Patients with cerebral hemorrhage often develop sudden onset due to emotional agitation and exertion, and have a high early mortality rate. Most of the survivors have sequelae such as motor disorders, cognitive impairments, and speech and swallowing disorders to varying degrees. The diagnosis of cerebral hemorrhage is generally made by head CT examination combined wi...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽娟张朦张国庆张睿
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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