Method for identifying biomarkers of asthma and detection kit

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: 2019-01-15
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

Method used

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  • Method for identifying biomarkers of asthma and detection kit
  • Method for identifying biomarkers of asthma and detection kit
  • Method for identifying biomarkers of asthma and detection kit

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 identifying biomarkers of asthma and a detection kit. The biomarkers are free mannose, free glucose in serum and the ratio of the glucose to the mannose, wherein the free mannose and the free glucose are obtained through pre-column PMP derivation high performance liquid chromatography. A detection method is the pre-column 1-phenyl-5-methyl pyrazolone (PMP) derivation high performance liquid chromatography. According to the technical scheme, the advantages that pretreatment is simple, the analysis time is short, the price of instruments is reasonable, conventional use is met, the operation steps are simple and easy to learn, the detection results have high accuracy, healthy person and asthmatic patients can be distinguished only through blood collection,the very little serum amount is required, and the blood collection amount is less than 1 mL are achieved. The obtained results show that the analysis method can rapidly quantify the free mannose and the free glucose in the serum of the asthma patients, and has great significance to study the relationship between the free mannose in the serum and asthma as well as the free glucose in the serum andasthma and to search for novel asthma clinical detection markers.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying an asthma biomarker and a detection kit thereof. Background technique [0002] Asthma, also known as bronchial asthma, is a common respiratory system disease; it is a chronic inflammatory disease of the airway involving a variety of cells and cell components. There are about 300 million asthma patients in the world, and it has become a major chronic disease that seriously threatens public health. Clinically, physical examination is one of the ways to check for asthma. Clinically, blood routine examination has elevated eosinophils, sputum checks whether eosinophils have degenerated to form sharp crystals, mucus plugs and transparent asthma beads, and pulmonary function tests are the largest Both expiratory flow and peak expiratory flow are reduced as a test for asthma. In addition, blood gas analysis to see if there is respiratory alkalosis,...

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 张丽娟窦怀乾唐立岷
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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