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Method and detection kit for identifying degenerative osteoarthropathy biomarkers

A technology for biomarkers and osteoarthritis, applied in the field of medicine, can solve the problems of unsuitable for routine purposes, cumbersome processing process, large volume of serum samples, etc., and achieve the effects of simplified operation, short analysis time, and simple pretreatment.

Inactive Publication Date: 2019-03-01
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
Simultaneous detection of free mannose and glucose in serum of patients with degenerative osteoarthritis by pre-column 1-phenyl-5-methylpyrazolone (PMP)-derived high-performance liquid chromatography has not been reported in the literature

Method used

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  • Method and detection kit for identifying degenerative osteoarthropathy biomarkers
  • Method and detection kit for identifying degenerative osteoarthropathy biomarkers
  • Method and detection kit for identifying degenerative osteoarthropathy biomarkers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] (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;

[0054] (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;

[0055] (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;

[0056](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;

[0057] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chlorof...

Embodiment 2

[0082] (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;

[0083] (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;

[0084] (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;

[0085] (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;

[0086] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0087] (6) Centrifuge the sample at 13000r / min for 10min, take 80μL su...

Embodiment 3

[0109] (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;

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0114] (6) Centrifuge the sample at 130...

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Abstract

The invention provides a method and a detection kit for identifying degenerative osteoarthropathy biomarkers. The biomarkers are free mannose and free glucose, which are obtained by subjecting serum to pre-column 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP) derivatization high-performance liquid chromatography, and a ratio of the free glucose to the free mannose. A detection method is pre-column PMPderivatization high-performance liquid chromatography. According to the technical scheme, the method and the detection kit for identifying the degenerative osteoarthropathy biomarkers have the advantages that pre-treatment is simple, the analysis time is short, the instrument price is reasonable, the conventional use is met, the operation steps are simple and easy to learn, the detection result ishigh in accuracy, only blood sampling is needed, moreover, the required serum amount is very few, the amount of sampled blood is less than 1 mL, and the like. An obtained result shows that by means of the analysis method, the free mannose and the free glucose in the serum of a degenerative osteoarthropathy patient can be fast quantified, so that the method and the detection kit for identifying the degenerative osteoarthropathy biomarkers have significant meaning in studying a relation between free mannose and free glucose in the serum and degenerative osteoarthropathy and finding a novel degenerative osteoarthropathy 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 degenerative osteoarthritis and a detection kit thereof. Background technique [0002] Degenerative osteoarthritis is a chronic joint disease that is caused by the aging of the human body and long-term wear and tear of the bones and joints. It is also known as osteoarthritis and degenerative arthritis, and it is more common in the middle-aged and elderly people. The disease is characterized by progressive deterioration of hyaline cartilage accompanied by changes in surrounding tissues including ligaments, synovium and subchondral bone, resulting in affected joints or the spine. As the population ages and life expectancy increases, the burden of bone and joint disease will increase The heavier it is, so it is especially important to find an early diagnosis method. [0003] For degenerative osteoarthropathy clinically, it mainly ...

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 张丽娟李秀莲马学晓
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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