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Method for identifying biomarkers of gouty arthritis and detection kit thereof

A technology for gouty arthritis and biomarkers, applied in the field of medicine, can solve the problems of unsuitability 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: 2019-04-12
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 the serum of patients with gouty arthritis 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 for identifying biomarkers of gouty arthritis and detection kit thereof
  • Method for identifying biomarkers of gouty arthritis and detection kit thereof
  • Method for identifying biomarkers of gouty arthritis and detection kit thereof

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 for identifying biomarkers of gouty arthritis and a detection kit thereof. The biomarkers are free mannose and glucose obtained by high performance liquid chromatography derived from pre-column PMP in serum, and the ratio of glucose to mannose. The detection method is the pre-column 1-phenyl-5-methyl-pyrazolone (PMP) derived high performance liquid chromatography. The method has the advantages that pretreatment is simple, analysis time is short, the instrument price is reasonable, conventional use is conformed to, operation steps are simple and easy to learn, the accuracy of detection results is high, only blood collection is needed, the amount of serum required is extremely small, the blood collection is less than 1 mL, and the like. The results show that the analysis method can quickly quantify the free mannose and glucose in the serum of patients with gouty arthritis, and has great significance to study the relationship between free mannose and glucose in serum and gouty arthritis and to find new clinical detection markers of gouty arthritis.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying biomarkers of gouty arthritis and a detection kit thereof. Background technique [0002] Gouty arthritis is caused by gout. When uric acid is in a supersaturated solution, monosodium urate crystals are formed, and most of them are located in the joint fluid and around the joints. Patients with gouty arthritis will deposit monosodium urate (MUS) in the joints, which is caused by long-term hyperuricemia, leading to acute gouty arthritis, joint deformities, and repeated attacks. The disease often has associated complications (diabetes, cardiovascular disease, chronic kidney disease or obesity), so finding a way to diagnose it early is especially important. [0003] The diagnostic criteria for gout are needle-like monosodium urate (MSU) crystals in tissue or synovial fluid that can exhibit polar birefringence under the microscope, but other join...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽娟李秀莲李长贵张朦
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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