Method of authenticating pancreatitis biomarker and detection kit thereof

A biomarker, pancreatitis technology, applied in the field of medicine, can solve the problems of unsuitable for routine purposes, expensive instruments, cumbersome processing procedures, etc., and achieve the effects of simplified operation, reasonable instrument price, 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

Method used

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  • Method of authenticating pancreatitis biomarker and detection kit thereof
  • Method of authenticating pancreatitis biomarker and detection kit thereof
  • Method of authenticating pancreatitis 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 of authenticating a pancreatitis biomarker and a detection kit thereof. The biomarker is free mannose and glucose that are obtained from serum through pre-column PMP-derived high performance liquid chromatography. The detection method refers to the pre-column PMP-derived high performance liquid chromatography. The method has the advantages that preprocessing is simple, analysis time is short, instrument price is reasonable and can be used routinely, operation steps are simple to learn, the detection result is highly accurate, a normal person and a patient suffering pancreatitis can be distinguished by sampling blood, and very little serum, less than 1mL, is needed. The obtained result shows that the analysis method can rapidly quantify free mannose and glucose in serum of a pancreatitis patient, and the method is significant for research of a relationship between free mannose and glucose in serum and pancreatitis and seeking of novel pancreatitis 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 pancreatitis biomarkers and a detection kit thereof. Background technique [0002] Pancreatitis is a disease of the pancreas caused by the autodigestive action of trypsin. Clinically, various symptoms such as abdominal pain, abdominal distension, nausea, vomiting, fever, and elevated amylase levels in blood and urine will appear. At present, the main diagnostic methods are white blood cell count, blood and urine amylase determination, serum lipase determination, serum calcium determination, serum metferritin (MHA) determination, X-ray examination, B-ultrasound and CT. The use of optical imaging can help to clarify the development of the disease, but the high cost is also one of the restrictive factors in the diagnosis of pancreatitis. Moreover, it is difficult to evaluate the disease of acute pancreatitis with a single method in the current ...

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 张丽娟窦怀乾杨乃龙欧阳汤鹏隋爱华
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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