Method of biomarker for identifying renal transplantation prognosis and detection kit for identifying renal transplantation prognosis

A biomarker and kidney transplantation technology, applied in the field of medicine, can solve the problems of being unsuitable for routine purposes, cumbersome processing, and large volume of serum samples, and achieve the effects of simplified operation, short analysis time, and simple pretreatment

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
There is currently no literature reporting the simultaneous detection of free mannose and glucose in serum of patients after renal transplantation by pre-column 1-phenyl-5-methylpyrazolone (PMP)-derived HPLC

Method used

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  • Method of biomarker for identifying renal transplantation prognosis and detection kit for identifying renal transplantation prognosis
  • Method of biomarker for identifying renal transplantation prognosis and detection kit for identifying renal transplantation prognosis
  • Method of biomarker for identifying renal transplantation prognosis and detection kit for identifying renal transplantation prognosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] (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;

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chlorofo...

Embodiment 2

[0080] (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;

[0081] (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;

[0082] (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;

[0083] (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;

[0084] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0085] (6) Centrifuge the sample at 13000r / min for 10min, take 80μL su...

Embodiment 3

[0106] (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;

[0107] (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;

[0108] (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;

[0109] (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;

[0110] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0111] (6) Centrifuge the sample at 130...

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Abstract

The invention provides a method of a biomarker for identifying renal transplantation prognosis and a detection kit for identifying the renal transplantation prognosis. The biomarker is a ratio of freemannose and glucose obtained by pre-column 1-phenyl-5-methyl pyrazolone (PMP) derivatization high performance liquid chromatography in serum. The detection method is a pre-column PMP derivatization high performance liquid chromatography method. The technical scheme has the advantages that the pretreatment is simple, the analysis time is short, the instrument price is reasonable, the conventionaluse requirements are met, the operation steps are simple and easy to learn, the accuracy of a detection result is high, only blood collection is needed, the needed serum amount is extremely small, theblood collection amount is smaller than 1 mL, and the like. The obtained result shows that the analysis method can quickly quantify the free mannose and glucose in the serum of a renal transplantation prognosis patient, and has very important significance for researching a relation between the free mannose and glucose in the serum and the renal transplantation prognosis, and finding a novel clinical prognosis monitoring marker for the renal transplantation prognosis.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying prognostic biomarkers after kidney transplantation and a detection kit thereof. Background technique [0002] The kidney is an important organ of the urinary system. Its main function is to produce and excrete urine, and to excrete the metabolic waste of the human body. generation and bone growth etc. Therefore, kidney disease has a great impact on the function of the human body. The survival rate of kidney transplantation is affected by chronic graft deterioration, and the cause of chronic graft deterioration is difficult to detect in the early stage of kidney transplantation. However, early intervention treatment is the most effective period to improve the survival rate of kidney transplantation. At present, monitoring the clinical characteristics and morphology of grafts through renal biopsy is still the basis for detecting renal failure...

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 张丽娟何燕利徐岩
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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