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Method for identifying kidney cancer biomarker and detection kit of kidney cancer biomarker

A biomarker and kit 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: 2019-03-26
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 a pre-column 1-phenyl-5-methylpyrazolone (PMP)-derived HPLC method for the simultaneous detection of free mannose and glucose in the serum of patients with renal cancer

Method used

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  • Method for identifying kidney cancer biomarker and detection kit of kidney cancer biomarker
  • Method for identifying kidney cancer biomarker and detection kit of kidney cancer biomarker
  • Method for identifying kidney cancer biomarker and detection kit of kidney cancer biomarker

Examples

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 chloro...

Embodiment 2

[0079] (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;

[0080] (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;

[0081] (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;

[0082] (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;

[0083] (5) Extraction: add 500 μL chloroform to each tube, vortex, centrifuge, remove the lower layer of chloroform, keep the supernatant, repeat 3 times;

[0084] (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 for identifying a kidney cancer biomarker and a detection kit of the kidney cancer biomarker. The biomarker is a concentration ratio of free mannose and glucose obtained by high performance liquid chromatography for performing pre-column 1-phenyl-5-methylpyrazolone (PMP) derivatization in serum. The detection method disclosed by the invention is the high performanceliquid chromatography of pre-column PMP derivatization. The technical scheme of the invention has the advantages that the pretreatment is simple, the analysis time is short, the instrument price is reasonable, the conventional application is met, the operating steps are simple and easy to learn, the detection result accuracy is high, normal people and kidney cancer patients can be distinguished by virtue of blood sampling only, the needed serum amount is extremely small, the blood sampling amount is less than 1mL, and the like. The obtained results show that, the analytical method is capableof rapidly quantifying the free mannose and glucose in serum of kidney cancer patients, and has very important significance for researching the relationship between the free mannose and glucose in theserum and the kidney cancer and searching a novel clinical detection marker for kidney cancer.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for identifying a renal cancer biomarker 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. Diagnosis of kidney disease generally requires urine tests, blood tests, imaging tests and pathological tests, combined with clinical manifestations for diagnosis. These diagnostic methods will bring certain disadvantages. For example, imaging examinations have an accuracy rate of more than 90% in diagnosing kidney cancer, but imaging examinations will have radiation, which will have an impact on the human body. In addition, at present, there is no recognized tumor biomarker that can be u...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/067
Inventor 张丽娟何燕利徐岩
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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