A liver cancer biomarker and kit for detecting liver cancer

A reagent and biotin technology, applied in the field of biomarkers and kits, to achieve the effect of specific diagnosis

Active Publication Date: 2022-08-05
LIAONING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sialic acid is an important part of the modification of cell membrane glycoproteins, but so far there is no relevant research report on the use of sialic acid (Neu5Gc) modified IgG in serum as a diagnostic marker for liver cancer

Method used

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  • A liver cancer biomarker and kit for detecting liver cancer
  • A liver cancer biomarker and kit for detecting liver cancer
  • A liver cancer biomarker and kit for detecting liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The IgG polyclonal antibody involved in the present invention is prepared by the laboratory, and the separation and purification steps of the mouse IgG polyclonal antibody are as follows:

[0027] (1) Dilute mouse serum in PBS at a ratio of 1:10, filter it through a 0.22 μm filter, and incubate with Protein G agarose column at 4°C for 6 hours;

[0028] (2) Wash the purification equipment with about 30 mL of 20% aqueous ethanol;

[0029] (3) Use Binding Buffer (0.1M NaCl, 20mM NaHPO 4 , pH=7.0-7.4) balance the purification equipment, after the purification equipment shows stable, adjust the reading to "0";

[0030] (4) Loading: Load the sample obtained in step (1) at a speed of 1 mL / min (the injection speed is adjusted to 12-18), and after the serum premix in the column is loaded (the device to be purified indicates the number of samples) The effluent can be received when it rises rapidly, that is, "flow through"), use Binding Buffer to balance the purification equipme...

Embodiment 2

[0037] The preparation of the lamprey immune protein LIP used in this example refers to the patent number 201310501366.2, which is a Chinese invention patent titled "lamprey plum protein, preparation method and application in the preparation of drugs for preventing and treating tumor diseases". The lamprey immune protein LIP can be obtained by separating it from the culture medium of the lamprey Lee's body, or obtaining it through a heterologous expression system, such as an E. coli prokaryotic expression system, preferably a pColdI expression vector, to efficiently obtain the recombinant protein rLiprotein.

[0038] The preparation of the isolation of lamprey immune protein LIP from the culture medium of lamprey Lee corpuscles is carried out as follows:

[0039] a. Take fresh lamprey Li's body and put it into trypsin for overnight digestion at 4°C;

[0040] b. Collect the digested cells and wash with PBS twice;

[0041] c. Transfer to serum-free 1640 medium for 72 hours;

...

Embodiment 3

[0052] The invention provides a kit for detecting liver cancer, comprising a coating reagent, a washing solution, a capture reagent, a detection reagent, a color developing reagent and a termination reagent, the coating reagent is IgG polyclonal antibody, the washing solution is TBST, and its components It is: Tris 10mM, NaCl 150mM, Tween-20 0.05% (V / V), HCl to adjust pH to 7.6, the capture reagent is biotin-conjugated lamprey immune protein (LIP-Biotin), which can specifically recognize IgG in serum The modification site of glycoprotein Neu5Gc, the detection reagent is streptavidin coupled with horseradish peroxidase (HRP), the color reagent is TMB chromogenic solution, and the stop reagent is sulfuric acid solution. Neu5Gc-modified IgG can be accurately quantified by the Hexin reaction system.

[0053] First, a standard curve for detecting Neu5Gc content was established. The specific method is as follows:

[0054] (1) The pure Neu5Gc was serially diluted in PBS buffer conta...

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Abstract

The invention discloses a liver cancer biological marker and a kit for detecting liver cancer, and belongs to the technical field of biological markers and kits. The present invention discovers for the first time that IgG modified by Neu5Gc is abnormally highly expressed in the serum of patients with liver cancer, and uses it as a biomarker for diagnosing liver cancer. The present invention also provides a kit for detecting liver cancer, comprising a coating reagent, a washing solution, and a capture reagent , detection reagent, chromogenic reagent and termination reagent, the capture reagent is biotin-conjugated lamprey immune protein, which can specifically identify the Neu5Gc modification site of IgG glycoprotein in serum, and the detection reagent is coupled horseradish peroxidase (HRP) streptavidin, Neu5Gc-modified IgG can be accurately quantified by the biotin-streptavidin reaction system. The kit of the invention can efficiently and specifically diagnose liver cancer, has monitoring effect on the effectiveness of the patient's treatment means, and has indication significance for the patient's prognosis monitoring.

Description

technical field [0001] The invention belongs to the technical field of biomarkers and kits, and particularly relates to a liver cancer biomarker and a kit for detecting liver cancer. Background technique [0002] Liver cancer is a cancer with a high mortality rate, and primary liver cancer or liver metastases from other tumors can cause severe liver damage. Most primary liver cancers in adults belong to one of two types: hepatocellular carcinoma and cholangiocarcinoma / cholangiocarcinoma, which are one of the eight most common cancers in the world. Hepatocellular carcinoma is one of the common malignant tumors in China. The second leading cause of cancer deaths in my country. The incidence of liver cancer in men is at least 2 to 3 times higher than in women, the early symptoms are not obvious and atypical, and the time between the start of tumor growth and the first signs is long, which is the main cause of high mortality. It belongs to the middle and late stage, and the rec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N33/57438G01N33/57488G01N33/68
Inventor 逄越李庆伟曹旭韩英伦
Owner LIAONING NORMAL UNIVERSITY
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