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Antibodies and kits for combined detection of liver cancer markers in serum

A combined detection and marker technology, applied to cells modified by the introduction of foreign genetic material, measuring devices, fusion cells, etc., can solve the problems of low early tumor detection rate, low specificity, and large serum volume, etc. Achieve the effects of reduced plasma consumption, low detection cost, and stable reaction system

Active Publication Date: 2019-08-16
马杰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing methods for detecting tumor markers are all single-index detection methods, which have shortcomings such as low specificity and low sensitivity, especially for early-stage tumors. The detection rate is not high
However, the cost of multi-index analysis for each sample is very expensive, and the amount of serum required is relatively large.

Method used

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  • Antibodies and kits for combined detection of liver cancer markers in serum
  • Antibodies and kits for combined detection of liver cancer markers in serum
  • Antibodies and kits for combined detection of liver cancer markers in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Preparation of monoclonal antibodies to liver cancer-related tumor markers

[0089] (1) Antigen preparation

[0090] 1. Antigen Preparation of Golgi Glycoprotein 73 (GP73)

[0091] The design and preparation methods of recombinant antigens are described in: Pang Lijun, Gene Cloning and Prokaryotic Expression of Golgi Proteins, Journal of Medicine Tribune, 2015(9):1-2. The high-purity antigen GP73-F3R3 was prepared according to the method described in the article, and its expression strain was preserved in Beijing Qingyuan Shengkang Biomedical Technology Co., Ltd.

[0092] 2. Antigen preparation of α-L-fucosidase (AFU)

[0093] 2.1 Antigen design

[0094] The full length of α-L-fucosidase is 466 amino acids, and the amino acid sequence of α-L-fucosidase (NCBI sequence number: NP_000138.2) was analyzed using DNA Star sequence analysis software to obtain the secondary structure, affinity and affinity of the protein. Aqueous, antigenicity and other informatio...

Embodiment 2

[0203] Example 2. Antibody pairs for double-antibody sandwich detection

[0204] (1) ELISA double-antibody sandwich pairing experiment

[0205] According to the following steps, pairing experiments were performed on the monoclonal antibodies of each protein purified in Example 1:

[0206] 1. Coating: The coating antibody (one of the monoclonal antibodies purified in Example 1) was diluted to 5 ug / ml with PBS (PH=7.4). 100ul per well, coated at 37°C for 1h.

[0207] 2. Plate washing: Wash 5 times with PBST and pat dry.

[0208] 3. Blocking: Blocking solution (containing 3% BSA and 4% sucrose in PBS), 200ul per well, blocked at 37°C for 1h.

[0209] 4. Drying: Do not wash the board, pat dry the residual liquid, and air dry for 30 minutes.

[0210] 5. Add antigen: the antigen (the antigen purified in Example 1) is diluted to 2ug / ml with PBS (PH=7.4) containing 1%BSA, added to a microtiter plate, and doubled from the first well (2ug / ml) Dilute to well 11 (0.2ng / ml), 100ul per...

Embodiment 3

[0235] AFP: Coating antibody 3B11H5 (CGMCC NO: 13581), detection antibody 2G5E8 (CGMCC NO: 13582). Example 3. Coupling and labeling of antibodies

[0236] 1. Coupling of magnetic beads and coated antibodies

[0237] (1) Wash magnetic beads: suspend uncoupled magnetic beads (MC10030-01, MC10034-01, MC10036-01, MC10038-01, MC10044-01), transfer 5.0×10 6 Put magnetic beads into a centrifuge tube, put the centrifuge tube on a magnetic separator for separation for 30-60s, suck out the supernatant, add 100ul dH 2 O Vortex to resuspend the magnetic beads, separate on a magnetic separator for 30-60s, and aspirate the supernatant.

[0238] (2) Activate magnetic beads: add 80ul 0.1M NaH 2 PO 4 (Ph6.2), vortex to suspend magnetic beads; then add 10ul 50mg / ml sulfo-NHS (N-hydroxysulfosuccinimide) and 10ul 50mg / ml EDC (1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide hydrochloride), vortexed and incubated at room temperature for 20 minutes to activate the microspheres. Then wash twice ...

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Abstract

The invention relates to serum antigen detection technology, in particular to an antibody for joint detection of a hepatoma marker in serum and a kit. An antibody composition for the joint detection of the hepatoma marker in the serum is provided, including at least two of an antibody pair against AFP, an antibody pair against GP73, an antibody pair against VEGF, an antibody pair against CD147 and an antibody pair against AFU, and the antibody pairs consist of first antibodies functioning as coating antibodies and second antibodies functioning as detection antibodies. The antibody composition is strong in specificity, and the AFP, the GP73, the AFU, the VEGF and the CD147 do not interfere with one another when detected simultaneously; the sensitivity is high, the sensitivity of the AFP, the GP73, the AFU and the VEGF can be up to 250pg / ml through detection, the sensitivity of the CD147 can be up to 64pg / ml through detection, and the antibody provided by the invention can be used for accurately quantifying the hepatoma marker in the serum.

Description

technical field [0001] The invention relates to a serum antigen detection technology, in particular to an antibody and a kit for combined detection of liver cancer markers in serum. Background technique [0002] The incidence of cancer worldwide is on the rise. On February 3, 2014, the "Global Cancer Report 2014" published by the World Health Organization showed that the number of new cancer cases in China ranked first in the world, and the number of new cases and deaths of liver cancer ranked first in the world. Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the most common malignant tumors in the world. my country is a big country with hepatitis B, and the incidence of liver cancer accounts for 55% of the global total. There is basically no effective treatment for advanced liver cancer. Screening and early diagnosis of liver cancer tumor markers for high-risk groups such as hepatitis B and cirrhosis are the most effective measures to solve the high mor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/12
CPCG01N33/57438G01N33/57488
Inventor 马杰吴云
Owner 马杰
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