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Antibody and kit for detecting Golgi glycoprotein 73 in blood serum

A glycoprotein and kit technology, which is applied in the field of antibodies and kits for detecting Golgi glycoprotein 73 in serum, can solve problems such as missed diagnosis of liver cancer, and achieve the effects of maintaining activity, reliable and accurate detection results, and stable reaction system.

Inactive Publication Date: 2017-08-04
马杰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the positive rate of AFP is about 60-70%. Not all hepatocellular carcinomas secrete a large amount of AFP. About 40% of patients with early primary liver cancer and 15%-20% of patients with advanced primary liver cancer have normal serum AFP levels, so There is still a possibility of missed diagnosis in the diagnosis of liver cancer only by AFP, resulting in 80% of the patients being diagnosed in the middle and advanced stage

Method used

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  • Antibody and kit for detecting Golgi glycoprotein 73 in blood serum
  • Antibody and kit for detecting Golgi glycoprotein 73 in blood serum
  • Antibody and kit for detecting Golgi glycoprotein 73 in blood serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Preparation of monoclonal antibodies to liver cancer-related tumor markers

[0077] (1) Antigen preparation

[0078] 1. Antigen Preparation of Golgi Glycoprotein 73 (GP73)

[0079] 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.

[0080] 2. Antigen preparation of α-L-fucosidase (AFU)

[0081] 2.1 Antigen design

[0082] 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

[0190] Example 2. Antibody pairs for double-antibody sandwich detection

[0191] (1) ELISA double-antibody sandwich pairing experiment

[0192] According to the following steps, pairing experiments were performed on the monoclonal antibodies of each protein purified in Example 1:

[0193] 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.

[0194] 2. Plate washing: Wash 5 times with PBST and pat dry.

[0195] 3. Blocking: Blocking solution (containing 3% BSA and 4% sucrose in PBS), 200ul per well, blocked at 37°C for 1h.

[0196] 4. Drying: Do not wash the board, pat dry the residual liquid, and air dry for 30 minutes.

[0197] 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

[0223] Example 3. Coupling and labeling of antibodies

[0224] 1. Coupling of magnetic beads and coated antibodies

[0225] (1) Washing 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 2O Vortex to resuspend the magnetic beads, separate on a magnetic separator for 30-60s, and aspirate the supernatant.

[0226] (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 with 250ul 50mM Ph5.0MES (2-(N-morpholine)ethanesulfonic acid) solution to remove unconjuga...

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Abstract

The invention relates to the antigen detection technology of blood serum and in particular relates to an antibody and a kit for detecting a Golgi glycoprotein 73 in blood serum. An antibody pair provided for detecting a liver cancer marker Golgi glycoprotein 73 in to-be-detected blood serum is characterized by being composed of a first antibody and a second antibody; and the first antibody serves as a coating antibody which is secreted from hybridoma, the preservation number of which is 8F10D1, and the second antibody serves as a detection antibody which is secreted from hybridoma, the preservation number of which is 10B9F11. The antibodies are high in specificity and can be combined in use with other markers to detect GP73 in blood serum without interference. The antibody is high in sensitivity, can detect GP73, the concentration of which is low to 250pg / ml, and can be used for accurately quantifying GP73 protein in the blood serum.

Description

technical field [0001] The invention relates to serum antigen detection technology, in particular to an antibody and kit for detecting Golgi glycoprotein 73 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. The clinical diagnosis of liver cancer is mainly through serum detection of alpha-fetoprotein (Alpha-fetoprotein, AFP), B-ultrasound to find liver nodules, and then CT and MRI examination. However, the positive rate of AFP is about 60-70%. Not all hep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N33/57484G01N33/57438G01N33/68
Inventor 马杰吴云
Owner 马杰