Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit

An antibody and antibody light chain technology, applied in biochemical equipment and methods, applications, antibodies, etc., can solve the problems of low specificity, specificity and sensitivity that cannot meet clinical needs, etc.

Inactive Publication Date: 2010-03-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CA19-9 has the strongest sensitivity (80%) in pancreatic cancer, but its specificity is very low (43%). The combination of CEA and CA242 can increase the specificity to 92%, but the sensitivity is lower after the combination of the three. Only 2

Method used

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  • Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit
  • Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit
  • Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0045] Example 1. Establishment of 3P9 Monoclonal Antibody Hybridoma Cell Line and Production of Monoclonal Antibody

[0046] Materials: 1. Cells SW1116 colon cancer cell line, purchased from ATCC (American TypeCulture Collection, ATCC No. CCL-233), SP2 / 0 mouse myeloma cells (ATCC No. CRL-1581). 2. Medium Serum-free DMEM, high-glucose DMEM, HAT, and HT medium were purchased from Gibco, fetal bovine serum was purchased from Wuhan Sanli Company, and cell culture plates were Corning products. 3. Reagent polyethylene glycol PEG (MW 4000) Purchased from Sigma Company, other reagents were domestic analytical grade 4. Animals BALB / c mice were purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences. 5. Antibody identification The mouse monoclonal antibody typing reagent Sigma ImmunoType Kit antibody subtype detection kit was used.

[0047] Method: 1. Mice were immunized with 1×10 7 Mix and emulsify SW1116 cell suspension with 0.5ml of Freund's complet...

Embodiment 2 3P9

[0054] Example 2. Purification and biochemical properties of 3P9 monoclonal antibody

[0055] Materials: 3P9 monoclonal antibody ascites was obtained by inoculating BALB / c mice with hybridoma cells intraperitoneally; prepacked chromatography column Sephadex-200, column volume 100ml, Phamacia product, horseradish peroxidase (HRP) goat anti-mouse The IgM antibody was purchased from Sant Cruz Company, and the ECL fluorescence detection kit was purchased from Pierce Company, and was purified using the AKTA FPLC protein chromatography system.

[0056] Method: Thaw the above ascites at 4°C, centrifuge at 15,000g for 20 minutes, collect the supernatant, and sterilize with a 0.2um membrane; equilibrate the prepacked column with 0.01M PBS at pH 7.2, load 5ml of ascites; wash with 0.01M PBS at pH 7.2 The flow rate was 1ml / min, and each elution peak was collected; 10ul samples of the elution peak were spotted on a nitrocellulose membrane, and after natural air drying, dot hybridization...

Embodiment 3 3P9

[0059] Example 3. Binding of 3P9 monoclonal antibody to bovine submandibular gland mucin

[0060] Materials: 3P9 monoclonal antibody was purified from ascites antibody by Sephadex-200; purified bovine submaxillary mucin (BSM, bovine submaxillary mucin) (Sigma), horseradish peroxidase (HRP) goat anti-mouse IgM antibody was purchased from Sant Cruz Company; 96-well enzyme-linked immunosorbent assay plate was purchased from Corning Costar, USA.

[0061] Method: Dilute BSM to 50ng / ml in the coating solution, add 100ul to each plate well, room temperature for 2 hours or overnight at 4°C, shake off the coating solution, wash with PBS, block with 2% BSA-PBS for 1 hour, add 50ul for serial dilution and purification The 3P9 monoclonal antibody was incubated at 37°C for 1 hour, washed with PBS or PBST, added 50ul horseradish peroxidase (HRP) goat anti-mouse IgM antibody (diluted 2,000 times in PBS) and incubated at 37°C for 1 hour, washed with PBS or PBST Wash, add 200ul color develo...

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Abstract

The invention provides a monoclonal antibody, which has the following characteristics: 1) the antibody is IgMs immunoglobulin and the light-chain type of the antibody is k-typed; (2) the antigen is identified into a sugar-chain structure with sialylation mucoprotein surface; the monoclonal antibody can specifically identify colorectal adenocarcinoma, endometrial adenocarcinoma and pancreatic carcinoma tissues, can not identify the corresponding normal tissues. Specifically, the monoclonal antibody is formed by the secreting of hybridomas cells with the accession number of CGMCC No. 2643, the amino acid sequence in the heavy-chain variable zone is shown as SEQ ID NO: 1, the amino acid sequence in the light-chain variable zone is shown as SEQ ID NO: 2. The invention also provides a use of the monoclonal antibody in the preparation of a medicament or a kit, wherein the medicament or the kit can be used for the screening of cancer, early-stage diagnosis, treatment monitoring and tumour imaging. In addition, the invention provides a new antibody for inhibitting the growth of the tumor cells of colorectal carcinoma.

Description

technical field [0001] The present invention belongs to the technical field of biomedicine. Specifically, the present invention provides a monoclonal antibody that specifically recognizes sugar chain antigens of colorectal adenocarcinoma, endometrial adenocarcinoma, and pancreatic cancer cells, and can inhibit the growth of colon tumor cells, and secretes the antibody. Hybridoma cells, and the application of the antibody in the preparation of medicines and kits for treating colon tumors, the present invention also provides medicines and kits, which contain the monoclonal antibody as an active ingredient. Background technique [0002] Colorectal cancer refers to malignant changes in the endothelial cells of the large intestine, including abnormal differentiation, abnormal proliferation, and abnormal cell metabolism and secretion. Clinically, the vast majority (>95%) of colorectal cancers are adenocarcinomas, and there are few other types of colorectal cancers, including ly...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N5/20A61K39/395A61P35/00
Inventor 阎锡蕴韩伟冯静杨东玲
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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