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Human ovary carcinoma resisting monoclonal antibody hybridoma cell line and its monoclonal antibody and application

A technology of hybridoma cell line and monoclonal antibody, applied in the field of biotechnology and cell engineering

Inactive Publication Date: 2005-04-27
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no radioimmunoimaging kit for ovarian cancer in China

Method used

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  • Human ovary carcinoma resisting monoclonal antibody hybridoma cell line and its monoclonal antibody and application

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Embodiment 1

[0011] 1. Example 1-Establishment of cell lines

[0012] 1. Antigen Preparation

[0013] Fresh tissue specimens of ovarian serous papillary cystadenocarcinoma were aseptically collected in the operating room, washed with sterile saline in an ultra-clean bench to remove bloody substances, weighed 22 grams, cut into pieces, and placed in a 50ml sterile plastic centrifuge tube. Add 30ml of sterile saline and freeze at -20°C. After repeated freezing and thawing 3 times, homogenize at 1,000 rpm for 1 minute; sonicate (A20) for 10 minutes; centrifuge at 10,000 g at 4°C for 60 minutes; take the supernatant and freeze at -20°C. This ovarian cancer crude antigen is called OVCA antigen. The antigen was negative in ELISA reaction with anti-CEA antibody (kit of Beijing Institute of Biological Products), and negative in double-diffusion reaction with anti-aFP antibody (Changchun Institute of Biological Products).

[0014] 2. Antigen Immunization

[0015] Several BALB / c female mice (Ins...

Embodiment 2

[0039] 2. Example 2 - Preparation and Identification of Monoclonal Antibody

[0040] 1. Production of monoclonal antibodies by mouse ascites method

[0041] BALB / c mice were intraperitoneally injected with pristane 0.5ml, and 2-3×10 6 For the above-mentioned hybridoma cells, closely observe the animal’s health status and signs of ascites. After 7 to 10 days, ascites appears. When the ascites is as large as possible and the mice die frequently, the mice are killed, and the skin of the abdomen is sterilized with 75% alcohol, and the skin is cut open. , expose the peritoneum, and cut the peritoneum about 0.5 cm with sterile scissors, suck out the ascites with a sterile dropper, let it stand at room temperature for 30 minutes, centrifuge at 1,000 rpm for 10 minutes, and collect the supernatant. Generally, 2-5ml ascites can be obtained from one mouse.

[0042] 2. Production of monoclonal antibodies by in vitro serum-free culture method

[0043] OVCAMab hybridoma cells grown stab...

Embodiment 3

[0054] 3. Example 3-Radioimmunoimaging of Monoclonal Antibody

[0055] 1.131I-labeled OVCAMab for radioimmunoimaging of patients

[0056] OVCAMab was tested using the modified chloramine T method. 131 I labeled, passed through Sephadex G50, and collected the labeled antibody peak. The radiochemical purity and labeling rate were determined by paper chromatography, and the antibody activity after labeling was determined by cell binding assay.

[0057] Intraperitoneal administration: 131 I-OVCAMab 2mg was dissolved in 500ml of normal saline, and was dripped into the peritoneal cavity within 20 minutes through the abdominal catheter. At 24, 48, 72 and 96 hours after injection, images were visualized with a gamma camera and high-energy collimator. At the same time, a computer was used to quantitatively record the radiation intensity of each important organ, pelvic mass, and tissue adjacent to the mass, which was used to calculate the ratio of radiation intensity of tumor to non...

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Abstract

The present invention belongs to the field of biotechnology and cell engineering, and relates to monoclonal antibody hybridoma cell line and its construction process. Technology, the present invention features that soluble immunogen is first prepared with human ovary carcinoma tissue sample and then used to immunize BALB / c mouse, and through fusion, screening and repeated cloning in hybridoma technology, hybridoma cell line capable of secreting stably human ovary carcinoma resisting monoclonal antibody is obtained. The hybridoma cell line, named as BUPH: OVCAMab, is preserved in the preservation number of CGMCC No. 1225. The present invention also discloses the method of preparing monoclonal antibody OVCAMab with the cell line and purifying the antibody. The antibody is used in early diagnosis, intracorporeal locating, detection, metastatic focus detecting, etc. of ovary carcinoma in radioimmunoassay.

Description

technical field [0001] The invention belongs to the field of biotechnology and cell engineering, and relates to a hybridoma cell line secreting an anti-human ovarian cancer monoclonal antibody, the monoclonal antibody secreted thereto, and the application of the monoclonal antibody, especially in radioimmunoimaging application. Background technique [0002] The incidence of ovarian cancer accounts for the third place among gynecological malignancies in my country, but the death rate ranks first. Because of its insidious onset, it is mostly in the late stage when it is diagnosed, and it often recurs even if it is completely relieved by treatment. Early diagnosis and timely treatment; early detection of recurrence and recurrence site, and selection of appropriate timing for retreatment are key factors to improve prognosis. Because the monoclonal antibody is an antibody against an antigenic epitope, it has high specificity and can specifically recognize the corresponding anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00C07K16/18C12N5/22G01N33/534G01N33/574
Inventor 冯捷钱和年崔恒付天云成夜霞程洪艳叶雪李小平姚煜昌晓红
Owner PEOPLES HOSPITAL PEKING UNIV
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