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Anti-human ovarian carcinoma anti-anti-idiotype antibody hybridoma cell line, and monoclonal antibody and application thereof

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

Inactive Publication Date: 2008-11-19
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although in most animal and clinical trials using anti-idiotypic antibody Ab2 (including ACA125) immunotherapy, it has been shown that the successful induction of Ab3 antibody immune response that specifically binds the antigen is associated with tumor shrinkage and prolongation of tumor-bearing host survival. , but so far the preparation and in-depth study of the ovarian cancer monoclonal antibody Ab3 have not been reported at home and abroad

Method used

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  • Anti-human ovarian carcinoma anti-anti-idiotype antibody hybridoma cell line, and monoclonal antibody and application thereof
  • Anti-human ovarian carcinoma anti-anti-idiotype antibody hybridoma cell line, and monoclonal antibody and application thereof
  • Anti-human ovarian carcinoma anti-anti-idiotype antibody hybridoma cell line, and monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 - Establishment of Cell Lines

[0020] 1. Antigen Preparation

[0021] The hybridoma cells secreting ovarian cancer anti-idiotypic antibody 6B11 established in our laboratory were cultured without serum, and the antibody was prepared by affinity chromatography. The relationship between 6B11 and COC166-9 (ovarian cancer monoclonal antibody Ab1) was determined by direct ELISA method. Very good binding activity. Then 2ml of 6B11 (5mg / ml) was mixed with 2ml of KLH (keyhole limpet hemocyanin 5mg / ml), 40ul of 50% (w / v) carbodiimide was added, mixed at room temperature for 2 hours, and overnight at 4°C. The reactant was dialyzed against 0.9% NaCl overnight, and the medium was changed every 4 hours. Genequant measures protein concentration. The immunogen 6B11-KLH was prepared by sterile filtration with a needle filter. Competitive inhibition test showed that 6B11-KLH basically retained the antibody activity of 6B11.

[0022] 2. Immunization protocol

[0023] The...

Embodiment 2

[0049] Example 2 - Monoclonal Antibody Preparation

[0050] 1. Production of monoclonal antibodies by mouse ascites method

[0051] 8-week-old BALB / c mice were pretreated with intraperitoneal injection of 0.5ml pristane, and 2-2.5×10 6 The above-mentioned hybridoma cells were closely observed for the health status and ascites signs of the animals. After 7-10 days, when the abdominal swelling of the mice showed obvious consumption, the blood was collected from the eyeballs, and the mice were killed by pulling the neck. The skin of the abdomen was disinfected with 75% alcohol, and the aseptic conditions Expose the peritoneum of the mouse, suck out the ascites with a sterile dropper, centrifuge at 3000 rpm for 15 minutes, discard the upper layer of fat, precipitate the cells and add complete 1640 culture. The ascites in the middle layer was packed and stored at -20°C. Generally, 2-5ml ascites can be obtained from each mouse.

[0052] 2. Identification of monoclonal antibodies ...

Embodiment 3-6B11

[0063] Example 3-6B11Ab3 and COC166-9(Ab1) double antibody sandwich method for detection

[0064] Using ELISA method, COC166-9 (Ab1) was coated on the ELISA plate, and the patient's serum antigen or tissue antigen was added for reaction, then 6B11Ab3 labeled with horseradish peroxidase or biotin was added, and finally the reaction substrate was added. The color was developed, and the light absorbance value was read. Through the detection of large samples, parameters such as sensitivity, specificity, positive predictive value and negative predictive value of the detection method are obtained, and it is possible to make a kit for clinical early detection of ovarian cancer.

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Abstract

The invention belongs to the biotechnology and cell engineering field and relates to the establishment of two hybridoma cell lines secreting anti-oophoroma anti-idiotype monoclonal antibodies. The invention is mainly technically characterized by taking a murine anti-oophoroma anti-idiotype monoclonal antibody as an immunogen to immunize a Balb / C mouse and using the hybridoma technology to obtain the hybridoma cell lines stably secreting anti-oophoroma anti-idiotype monoclonal antibodies through fusion, screening and repeated cloning. The two hybridoma cell lines which are respectively named as OVCAMab3D12 and OVCAMab32F2 are preserved by the common microorganism center of China Committee of Culture Collection for Microorganisms with respective collection numbers of CGMCCNo.2144 and CGMCCNo.2145. The invention also discloses monoclonal antibodies 6B11Ab3(IgM) and 6B11Ab3(IgG) respectively through the two hybridoma cell lines. The antibodies can be applicable to an ELISA kit capable of detecting oophoroma associated antigen for early diagnosis of oophoroma. Simultaneously the antibodies can be used for the development of oophoroma curative medicines, particularly targeted therapeutic medicines, and for the therapy of oophoroma.

Description

technical field [0001] The invention belongs to the fields of biotechnology and cell engineering, and relates to a hybridoma cell line secreting ovarian cancer anti-anti-idiotype monoclonal antibody, the secreted monoclonal antibody (6B11Ab3) and its possible application in the diagnosis and treatment of ovarian cancer. Background technique [0002] Ovarian cancer is a highly malignant gynecological tumor. Its incidence rate is the second among malignant tumors of the female reproductive tract, but its mortality rate is the first. Although cytoreductive surgery and multi-drug combination chemotherapy have improved the 5-year survival rate of patients, there are still serious problems such as difficulty in early diagnosis, high recurrence rate, and refractory rate. With the rapid development of tumor biotherapy, immunotherapy may break through the limitations of traditional treatment and solve the bottleneck of high recurrence and refractory tumors. [0003] In terms of immu...

Claims

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Application Information

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IPC IPC(8): C12N5/18C07K16/42G01N33/577A61K39/395A61P35/00
Inventor 崔恒昌晓红张超冯捷倪俊付天云叶雪程洪艳成夜霞
Owner PEOPLES HOSPITAL PEKING UNIV
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