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Hybridoma cell capable of secreting anti-cystatin S monoclonal antibody as well as monoclonal antibody and application of hybridoma cell

A technology of monoclonal antibody and hybridoma cells, which is applied in the field of chemistry, can solve the problems of poor specificity, low sensitivity, and a ratio of less than 3%, and achieve the effect of high specificity and good sensitivity

Inactive Publication Date: 2015-04-29
SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Shanghai, the screening rate for fecal occult blood is less than 5%, and the proportion of those who find problems and then undergo colonoscopy is even less than 3%.
But so far, the monoclonal antibody of Cystatin S has low sensitivity and poor specificity in detecting intestinal cancer, so it is urgent to obtain a specific antibody against the epitope of Cystatin S to improve the sensitivity and specificity of detection

Method used

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  • Hybridoma cell capable of secreting anti-cystatin S monoclonal antibody as well as monoclonal antibody and application of hybridoma cell
  • Hybridoma cell capable of secreting anti-cystatin S monoclonal antibody as well as monoclonal antibody and application of hybridoma cell
  • Hybridoma cell capable of secreting anti-cystatin S monoclonal antibody as well as monoclonal antibody and application of hybridoma cell

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, recombinant protein expression, purification and identification of Cystatin S

[0028] Using RT-PCR method from gastric cancer tissue, using the upstream primer of cloning primer: F: 5'-cccaagctt g ccaccatggcccggcctctg-3' (SEQ ID NO.1); the downstream primer is: 5'-cgcggatccttcttgacacctggaa-3' (SEQ ID NO.2) to clone the Cystatin S gene (SEQ ID NO.3), and then insert the cloned Cystatin S gene On the pc DNA3.1 vector containing His tag, Cystatin S-pc DNA3.1 was obtained. Then transform Cystatin S-pc DNA3.1 into DH5α, pick positive clones, expand and culture the picked positive clones, extract the recombinant plasmid Cystatin S-pc DNA3.1, and then perform enzyme digestion analysis with HindⅢ and BamHI, the results show Enzyme-digested fragments with the expected size were obtained. The correct recombinant Cystatin S-pc DNA3.1 was sequenced to confirm that the gene was correct, and then transfected into cos-7 cells with Lipofectamine2000. The positive clone...

Embodiment 2

[0030] Example 2, Preparation, Purification and Identification of Cystatin S Monoclonal Antibody

[0031]The Cystatin S recombinant protein purified in Example 1 was used to immunize BALB / c mice by subcutaneous injection. Each mouse was immunized with 50 μg of antigen each time, and the immune volume was 100 μl. The first immunization with cystatin S mixed with Freund's complete adjuvant 1:1 was immunized once every two weeks, and cystatin S was mixed with Freund's incomplete adjuvant 1:1 for a total of 3 immunizations; finally, after Cystatin S (without adjuvant ) intraperitoneal booster immunization (30 μg), 3 days later, the spleen cells of the immunized mice were taken, and then the spleen cells of the immunized mice and the myeloma cell line SP2 / 0 of the mouse were fused with PEG-4000. Hybridoma cells were selected on the culture plate, and cells producing anti-Cystatin S protein (Cystatin S monoclonal antibody) were identified by ELISA method; hybridoma cell line 5D2F2, ...

Embodiment 3

[0039] Example 3 Preparation, purification and identification of Cystatin S polyclonal antibody

[0040] Immunize rabbits with the Cystatin S recombinant protein purified in Example 1, 500 μg each, once every two weeks, three times in total; take the whole blood of the rabbits, and first use the ammonium sulfate precipitation method to preliminarily purify the immunoglobulin IgG from the rabbit serum , the specific steps are: first, centrifuge the rabbit serum at 4°C and 12000rpm for 20 minutes, and collect the supernatant; add an equal volume of saturated ammonium sulfate solution dropwise, stir at room temperature for 30 minutes, and then place it in a refrigerator at 4°C for 2 hours. During this process, a large amount of protein precipitated out. Centrifuge the solution at 12000 rpm for 20 minutes at 4°C, discard the supernatant, redissolve the precipitate with 4 times the original volume of serum in phosphate buffer, and centrifuge at 12000 rpm for 2 minutes at 4°C. Disc...

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Abstract

The invention discloses a hybridoma cell capable of secreting an anti-Cystatin S monoclonal antibody as well as a monoclonal antibody and an application of the hybridoma cell. The hybridoma cell strains comprise 5D2F2 and 5E4G5 and are preserved in China Center for Type Culture Collection in Wuhan University on June 17, 2014, and the preservation numbers are CCTCC NO:C201416 and CCTCC NO:C201415. The hybridoma cell can secrete the Cystatin S monoclonal antibody, can be specifically combined with Cystatin S recombinant protein, has high potency and good affinity, can be used for specifically detecting Cystatin S and can be used for intestinal cancer diagnosis.

Description

technical field [0001] The invention belongs to the field of chemistry, and specifically relates to a hybridoma cell secreting anti-Cystatin S monoclonal antibody, and also relates to the monoclonal antibody secreted by the hybridoma cell and its application. Background technique [0002] During the 17th National Cancer Prevention and Propaganda Week, the Beijing Municipal Health Bureau reported through the media that the annual incidence of colorectal cancer in Beijing, Shanghai and other big cities has reached 30 / 100,000-40 / 100,000, reaching or exceeding the average level of Western developed countries , Colorectal cancer has become the second most common malignant tumor after lung cancer. The rise of colorectal cancer in my country has three characteristics—younger, more rectal cancer, and more low rectal cancer. The age of high incidence of colorectal cancer is generally 50-60 years old, which is 10 years earlier than Western countries on average. [0003] At present, f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K16/00C12N5/20G01N33/577G01N33/574C12R1/91
Inventor 王弢周小进孙玉龙秦勇
Owner SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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