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Anti-GLIPR-2 monoclonal antibody hybridoma cell and anti-GLIPR-2 monoclonal antibody generated from same

A hybridoma cell and monoclonal antibody technology, applied in the field of anti-GLIPR-2 monoclonal antibodies, can solve the problems of unclear molecular mechanism and lack of test tools, and achieve the effects of stable cell chromosomes and high antibody titers

Inactive Publication Date: 2014-04-02
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular mechanism of the role of GLIPR-2 in fibrosis is still unclear, mainly due to the lack of effective experimental tools, especially the specific antibody against GLIPR-2

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Immunization of mice

[0027] 1. Materials

[0028] Antigen: GLIPR-2 protein, according to the literature "Cloning and Soluble Expression of Human GLIPR-2 Gene in Prokaryotes" (Huang Shaoguang, Li Qiang, Niu Qin, Ni Danni, Pu Xiaoyun, Immunological Impurities, Volume 26, Issue 5, May 2010 ) method of preparation;

[0029] Experimental animals: 6-week-old, 17g BALB / C mice (provided by the Experimental Animal Center of Third Military Medical University);

[0030] Reagents: Freund's complete adjuvant and incomplete adjuvant (purchased from Promega);

[0031] 2. Method

[0032] BALB / C mice were immunized by intradermal injection of antigen in the hind leg muscles and back, firstly 100 μg with the same amount of Freund’s complete adjuvant, 100 μg with the same amount of Freund’s incomplete adjuvant after 3 weeks, and 500 μg without adjuvant after 7 weeks 4 days after intraperitoneal injection, prepare for cell fusion.

Embodiment 2

[0033] Example 2: Cell Fusion

[0034] 1. Materials

[0035] Myeloma cells: SP2 / 0 cells (provided by China Center for Type Culture Collection);

[0036] Medium: fetal bovine serum (purchased from Invitrogen); HAT selective medium (purchased from Sigma);

[0037] Feeder cells:

[0038] 2. Method

[0039] On the 4th day after intraperitoneal injection, splenocytes from BALB / C mice were fused with SP2 / 0 cells under the action of 50% polyethylene glycol. The fused hybridoma cells were mixed with HAT selective medium containing 20% ​​imported fetal bovine serum and added to a 96-well plate with feeder cells for culture. After 2 weeks, clone wells were selected under an inverted microscope.

[0040] Seven 96-well cell culture plates were inoculated with a total of 660 wells (except the control wells), of which 420 wells had clonal growth, and the fusion rate was 64% (420 / 660).

Embodiment 3

[0041] Embodiment 3: ELISA detection

[0042] GLIPR-2 protein coated ELISA plate, after sealing, add hybridoma cell culture supernatant and incubate at 37°C for 20min, after washing, add HRP-labeled goat anti-mouse IgG (working concentration 1:2000), incubate at 37°C for 20min, then wash. The reaction was terminated after adding o-phenylenediamine (OPD) substrate for color development for 15 minutes. At the same time, negative, positive and blank controls are set up to screen out hybridoma cells that can secrete antibodies. The results showed that 16 wells were positive for the antibody, and the positive rate was 2.4% (16 / 660).

[0043] Example 3: Cloning culture and chromosome identification

[0044] The positive hybridoma cells screened above were cloned and cultured multiple times by the limiting dilution method until the positive rate of cloned cell antibodies was 100%, and 10 hybridoma cells that could stably secrete specific antibodies were obtained, and one of the mos...

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Abstract

The invention relates to the technical field of biology, and discloses an anti-GLIPR-2 monoclonal antibody hybridoma cell and an anti-GLIPR-2 monoclonal antibody generated from the same. The collection number of the anti-GLIPR-2 monoclonal antibody hybridoma cell is CCTCC NO:C201221. The hybridoma cell can stably generate the anti-GLIPR-2 monoclonal antibody, has the advantages of stable cell chromosome and high antibody titer, and can be completely used in researching the molecular action mechanism of GLIPR-2.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-GLIPR-2 monoclonal antibody hybridoma cell and an anti-GLIPR-2 monoclonal antibody produced therefrom. Background technique [0002] GLIPR-2 (glioma pathogenesis related-2, GLIPR-2) belongs to the PR-1 (pathogenesis related-1) family and is commonly expressed in mammalian peripheral blood mononuclear cells and spleen. Recent studies have found that GLIPR-2 expression gene is increased in renal tubular epithelial cells in renal fibrosis, suggesting that it plays an important role in this pathological process. [0003] Renal fibrosis is a pathophysiological change, which is a gradual process in which the function of the kidney changes from healthy to damaged, then damaged, until the loss of function, mainly including renal interstitial fibrosis and glomerulosclerosis. In the past, it was believed that renal dysfunction was caused by glomerular lesions. However, since Schainuch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/18
Inventor 蒲晓允黄绍光蒋栋能刘飞
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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