Anti-GLIPR-2 monoclonal antibody hybridoma cell and anti-GLIPR-2 monoclonal antibody generated from same
A hybridoma cell and monoclonal antibody technology, which is applied in the field of anti-GLIPR-2 monoclonal antibody, can solve the problems of lack of test tools and unclear molecular mechanism, and achieve the effect of high antibody titer and stable cell chromosome
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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 muscle and back, firstly 100 μg with the same amount of Freund’s complete adjuvant, 3 weeks later with 100 μg with the same amount of Freund’s incomplete adjuvant, and 7 weeks later with 500 μg without adjuvant 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 above-screened positive hybridoma cells 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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