Monoclonal Antibody Recognizing Alternaria and Its Hybridoma Cell Line aac5
A hybridoma cell line and monoclonal antibody technology, applied in the field of animals, can solve problems such as agricultural production losses, and achieve highly specific effects
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Embodiment 1
[0018] Embodiment 1: Preparation of hybridoma cell lines
[0019] (1) Pick single colonies of Alternaria and inoculate them in potato dextrose liquid medium respectively. After shaking and culturing at 25°C for 4-5 days, collect spores and hyphae in a 50mL centrifuge tube, centrifuge at 6000r / min for 20min, and use After washing with PBS for 2 times, ultrasonic crushing (power 200W, crushing 2s, intermittent 2s), the crushed solution was centrifuged at 6000r / min for 20min, the supernatant was collected, and the supernatant protein content was measured by the Coomassie brilliant blue method, and the protein concentration was adjusted to 1000μg / mL, as the initial antigen for immunization and later detection, the antigen solution was aliquoted in a small amount and stored in a freezer at -80°C. Take a small amount and store at -20°C before use.
[0020] (2) Three healthy BaL b / c mice aged 10-12 weeks were selected and injected intraperitoneally with 200 μL of antigen emulsified...
Embodiment 2
[0023] Embodiment 2: the production of monoclonal antibody
[0024] Take BaL b / c mice about 8 weeks old, inject 0.3mL pristane intraperitoneally, and inject 5-10×10 5 7-10 days after injection, the peritoneal cavity of the mouse was obviously swollen. The ascites was collected, centrifuged at 2000r / min for 3min, and the supernatant was collected, which was the ascitic monoclonal antibody. Monoclonal antibodies were purified by Protein A column chromatography and stored at -80°C. The monoclonal antibody prepared from the AaC5 cell line is a monoclonal antibody that can specifically recognize Alternaria.
Embodiment 3
[0025] Example 3: Titer detection experiment of monoclonal antibody
[0026] Antibody titer was determined by indirect ELISA method. Dilute 1000μg / mL Alternaria antigen 1000 times with the coating solution and coat the whole plate (1μg / mL), overnight at 4°C, make it adsorb to the wells of the polystyrene plate, wash with PBST three times Block with skimmed milk for 60 min. Add monoclonal antibody AaC 5-fold dilution to the coated wells, add 100 μL to each well, wash at 37°C for 1 h, wash three times with PBST, then add 100 μL of horseradish peroxidase-labeled rabbit anti-mouse (Sigma Company) diluted 5000 times according to the instruction manual, 37 ℃1h, after washing with PBST, add OPD substrate chromogenic solution to develop color, use 50μL 2M H 2 SO 4 After terminating the reaction, read the OD with a microplate reader 490nm , To determine the titer of monoclonal antibody ascites with the ratio of negative to positive greater than 2.
[0027] After testing, it was de...
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