Monoclonal antibody for recognizing fusarium solani and hybridoma cell strain FsD4 of monoclonal antibody
A hybridoma cell line and monoclonal antibody technology, applied in the field of animals, can solve the problems of early diagnosis restricting the scientific and effective control of Fritillaria root rot, and the difficulty of field symptoms, and achieve good development and application prospects, strong specificity, and detection sensitivity high effect
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Embodiment 1
[0018] Embodiment 1: Preparation of hybridoma cell lines
[0019] (1) Pick single colonies of Fusarium solani and inoculate them in potato glucose liquid medium respectively, and culture them with shaking 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 emul...
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 FsD4 cell line is a monoclonal antibody that can specifically recognize Fusarium solani.
Embodiment 3
[0025] Example 3: Titer detection experiment of monoclonal antibody
[0026] Antibody titer was determined by indirect ELISA method. Dilute 1000 μg / mL Fusarium solani antigen 1000 times with the coating solution and coat the whole microplate (1 μg / 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. Monoclonal antibody FsD was diluted 4 times and added to the coated wells, 100 μL per well, 37 ° C, 1 h, washed three times with PBST, and then 100 μL of horseradish peroxidase-labeled rabbit anti-mouse (Sigma Company) diluted 5000 times according to the instructions was added to each well, 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] ...
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