Monoclonal antibody capable of recognizing Alternaria tenuissima and hybridoma cell strain AtA8 of monoclonal antibody capable of recognizing Alternaria tenuissima
A hybridoma cell line and monoclonal antibody technology, applied in the field of animals, can solve problems such as agricultural production losses, and achieve the effect of strong specificity and good development and application prospects
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Embodiment 1
[0018] Embodiment 1: Preparation of hybridoma cell lines
[0019] (1) Antigen preparation: pick single colonies of Alternaria tenoides and inoculate them in potato dextrose liquid medium respectively, shake culture at 25°C for 4-5 days, collect spores and hyphae in a 50mL centrifuge tube, 6000r Centrifuge at 6000r / min for 20min, wash twice with PBS, ultrasonically crush (power 200W, crush for 2s, intermittent for 2s), centrifuge the crushed solution at 6000r / min for 20min, collect the supernatant, and measure the supernatant protein content with the Coomassie brilliant blue method , adjust the protein concentration to 1000 μg / mL, as the initial antigen for immunization antigen and later detection, aliquot the antigen solution in a small amount and store it 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 with an equal...
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 by the AtA8 cell line is a monoclonal antibody that can specifically recognize Alternaria tengella.
Embodiment 3
[0025] Example 3: Titer detection experiment of monoclonal antibody
[0026] Antibody titer was determined by indirect ELISA method. Dilute 1000 μg / mL Alternaria tenugarii antigen 1000 times with coating solution and coat the whole microplate plate (1 μg / mL), overnight at 4°C, let it adsorb to the wells of the polystyrene plate, wash with PBST After three times, it was blocked with skim milk for 60 min. Add monoclonal antibody AtA 8-fold dilution into the coated wells, add 100 μL to each well, wash at 37°C for 1 h, wash three times with PBST, add horseradish peroxidase-labeled rabbit anti-mouse (Sigma Company) diluted 5000 times according to the instructions, 100 μL wells, 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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