Metalaxyl monoclonal antibody hybridoma cell strain and application thereof
A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, material inspection products, instruments, etc., can solve the problems of low sensitivity, difficulty in obtaining accurate results, and many interfering substances, and achieve good specificity, The effect of good detection sensitivity
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Embodiment 1
[0022] Example 1 Preparation of metalaxyl monoclonal antibody hybridoma cell line 20170507
[0023] (1) Synthesis of hapten:
[0024]
[0025] 2 g of compound 1 was dissolved in 200 mL of water, and then 200 mg of KOH was added to the solution. The mixture was stirred overnight at 100°C. Water was added and the aqueous layer was extracted with EA. The aqueous phase was acidified with 6 mol / L aqueous hydrochloric acid until pH = 7, and concentrated to dryness. The crude product was purified by chromatography on a silica gel cartridge to afford compound 2, the desired product, as a white solid.
[0026] (2) Synthesis of complete antigen:
[0027] Preparation of the immunogen MET-BSA: Weigh 8.0 mg of MET, 18 mg of 1-ethylcarbodiimide hydrochloride, 8.0 mg of N-hydroxysuccinimide, and add 400 μL of anhydrous N,N-dimethylformamide Dissolved and stirred at room temperature for 4-5h (referred to as liquid A). Take bovine serum albumin BSA 10mg, add 3mL boric acid buffer solu...
Embodiment 2
[0034] Example 2 Preparation and Identification of Metalaxyl Monoclonal Antibody
[0035] Take 8-10-week-old BALB / c mice, and inject 1 mL of sterile paraffin oil into each mouse; 7 days later, inject 1×10 6 Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by octanoic acid-ammonium sulfate method. Under acidic conditions, n-octanoic acid can precipitate other miscellaneous proteins in ascites except IgG immunoglobulin, then centrifuge, and discard the precipitate; then use an equal amount of saturated ammonium sulfate solution to precipitate IgG-type monoclonal antibodies, centrifuge, and discard The supernatant was dissolved with 0.01 M PBS solution (pH 7.4), dialyzed and desalted, and finally the purified monoclonal antibody was stored at -20°C.
Embodiment 3
[0036] Example 3 Application of Metalaxyl Monoclonal Antibody
[0037] (1) Coating: Dilute the coated original MET-OVA with 0.05M pH9.6 carbonate buffer starting from 1µg / mL, 100µL / well, and react at 37°C for 2h;
[0038] (2) Washing: Pour off the solution in the plate, and wash with washing solution 3 times, each time for 3 minutes;
[0039] (3) Blocking: After patting dry, add 200 μL / well blocking solution and react at 37°C for 2 hours. After washing, dry it for later use;
[0040] (4) Adding samples: Dilute the antiserum starting from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes; after fully washing, add 1:3000 diluted HRP - Goat anti-mouse IgG, 100 μL / well, react at 37°C for 30 minutes;
[0041] (5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react in the dark at 37°C for 15 minutes;
[0042] (6) Termination and measurement: Add ...
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