[0072] Example 1 Preparation of Deoxynivalenol Hapten, Complete Antigen, Monoclonal Antibody, Goat Anti-mouse Secondary Antibody
[0073] 1. Preparation of vomitoxin hapten
[0074] The synthetic route of vomitoxin hapten of the present invention is as follows Figure 4 As shown, it specifically includes the following steps:
[0075] (1) Weigh 23.2 mg of vomitoxin in advance, add 10 mL of ethylene glycol dimethyl ether, dissolve it by ultrasonic, and set aside;
[0076] (2) Weigh 0.6g of sodium hydride (because sodium hydride contains kerosene, use 20mL of petroleum ether to stir magnetically for 30s, let it stand for 2min, pour off the petroleum ether; repeat the cleaning 2 to 3 times; after pouring off the petroleum ether for the last time, Blow dry with nitrogen);
[0077] (3) Add dissolved vomitoxin in sodium hydride, stir for 1 hour, add 0.5 g of succinic anhydride, stir for 3 hours, after reacting for 1 hour, slowly add 4 mL of methyl 3-bromopropionate dropwise, react for 3 hours, and obtain the product, Carry out extraction purification again, obtain the compound shown in formula (I):
[0078]
[0079]In the preparation of the vomitoxin hapten, in order to verify whether the hydrolysis is successful, the product of the above step (3) and vomitoxin are spotted together (chloroform:methanol=30:1); evaporate after filtration; add 2g lithium hydroxide and 15mL Three-grade water, stirred for 1 hour, if the dissolution is relatively clear, it proves that the hydrolysis is successful. After the hydrolysis is successful, add water and ethyl acetate to the hydrolyzate to extract 2 to 3 times, and take the product in the water layer; add concentrated hydrochloric acid to the product in the water layer dropwise until the pH value is 4 to 5. After the precipitated product appears, use ethyl acetate Ester extracts the product in the water layer back; finally, use ethyl acetate and anhydrous sodium sulfate as water removal agents to remove water, and rotary evaporation to obtain the deoxynivalenol hapten shown in formula (I) after purification.
[0080] 2. Preparation of complete antigen (coating source, immunogen)
[0081] (1) Preparation of coating original
[0082] 1) Dissolve 10mg of vomitoxin hapten in 1mL of dimethylamide (DMF); take 20mg of carbodiimide (EDC), fully dissolve it in 0.2mL of DMF, add it to the hapten solution, and stir at room temperature for 8 hours. Obtain reaction solution A;
[0083] 2) Weigh 36 mg of OVA and fully dissolve it in 3 mL of 0.1 mol/L carbonate buffer solution (pH=9.6) to obtain an OVA protein solution;
[0084] 3) The reaction solution A was slowly added dropwise to the OVA protein solution, and stirred at room temperature for 16 hours in the dark, and dialyzed with 0.01mol/L PBS at 4°C for 3 days, and the dialysate was changed twice a day to obtain the packaged solution. Be original.
[0085] (2) Preparation of immunogen
[0086] 1) Dissolve 10mg of vomitoxin hapten in 1mL of dimethylamide (DMF); take 20mg of carbodiimide (EDC), fully dissolve it in 0.2mL of DMF, add it to the hapten solution, and stir at room temperature for 8 hours. Obtain reaction solution A;
[0087] 2) Weigh 36 mg of BSA and fully dissolve it in 3 mL of 0.1 mol/L carbonate buffer solution (pH=9.6) to obtain a BSA protein solution;
[0088] 3) Slowly add the reaction solution A to the BSA protein solution drop by drop, and stir at room temperature for 16 hours in the dark, dialyze with 0.01mol/L PBS at 4°C for 3 days, change the dialysate twice a day, and obtain the immune Original.
[0089] 3. Preparation of DON Monoclonal Antibody
[0090] a. Animal immunization: inject the immunogen obtained in step 2 into BALB/c mice with an immunization dose of 100 μg/mouse to produce antiserum;
[0091] b. Cell fusion and cloning
[0092] Booster immunization: Booster immunization of mice once three days before formal cell fusion, injection concentration: 1mg/mL, injection volume: 100μL, no adjuvant required;
[0093] Prepare feeder cells: One day before formal cell fusion, take 1 Kunming mouse, kill it by bloodletting eyeballs, place it on a dissecting plate, take pleural macrophages and splenocytes as feeder cells, mix them with about 75mL of HAT medium, and add them to a multi-well culture plate , adjust the concentration at 2×10 5 cells/mL, take 24-well culture plate, 1 mL/well. Observe the growth status of the feeder cells before use the next day. If there are any abnormal phenomena such as bacterial contamination, they should be discarded.
[0094] Preparation of splenocyte liquid: first add 3mL of basal medium to a small cell dish, put a cell sieve on it, take out the spleen and put it in a cell sieve, grind the spleen with a syringe plunger; collect spleen cells into a 15mL centrifuge tube, and use RPMI-1640 basic Dilute the medium to 10mL and seal it; centrifuge at 1000rpm for 7min and discard the supernatant; take out Tris-ammonium chloride from the incubator, first add 1mL Tri-ammonium chloride, blow it gently, then add 2mL Tris-ammonium chloride to mix Homogenize (to lyse red blood cells), seal, and incubate at 37°C for 7 minutes; add RPMI-1640 basal medium to make up to 10 mL, seal and centrifuge at 1000 rpm for 7 minutes, discard the supernatant; resuspend to 10 mL with RPMI-1640 basal medium to obtain spleen cell fluid ,spare.
[0095] Preparation of myeloma cell liquid: collect myeloma cells into another 15 mL centrifuge tube, seal, centrifuge at 1000 rpm for 7 min, discard supernatant, resuspend with basal medium, and dilute to 10 mL to obtain myeloma cell liquid.
[0096] Mixed cell solution: mix spleen cell solution and myeloma cell solution in a 50mL centrifuge tube, add basal medium to 20mL, mix well, seal, centrifuge at 1000rpm for 7min, discard the supernatant, and suck up with a gun with the mouth of the centrifuge tube facing down excess medium.
[0097] Preparation for cell fusion: Gently tap the pelleted cell mass at the bottom of the centrifuge tube with your fingers to loosen the pellet and mix well, seal the tube, incubate at 37°C for 10 minutes, and prepare a timer for 4 minutes.
[0098] Fusion of cells: Take out PEG and 10mL complete medium from the incubator; add PEG within the first 1min, with gentle movements, accompanied by gentle stirring, do not blow away, so that the PEG is separated; seal, incubate at 37°C for 10min, and centrifuge at 800rpm 10min, discard the supernatant.
[0099] Plating: Take out the HAT from the incubator, add a small amount of HAT medium, use a pipette to gently absorb and release the liquid, stir gently, and then adjust the volume to 75mL; add it to a 24-well plate with feeder cells, 1mL/well, place Cell culture incubator.
[0100] Culture after fusion: check whether it is necessary to add HAT culture medium on the 4th day; change the medium for the first time on the 7th day, absorb 1/2 of the culture medium when changing the medium, add an equal amount of fresh HT culture medium, and overuse HT.
[0101] Positive wells were cloned using the limiting dilution method until hybridoma cell lines that can stably secrete monoclonal antibodies.
[0102] (3) Cell cryopreservation and recovery
[0103] Prepare hybridoma cells with cryopreservation solution to 1*10 6 cells/mL for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it in a 37°C water bath for instant dissolution, remove the cryopreservation solution by centrifugation, and transfer it to a culture bottle for culture.
[0104] (4) Preparation and purification of monoclonal antibodies
[0105] After the hybridoma cell lines are established, monoclonal antibodies can be produced in large quantities as needed. Ascites collection method: first inject 500 μL of liquid paraffin or pristane into the peritoneal cavity of BALB/c mice, and then inject 1×10 6 About 10 days after inoculation with hybridoma cells, abdominal swelling of the mice was seen. The mice were sacrificed before the mice died, and the ascites was extracted. The ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain the final vomitoxin monoclonal antibody.
[0106] 4. Preparation of goat anti-mouse secondary antibody
[0107] Goats were used as immunized animals and mouse-derived antibodies were used as immunogens to immunize pathogen-free sheep to obtain goat anti-mouse secondary antibodies.
