Synthetic method and application of carbofuran amination hapten
A synthesis method and hapten technology, applied in chemical instruments and methods, organic chemistry, animal/human proteins, etc., can solve problems such as harm to human health, environmental pollution, etc., to meet the needs of detection, and the effect of simple and effective reaction synthesis steps
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Embodiment 1
[0021] Example 1 Preparation of Budweiser Aminated Hapten.
[0022] (1) Place the four-neck flask on a magnetic stirrer and configure a thermometer. Dissolve 2.6g of furanol in 50mL of dichloromethane, add 1.7g of triethylamine under ice-cooling, stir for 10 minutes, dissolve 4.9g of bis(p-nitrophenyl)carbonate in 15mL of dichloromethane, slowly drop Into the above reaction system, gradually raise the temperature to 30°C and continue to stir for 1 hour, and monitor the completion of the reaction with a TLC plate. The developer is petroleum ether: ethyl acetate = 10:1, and the intermediate product (II) is obtained.
[0023] (2) Weigh 2.8g of 3-tert-butoxycarbonylaminopropylamine alkalized by triethylamine, dissolve it in 10mL of dichloromethane, and add it to the reaction solution of the above-mentioned intermediate product (II) under ice-bath conditions. Rise to room temperature and continue to stir overnight; wash with 50mL water and saturated brine twice respectively; after...
Embodiment 2
[0025] Example 2 Preparation of Budweiser artificial antigen.
[0026] (1) Weigh 21mg of hapten and 67mg of bovine serum albumin and dissolve it in 5mL of PBS buffer, stir it under magnetic force at room temperature until fully dissolved, react in the dark and add 14ul of 25% glutaraldehyde solution dropwise, and gently stir for 30 minutes Finally, sodium borohydride solution was added to the reaction solution to a final concentration of 10 mg / mL, and then reacted at 4°C in the dark for 2 hours to obtain an artificial antigen mixture.
[0027] (2) Transfer the artificial antigen mixture into the treated dialysis bag, dialyze with 0.01M PBS buffer solution under magnetic stirring at 4°C for 2 days, then dialyze with deionized water for 1 day, and change the dialysate 3 times a day; After the end, centrifuge to take the supernatant, and use the freeze-drying method to make it into powder to obtain the carbofuran artificial antigen: CAR-BSA.
Embodiment 3
[0028] Example 3 Preparation of Budweiser antiserum.
[0029] Use the artificial antigen prepared in Example 2 as the immunogen, mix it with Freund's complete adjuvant in an equal amount, select 2-month-old healthy female New Zealand white rabbits after emulsification is complete, and inject them subcutaneously at multiple points on the back of the neck, and boost the immunization once every 3 weeks . On the 7th day after the fourth immunization, blood was collected from the ear vein, and the antiserum was obtained by centrifugation.
[0030] Experimental results show that the antiserum obtained by immunizing rabbits with the carbofuran artificial antigen prepared by the present invention has a four-immunity titer of up to 128,000, and has good selective recognition ability for carbofuran.
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