A dihydrocapsaicin artificial hapten, artificial antigen and preparation method thereof
A dihydrocapsaicin and artificial hapten technology, applied in the field of immunochemistry, can solve the problems of long cycle, high detection cost, complicated operation, etc., and achieve the effect of high affinity, high sensitivity and strong specificity
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Embodiment 1
[0035] Preparation of artificial hapten of dihydrocapsaicin
[0036] Weigh 5.86g of N-(4-hydroxy-3-methoxybenzyl)nonanamide, 2.36g of maleic anhydride and 0.49g of N,N'-dicyclohexylcarbodiimide, and put 25mL of dichloromethane in React in the reaction bottle at room temperature for 0.5h, then place the above reaction solution in an ice bath to control the temperature within the range of 0-5°C, take 0.30g of 4-dimethylaminopyridine and 4mL of dichloromethane and place it in a constant pressure drop Mix well in the funnel, slowly add dropwise to the reaction bottle, and the dropwise addition is completed in 0.5h. After the dropwise addition is completed, keep the room temperature at 25°C and continue to stir for 6 hours, filter the reaction solution, and distill off the solvent under reduced pressure to obtain a light yellow oil. Thick product, then this crude product is passed through silica gel column chromatography, and column chromatography reagent: petroleum ether and ethyl...
Embodiment 2
[0040] Preparation of Dihydrocapsaicin Artificial Antigen
[0041]Weigh 6 mg of dihydrocapsaicin hapten (E)-4-[2-methoxy-4-(nonamidomethyl)phenoxy]-4-oxo-2-butenoic acid, and then weigh 10 mg of NHS, Dissolve 0.2mL DMF in the reaction device, and carry out the reaction at room temperature (25-27°C) for 1 hour under magnetic stirring; weigh 20 mg of DCC and dissolve it in 200 uLDMF, add dropwise to the above reaction device, react at room temperature for 4 hours, a white precipitate is formed, and stand still Overnight, 8000r / min, 5min centrifugation, take the supernatant;
[0042] The supernatant was slowly added dropwise to 10ml of 2mg / mL bovine serum albumin in phosphate buffered saline solution and stirred at room temperature for 12h; dissolved in saline buffer. Then dialyze with 0.01MPBS buffer solution for 72 hours, and change the dialysate every 4-8 hours to obtain the artificial antigen of dihydrocapsaicin. For the continuous scanning spectrum of UV-Vis spectrum, see...
Embodiment 3
[0044] Preparation of antiserum against dihydrocapsaicin
[0045] Taking 400uL of the dihydrocapsaicin artificial antigen prepared in Example 2 as the immunogen, mixed with an equal volume of Freund's complete adjuvant and emulsified completely, immunized 4 Balb / c mice, and injected subcutaneously at multiple points on the back. Freund's complete adjuvant was used for the first immunization, followed by incomplete adjuvant emulsification, each 100ug / time, the interval between the first immunization and the second immunization was 4 weeks, and the interval between each subsequent immunization was 3 weeks, a total of 4 immunizations. From the second immunization, blood was collected from the tail of the mice 7-10 days after each immunization, about 25 μL per mouse, and the blood samples were placed at 37°C for half an hour, centrifuged at 5000 r / min for 1 min, and the supernatant was mixed with an equal volume of glycerol. Store at -20°C for later use for titer testing.
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