Ketoconazole hapten and artificial antigen as well as preparation methods and application thereof
A technology of ketoconazole and hapten, which is applied in the field of ketoconazole artificial antigen and its preparation, can solve the problems of expensive instruments, cumbersome operation, and damage to the liver and gallbladder system, and achieve the effects of high yield, high purity, and simple synthesis method
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Embodiment 1
[0034] Embodiment 1, the preparation of ketoconazole hapten
[0035] 1. Preparation of ketoconazole hapten
[0036] Add 1000mg of deacetylketoconazole to a 50ml round bottom flask, stir 25ml of pyridine for 15min to completely dissolve, then add 411.52mg of succinic anhydride at 20-24°C, and react with magnetic stirring at 20-25°C for 5 hours. Afterwards, the reaction solution was added dropwise to 100 ml of ice water to precipitate a white solid, filtered, the filter cake was washed with 25 ml of water, and the collected filter cake was air-dried at 50° C. for 5-6 hours to obtain 1144.25 mg of the ketoconazole hapten.
[0037] The reaction equation is as follows:
[0038]
[0039] 2. Structural identification of ketoconazole hapten
[0040] The resulting 1144.25 mg ketoconazole hapten was detected by mass spectrometry ( figure 1 ), the results show that its chemical structure is shown in formula I (MW=589.47), which is the hapten of ketoconazole.
[0041]
[0042] F...
Embodiment 2
[0043] Embodiment 2, preparation and structural identification of ketoconazole artificial antigen
[0044] 1. Preparation of ketoconazole artificial antigen
[0045] 1. Synthesis of immunogen
[0046] (1) Dissolve 26.39mg of ketoconazole hapten in 1.5mL DMF, stir at 200rpm for 10min, add 21.5mg of EDC to dissolve, then add 13mg of NHS, stir at room temperature (500rpm) to activate for 2-3h.
[0047] (2) Weigh 50mg of BSA and dissolve it in 3.5mL of 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve, cool down in an ice bath to 0-4°C, stir at 1000rpm, add the reaction solution in step 1 dropwise (1mL / min), stirring at 500rpm for 24h.
[0048] (3) Put the reaction product into distilled water and rinse the dialysis bag (10cm), dialysis with 1L of 0.01M PBS (1×, pH7.2) at 4°C (100rpm) for 3 days, and change the solution 3 times a day (morning, middle and evening) , changing the medium 9 times in total, centrifuge the dialysis product at 5000rpm for 6...
Embodiment 3
[0055] Embodiment 3, ketoconazole artificial antigen immunization animal prepares monoclonal antibody
[0056] 1. Animal immunity
[0057] Use the immunogen (ketoconazole-BSA) prepared in Example 2 at 100 μg per mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and inject subcutaneously on the back of the neck to immunize 6-8 week-old Balb / c Female mice, on the 7th, 14th, and 28th day after the initial immunization, mix the immunogen and Freund's incomplete adjuvant in equal volumes, each additional immunization once, and 3 days before the fusion, the immune complex is 100 μg / rat, without Freund's Adjuvant booster immunization again.
[0058] 2. Cell fusion and cloning
[0059] According to the conventional method, the splenocytes of the immunized mice were mixed with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then the preheated fusion agent (PEG4000) was slowly added within 45s for fusion...
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