A kind of triptolide hapten and its preparation method and application
A technology for triptolide and hapten, which is applied in chemical instruments and methods, peptides, immunoglobulins, etc., can solve the problems of difficulty in applying rapid diagnosis and promotion of poisoning reactions, and cumbersome pretreatment of test samples, and achieves good application prospects. , the effect of high practical value and simple preparation process
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Embodiment 1
[0055] Synthesis and identification of embodiment 1 triptolide hapten
[0056] 1. Synthesis of triptolide hapten
[0057] Weigh 100mg triptolide and dissolve it in 10mL anhydrous dichloromethane, add 300mg succinic anhydride and 100mg 4-dimethylaminopyridine (DMAP) to the above solution, stir the reaction system at room temperature for 12 hours, then pour the reaction system into The separatory funnel was washed three times with dilute hydrochloric acid, the organic phase was collected, dried and concentrated. The white target compound was obtained by column chromatography using ethyl acetate:petroleum ether=1:1 system as developing solvent.
[0058] 2. Identification of triptolide hapten structure
[0059] The triptolide hapten was identified by mass spectrometry, and the identification results are shown in figure 2 . It can be clearly seen that [M+H] + Peak: m / z461.18005 (the error is -2.41ppm, its theoretical accurate mass number is 461.18116), and its elemental composi...
Embodiment 2
[0061] Preparation and Identification of Example 2 Triptolide Antigen
[0062] The triptolide hapten prepared in Example 1 was coupled with BSA and OVA respectively to obtain artificial antigens TPL-BSA and TPL-OVA, wherein TPL-BSA was used as an immunogen and TPL-OVA was used as a coating agent.
[0063] 1. Preparation and identification of triptolide immunogen
[0064] 1. Weigh 20 mg of hapten and dissolve it in 500 μL of DMF to obtain hapten solution.
[0065] 2. Take the hapten solution prepared in step 1, add 30 mg DCC and 20 mg NHS, place it on a magnetic stirrer, and react at room temperature at 300 rpm for 5 hours.
[0066] 3. Take 60 mg of BSA and dissolve it in 10 mL of PBS buffer to obtain a protein solution.
[0067] 4. Add the liquid phase completed in step 2 dropwise to the protein solution prepared in step 3, stir with a magnetic stirrer while adding dropwise, and react for 10 hours. Then it was transferred to a dialysis bag with a molecular weight cut-off of...
Embodiment 3B
[0074] The immunization of embodiment 3Balb / c mice
[0075] The protein concentration of TPL-BSA was determined by Coomassie brilliant blue (Bradford) method protein concentration assay kit.
[0076] For the first immunization, the TPL-BSA immunogen was diluted to 1mg / mL (diluted with 0.01mol / L PBS), mixed with an equal volume of Freund’s complete adjuvant, and fully emulsified, and multi-point subcutaneously inoculated with 6-week-old Balb / c on the back of the neck. Five mice were inoculated with an antigen dose of 100 μg / mouse, and a mouse injection dose of 0.2 ml / mouse. Afterwards, booster immunization was performed every 21 days, and the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant during booster immunization. The immunization dose of the immunogen was the same as that of the first immunization, and the number of booster immunizations was 3 times. Two weeks after the third booster immunization, the mice were enucleated to collect blood fr...
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