Thidiazuron antigen and preparation method and application thereof
A thidiazuron and antigen technology, which is applied in the field of thidiazuron antigen and its preparation, can solve the problems of inapplicability for rapid detection, expensive instruments and equipment, and high detection cost, and achieves simple and clear synthesis steps, low synthesis cost and good effect. Effect
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Embodiment 1
[0045] Example 1. Preparation of thiadiuron-ovalbumin (thiadiuron-OVA) antigen
[0046] Synthetic roadmap as figure 1 shown.
[0047] 1) Synthesis of compounds shown in formula I
[0048] Add 5-amino-1,2,3-thiadiazole (12mmol) and 30mL toluene into a three-necked flask, stir and dissolve, then add 4-isocyanato-benzoic acid ethyl ester (10mmol), continue the reaction at 70°C for 24h , TLC detected that the reaction of the raw materials was basically complete, then suction filtered, and the solid part was washed several times with a small amount of toluene to obtain a milky white powdery solid.
[0049] 1 H-NMR(DMSO):1.29(t,CH 3 ,3H),4.30(m,CH 2 ,2H),7.65(d,CH,2H),7.93(d,CH,2H),8.69(s,CH,1H),9.88(s,NH,1H),11.01(s,NH,1H).
[0050] Product by 1 Confirmed by H-NMR, it is the compound shown in formula I.
[0051]
[0052] Formula I
[0053] 2) Synthesis of compounds represented by formula II
[0054] Add the compound represented by formula I (5mmol) into concentrated hy...
Embodiment 2
[0069] Example 2, Preparation of Thidiazuron-Bovine Serum Albumin (Thiadizuron-BSA) Antigen
[0070] The reaction scheme is as follows figure 1 shown.
[0071] 1) The synthesis and activation of the thiadizuron hapten 4-(3-(2-chloro-4-pyridine)-urea)-benzoic acid are the same as those in Example 1, and will not be repeated here.
[0072] 2) slowly drop the compound shown in formula III obtained in step 3) of Example 1 into the carrier protein solution (the carrier protein solution is 157.5mg BSA dissolved in 10mL phosphate buffer saline (PBS) with a pH value of 7.5), Formula B compound 4-(2-(3-bromo-1-(3-chloro-2-pyridyl)-1H-pyrazole-5-carboxamido)-5-chloro-3-methylbenzamido ) The molar ratio of butyric acid to carrier protein was 15:1, and stirred overnight at 4°C.
[0073] 3) Dialysis: Dialyze the reaction liquid obtained in step 2) with a PBS solution with a pH value of 7.5 and a concentration of 0.1 mol / L for three days, and dilute the dialyzed reaction product solution...
Embodiment 3
[0077] Example 3, application of thiadiuron-bovine serum albumin (thiadiuron-BSA) antigen
[0078] 1. Antibody preparation using thiadiuron-bovine serum albumin (thiadiuron-BSA) antigen
[0079] (1) Take Bal b / c mice aged 8-10 weeks as experimental animals.
[0080] (2) Basic immunization: The diluted thiadizuron-BSA antigen solution (concentration: 1 mg / mL) obtained in Example 2 was filtered through a sterile filter and added with an equal volume of Freund's complete adjuvant, and the mixture was stirred with a magnetic stirrer. Stir well to emulsify until dripping into water does not spread. Bal b / c mice were injected subcutaneously with emulsified complete antigen at multiple points in the abdominal cavity and back, and the injection dose was 0.1 mg emulsified antigen / mouse.
[0081] (3) Booster immunization: 2 weeks after the basic immunization, take 1 mL of the above-mentioned diluted thiadizuron-BSA antigen solution, then add 1 mL of Freund's incomplete adjuvant, stir ...
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