Zonisamide derivative, homogeneous enzyme immunoassay reagent and preparation method
A technology of homogeneous enzyme immunoassay and zonisamine enzyme is applied in the field of zonisamide derivatives, homogeneous enzyme immunoassay detection reagents and preparation, and can solve the problem of high professional requirements of operators, high price of high-precision instruments, and microscopic latex. It can improve the detection sensitivity, be easy to implement and popularize and use, and achieve the effect of high accuracy.
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[0075] Example 1: Preparation of Zonisamide Derivatives
[0076] The synthetic route of zonisamide derivatives is as follows:
[0077] .
[0078] The preparation method of the zonisamide derivative comprises the following steps:
[0079] Compound 1 (7 g) was weighed and dissolved in toluene (70 ml), and then succinic anhydride (4 g) was added to prepare a reaction mixture solution, which was stirred overnight under reflux conditions. After the reaction, the above reaction mixture solution was concentrated, and then purified by flash chromatography to obtain 4 g of zonisamide derivatives.
[0080] pass 1 H NMR (Varian mercury plus 400 MHz) spectral scanning analysis (TMS as the internal standard) and LC-MS (Agilent 1200A) were used to identify the chemical structures of the obtained zonisamide derivatives. The results showed that the zonisamide derivatives were: That is, the zonisamide derivative whose structural formula is shown in formula (V).
[0081]
[0082] Form...
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[0083] Example 2: Preparation of zonisamide immunogen
[0084] The preparation method of zonisamide immunogen comprises the following steps:
[0085] (A1) Preparation of carrier solution: Dissolve the carrier protein in 0.2 mol / L potassium phosphate buffer (pH=8.5), and the final concentration of the carrier protein is 5.5 mg / mL to obtain a carrier solution;
[0086] (A2) Preparation of zonisamide derivative solution: 200 mg of the above zonisamide derivative, 4 mL of dimethylformamide, 4 mL of ethanol, 6 mL of potassium phosphate buffer (10 mmol / L, pH=7.0), 200 mg of 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and 60 mg of N-hydroxysulfosuccinimide were mixed, and stirred and dissolved for 120 min to obtain a zonisamide derivative solution;
[0087] (A3) Synthesis of zonisamide immunogen: The zonisamide derivative solution obtained in step (A2) was added to the carrier solution obtained in step (A1), and stirred overnight at 4°C, purified by dialysis, Zonisamide immunog...
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[0088] Example 3: Preparation of anti-zonisamide-specific antibodies
[0089] The preparation method of anti-zonisamide specific antibody comprises the following steps:
[0090] (B1) Dilute the above-mentioned zonisamide immunogen with 0.01mol / L sodium phosphate buffer (pH=6.5) to a final concentration of 2.0mg / mL to obtain an artificial antigen solution, and then mix the artificial antigen solution with an equal amount of Mixed with complete adjuvant, the experimental animals were injected into rabbits at multiple points;
[0091] (B2) After 3 weeks, the same artificial antigen solution and the same amount of incomplete Freund's adjuvant were used to inject the above experimental animal rabbits at multiple points, and then once every 3 weeks, for a total of 5 injections;
[0092] (B3) Blood is drawn from the experimental animal rabbits injected in step (B2), and separated and purified to obtain an anti-zonisamide specific antibody.
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