Preparation method and application of hapten and antigen of butralin
A technology of Zhongbuling and hapten, which is applied in the field of preparation of Zhongbuling hapten and antigen, to achieve high sensitivity, high antibody specificity and rapid detection effect
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Embodiment 1
[0020] The preparation of embodiment 1 secbutaline hapten
[0021] 1. Synthesis of Zhongbutaline hapten (see the appendix for the synthetic route figure 1 )
[0022] Take 1.00 g of 4-chloro-3,5-dinitrophenylacetic acid, add 50 mL of ethanol to dissolve, add 0.45 g of sodium carbonate, stir, add 0.31 g of butylamine, heat in an oil bath at 80°C, stir for 3 h, and detect the raw material The basic reaction is complete; stop the reaction, cool to room temperature, rotary evaporate, remove ethanol, add 80 mL of water, adjust the pH value to 4 with 1 mol / L hydrochloric acid, there is a lot of turbidity, add 80 mL of ethyl acetate to extract, wash with water, and apply silica gel The column was eluted and separated with dichloromethane / methanol with a volume ratio of 5:1 to obtain 1.05 g of the hapten product of sec-butyrin, with a yield of 92.11%.
[0023] 2. Identification of Zhongbuling hapten
[0024] NMR identification 1 H NMR (CDCl 3 , 300MHz) δ: 11.01 (1H, d, J=0.000), 8...
Embodiment 2
[0026] Example 2 Preparation of Zhongbuling Antigen
[0027] 1. Synthesis of Zhongbuling immune antigen
[0028] The immunogen was obtained by conjugating the secbutaline hapten with bovine serum albumin (BSA).
[0029] Take 11 mg of sec-butyrin hapten, add 0.2 mL of dimethyl sulfoxide (DMSO) to dissolve, add 20 μL of triethylamine, stir, mix well, add 6 mg of isobutyl chloroformate, stir for 2 h, and obtain the hapten Activation solution A solution; take 50 mg BSA and add 0.1 mol / L PB buffer solution with a pH value of 7.2 to dissolve to obtain B solution, slowly add A solution to B solution, continue to stir for 5 h, stop the reaction, 0.02mol / L L PBS was dialyzed for 3 days, and the medium was changed 3 times a day to obtain the secbutamyl-BSA conjugate, which was the immunogen, which was aliquoted and stored at -20°C.
[0030] 2. Synthesis of Zhongbuling-coated antigen
[0031] The sec-butyrin hapten was coupled with ovalbumin (OVA) to obtain the coating source.
[003...
Embodiment 3
[0035] Example 3 Preparation of secbutaline monoclonal antibody
[0036] 1. Obtaining hybridoma cells
[0037] 1) First immunization: fully emulsify the secbutyrine hapten-BSA conjugate (immunogen) with the same amount of complete Freund's adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each;
[0038] 2) Booster immunization twice: from the first immunization, booster immunization once every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the method and dosage are the same as the first immunization;
[0039] 3) One week after the last booster immunization, the fundus vein blood was collected to measure the titer and inhibition. When there was inhibition and the titer reached more than 1:10000, the following last immunization was carried out: intraperitoneal injection of 0.1 mL of the immunogen solution without any adjuvant, and then executed three days later Mice, whose spleen was fused with myeloma cells;
[0040] 4) Us...
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