A kind of metolachlor chiral hapten, artificial antigen and antibody and its preparation method and application
A metolachlor and artificial antigen technology, applied in chemical instruments and methods, thioether preparation, animal/human protein, etc., can solve problems such as accurate detection of metolachlor, achieve good detection effect and wide application Prospective, specific effects
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Embodiment 1
[0050] The synthesis of embodiment 1 metolachlor hapten
[0051] 1. Synthesis of metolachlor hapten
[0052] The synthetic route of small molecule hapten is as follows: figure 1 As shown, the specific reaction process is as follows:
[0053] (1) Add 0.4g of 3-mercaptopropionic acid (3-MPA) and 1g (about 1mL) metolachlor to a round bottom flask containing 50mL of ethanol, and then add 0.66g of KOH; Reflux and stir in an oil bath (80°C) for 6 hours;
[0054] (2) Use thin-layer chromatography to monitor the reaction process every 2 hours; the developer is: dichloromethane plus two drops of ammonia water, or n-hexane: ethyl acetate: acetic acid mixed in a volume ratio of 2:1:0.1 after the solution;
[0055] (3) After the reaction is complete, spin dry to about 5mL, inhale into a separatory funnel, add a small amount of ethyl acetate to wash the reaction flask; extract twice with saturated saline, pour the organic phase into flask A, and use HCl for the aqueous phase and to a ...
Embodiment 2
[0063] Example 2 Hapten separation and identification
[0064] 1. Splitting of haptens
[0065] The CHIRALPAK AD-H chiral column was used to resolve metolachlor derivatives, the mobile phase was n-hexane:ethanol:acetic acid=90:10:0.1, the flow rate was 1mL / min, the column temperature was 35°C, and the detection was performed at 220nm , split the spectrum as figure 2 shown.
[0066] 2. Identification of enantiomers
[0067] Dissolve the four haptens in acetonitrile and prepare 1 mg / mL solutions respectively. The Chirascan circular dichroism spectrum was used for measurement, scanning at 190-300 nm, with a bandwidth of 1 nm, a step of 1 nm, and a sampling time of 0.5 s to obtain the experimental ECD spectrum. Then through the Gaussian software calculation, the calculation map is obtained, and the comparison between the experiment and the calculation map is as follows: image 3 As shown, the order of the peaks can be determined as: αRS, αRR, αSS, αSR.
Embodiment 3
[0068] Synthesis and identification of embodiment 3 artificial antigen
[0069] 1. Synthesis of artificial antigen
[0070] (1) Accurately weigh 3.5 mg of metolachlor, dissolve in 200 μL of dimethylformamide (DMF), and make solution A;
[0071] (2) Add 10mg of bovine serum albumin (BSA) to 1.7mL of PBS (0.1moL / L, pH 7.4) to form solution B;
[0072] (3) Dissolve 4.22 mg of carbodiimide in 100 μL of dimethylformamide (DMF) to prepare liquid C;
[0073] (4) Add liquid A dropwise to liquid B, then slowly add liquid C dropwise, stir while adding, and react at -4°C for 9 hours;
[0074] (5) Dialyze with phosphate buffered saline for two days, 4 times a day, and obtain racemic metolachlor artificial antigen after dialysis.
[0075] Phosphate buffer (0.01M, pH7.4): Na 2 HPO 4 12H 2 O 2.90g, NaCl 8.50g, KCl 0.20g, KH 2 PO 4 0.20g, add deionized water to make up to 1000mL.
[0076] 2. Identification
[0077] The artificial antigen synthesized above was taken for ultraviolet ...
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