Organic chlorine pesticide benzoepin artificial antigen and antibody, their preparation and use thereof
A technology of artificial antigen and organochlorine, applied in the field of application in the establishment of immunoassay methods
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Embodiment 1
[0068] 1. Hapten Synthesis
[0069] Synthesis of Haptens A and B:
[0070] Add succinic anhydride (1-1.5mmol) to 1-hydroxychlordene (1-1.5mmol) in 2 mL of anhydrous pyridine solution, then add dimethylaminopyridine (DMAP), and stir the mixed system overnight. 30 mL of ethyl acetate was added, and the organic layer was washed with acid (HCl), water and brine, and dried over magnesium sulfate. The solution was concentrated, and the residue was passed through a silica gel column (methanol / chloroform / acetic acid 1-3:90-100:0.1-0.5) to obtain product 1 as a white solid.
[0071] 1) Add N-hydroxysuccinimide (0.5-1 mmol) to a solution of compound 1 (0.5-1 mmol) in 10 mL of DCM, and then add DMAP. The mixed system was stirred overnight, filtered and evaporated, and the residue was passed through a silica gel column (acetone / chloroform=1-5:5-10) to obtain a white solid: recrystallized from dichloromethane / petroleum ether to obtain hapten A.
[0072] 2) Treating 1-hydroxychlordene ac...
Embodiment 2
[0110] During the immunoassay, extract the samples using the methanol extraction method ① that is: use 10g of green grapes, carrots, spinach, tea, tobacco and other five samples as the test items, soak in 50ml of methanol and shake for 60 minutes, let it stand, and take it out. solution to obtain the sample to be tested. The resulting soaking solution is the sample to be tested. Others are with embodiment 1.
Embodiment 3
[0112] The detection method adopts the laboratory detection method (the test uses 8-well enzyme labeling strip):
[0113] Coating: Dissolve the prepared antibody in 50 mmol, pH 9.6 carbonic acid buffer to prepare a 10 mg / mL coating solution. Add 100 μl of coating solution to each well of the enzyme-labeled strip overnight. Each well of the coated enzyme-labeled strip was washed three times with PBST (phosphate buffer saline 0.05% (v / v), Tween 20) washing solution.
[0114] Blocking: add 150 μl of 1% bovine serum albumin (BSA) / PBS blocking solution to each well, and block for one hour.
[0115] ·Sampling: Dissolve the sample to be tested in PBS containing 1% BSA, and the endosulfan standard sample is also dissolved in PBS containing 1% BSA. When adding samples, add 50 μl of the above-mentioned sample or standard sample to each well.
[0116] Competitive reaction: dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the sample or standard sample to ...
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