Hapten for isoprothiolane content detection and preparation method and application thereof
A technology of rice blastin and hapten, which is applied in chemical instruments and methods, biological testing, measuring devices, etc., can solve the problem of lack of hapten, etc., and achieve the effect of strong specificity, efficient detection method, and good affinity
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Embodiment 1
[0061] Preparation of the above-mentioned hapten comprises the following steps:
[0062] Take 1.88g of diisopropyl malonate, add 20mL of carbon disulfide, stir at room temperature, mix thoroughly, add 13mL of 2mol / L KOH solution, stir at room temperature for 1h, then add 1.56g of 3,4-dichlorobutyric acid, add 2mol / L KOH solution 5mL, tetrabutylammonium bromide 3.8g, acetonitrile 80mL, heat to reflux for 4h, stop the reaction, rotary evaporate, remove acetonitrile and carbon disulfide, add water 80mL, add 6mol / L HCl to adjust the pH value to 6, add acetic acid Ethyl ester 100mL×3 extracted three times, combined the organic phases, washed with water, dried over anhydrous sodium sulfate and evaporated to dryness to obtain a yellow oil, put it on a silica gel column, and eluted and separated it with a mixed solution of petroleum ether-ethyl acetate with a volume ratio of 3:1. After purification, 0.76 g of acetic acid-blastidin was obtained, which was the above-mentioned rice blas...
Embodiment 2
[0064] The preparation of the antigen for the detection of rice blastin content comprises the following steps:
[0065] Take 12.9 mg of the hapten prepared in Example 1, add 1 mL of tetrahydrofuran to dissolve and clarify, add N-hydroxysuccinimide (NHS) 8.5 mg, 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride (EDC) 14.8 mg, fully dissolved and mixed, reacted at room temperature for 2 hours to obtain hapten activation solution A liquid; take 50 mg of bovine serum albumin (BSA), add 0.1mol / L CB9.1 8mL to dissolve, and obtain Liquid B: Add liquid A dropwise to liquid B, react at room temperature for 4 hours, stop the reaction, dialyze and purify with 0.02mol / L PBS buffer for 3 days, change the liquid 3 times a day, and centrifuge to obtain the bovine serum albumin conjugate. Linked rice blastin artificial antigen, aliquoted and stored at -20°C.
Embodiment 3
[0067] The preparation of the antigen for the detection of rice blastin content comprises the following steps:
[0068] Take 7.7 mg of the rice blastin hapten prepared in Example 1, add 1 mL of dimethyl sulfoxide (DMSO) to dissolve and clarify, add NHS 5.1 mg, EDC 9.7 mg, fully dissolve and mix, and react at room temperature for 2 hours to obtain a hapten activation solution Liquid A; take 50 mg of ovalbumin (OVA), add 0.1mol / L CB9.1 7mL to dissolve, and obtain liquid B; add liquid A dropwise to liquid B, react at room temperature for 4 hours, stop the reaction, and use 0.02mol / L L PBS buffer was purified by dialysis for 3 days, the medium was changed 3 times a day, and centrifuged to obtain the artificial antigen of rice blastilin coupled with ovalbumin, which was aliquoted and stored at -20°C.
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