Spinosad hapten and preparation method and application thereof

A spinosyn and hapten technology, applied in the field of hapten and its preparation, can solve the problems of many processing steps and low recovery rate, and achieve the effects of low detection cost, good affinity and convenient use

Inactive Publication Date: 2017-07-21
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High performance liquid chromatography is accurate and reliable, with high sensitivity, and the detection limit can reach 0.01μg / g, but there are many pretreatment steps and the recovery rate is low

Method used

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  • Spinosad hapten and preparation method and application thereof
  • Spinosad hapten and preparation method and application thereof
  • Spinosad hapten and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Synthesis of spinosad haptens

[0025] step 1:

[0026] Weigh 1g, that is, 1.37mmol spinosad in a 50ml eggplant-shaped flask, dissolve it in 30ml 1N sulfuric acid, heat to 80°C, stir the reaction with a magnetic force, and monitor the completion of the reaction on a TLC board (developing agent is ethyl acetate:petroleum ether=7: 3); wash the precipitate with 50ml of dilute sulfuric acid, add 30ml of dichloromethane to dissolve, transfer to a separatory funnel, wash twice with saturated sodium chloride solution, and collect the organic phase; after drying with anhydrous sodium sulfate, filter and spin to dry the solvent, The hapten intermediate was purified by 200-300 mesh silica gel column chromatography, the eluent was ethyl acetate:petroleum ether, the volume ratio was 7:3, and the yield was 71.4%.

[0027] Step 2:

[0028] Weigh 600mg of the hapten intermediate into a brown bottle, add 750mg of succinic anhydride and 122.2mg of 4-dimethylaminopyridine, d...

Embodiment 2

[0029] Example 2: Identification of spinosad haptens

[0030] The product of the above-mentioned Example 1 was measured by H NMR and C NMR, and the NMR data were as follows, indicating that the hapten was successfully synthesized:

[0031] 1H NMR (500MHz, CD3OD) δ7.09(s, 1H), 5.93(d, J=9.8Hz, 1H), 5.86(dt, J=9.8, 2.8Hz, 1H), 5.03–4.95(m, 1H) ,4.87(d,J=1.7Hz,2H),4.70–4.61(m,1H),4.34(dd,J=12.7,7.1Hz,1H),3.60–3.52(m,3H),3.52(s,3H ),3.48–3.44(m,9H),3.43(t,J=3.3Hz,1H),3.35(s,6H),3.08(dt,J=18.8,7.2Hz,2H),2.98-2.96(m, 1H),2.91–2.83(m,1H),2.64–2.58(m,4H),2.47(dd,J=13.4,3.2Hz,1H),2.41–2.30(m,1H),2.22–2.12(m, 1H),2.04–1.93(m,1H),1.66–1.27(m,11H),1.24(d,J=6.2Hz,3H),1.09(d,J=6.7Hz,3H),0.99–0.88(m ,1H),0.83(t,J=7.5Hz,3H).

[0032] 13C NMR(126MHz,MeOD)δ203.44,175.96,173.93,173.92,150.37,145.06,130.66,129.74,97.06,83.55,82.52,78.63,77.96,77.91,76.29,69.10,61.14,59.15,57.72,51.06,49.85,47.46 ,46.72,42.87,42.55,38.54,37.44,35.01,33.58,31.11,30.27,29.84,29.28,22.34,18.13,16.45,9.62.

Embodiment 3

[0033] Example 3: Spinosyn antigen

[0034] Add 0.6ml of anhydrous DMF to 0.09mmol of the succinyl spinosyn hapten, mix well, add 0.12mmol of DCC and 0.12mmol of NHS to activate the carboxyl group on the hapten, stir overnight, then centrifuge To remove precipitation, divide the solution into three parts on average, and add them dropwise to different carrier protein solutions. The carrier protein is BSA, OVA, KLH, etc., and the buffer for dissolving the protein is carbonate buffer or boron Salt buffer solution, the amount of protein used is calculated according to the molar ratio of hapten to protein 15-40:1. After the coupling reaction between the carboxyl-activated hapten and the amino group on the protein molecule overnight, the reaction solution was transferred to a semi-permeable membrane, and dialyzed at 4°C for 3-5 days using 10 mM PBS buffer solution of pH 7.2 to obtain a coating Originally, the spinosad hapten was successfully coupled to the carrier protein after ult...

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Abstract

The invention discloses hapten and a preparation method and application thereof and particularly relates to spinosad hapten and a preparation method and application thereof. The spinosad hapten has the advantages that the chemical structure of spinosad is retained to a maximum degree, -COOH capable of being coupled with protein is introduced through chemical synthesis modification, and the synthesizing method is simple and high in product purity and yield; an antigen system suitable for animal immunization is prepared by using the spinosad hapten as the raw material, an antibody obtained after the animal immunization is good in titer, specificity and affinity, and the antibody can be used for preparing an enzyme linked immunosorbent assay kit; the kit is simple to operate, low in detection cost, efficient, accurate and fast in detection method, capable of simultaneously detecting a large batch of samples and applicable to the field monitoring of spinosad residues in animal-derived food and the screening of a large amount of samples; the spinosad hapten plays an important role in spinosad detection.

Description

technical field [0001] The invention relates to a hapten and its preparation method and application, in particular to a spinosyn hapten and its preparation method and application. Background technique [0002] Spinosad is a broad-spectrum insecticide isolated from the soil actinomycete Saccharopolyspora spinosa. It has the advantages of high insecticidal activity, easy degradation, and low toxicity to beneficial organisms. It is widely used in the control of various crop pests all over the world, and the excess of spinosad residues may affect food safety and environmental safety. Therefore, it is urgent to establish a detection method for spinosad with high sensitivity, strong specificity, and easy operation. [0003] Spinosad detection methods include high performance liquid chromatography, enzyme-linked immunoassay. High performance liquid chromatography is accurate and reliable, with high sensitivity, and the detection limit can reach 0.01 μg / g, but there are many pretre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/08C07H1/00C07K14/765C07K14/77C07K14/795C07K16/44G01N33/535G01N33/52
CPCC07H1/00C07H17/08C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/52G01N33/535
Inventor 韩谦赵洪伟兰坚强范咏梅刁晓平
Owner HAINAN UNIVERSITY
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