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Synthesis method and application of coumafuryl hapten

A hapten and rodent control technology, which is applied in the field of public health, can solve the problems of complex sample processing, high detection cost, and inability to meet rapid screening, and achieve the effects of high sensitivity, high practical value, and good application prospects

Inactive Publication Date: 2019-12-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the instrumental analysis method is a confirmatory method, there are disadvantages such as high detection cost, the need for specialized personnel to operate, and complicated sample processing, which cannot meet the requirements of rapid screening

Method used

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  • Synthesis method and application of coumafuryl hapten
  • Synthesis method and application of coumafuryl hapten
  • Synthesis method and application of coumafuryl hapten

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, the preparation and identification of the gram rodent hapten CF-CMO

[0053] Weigh 20 mg of Kemieratine and 15 mg of carboxymethylhydroxylamine hemihydrochloride (CMO), dissolve them in 3 mL of pyridine solution, transfer them to a round-bottomed flask with a condensing device, and react with magnetic stirring at 60 ° C for 8 h. Blow dry (60° C.) under a water bath nitrogen instrument. Add 3 mL of 0.1M NaHCO 3solution, vortex to dissolve the residue. And extraction was performed 3 times with 5 mL of ethyl acetate. Afterwards, the aqueous phase was adjusted to pH=3 with 0.1M HCl, and extracted three times with 5 mL of ethyl acetate. The organic phase was blown dry (30° C.) with a water bath nitrogen apparatus to obtain the hapten CF-CMO (23 mg). (Synthetic route diagram see figure 1 )

[0054] The purified haptens were identified by mass spectrometry (see figure 2 ), CF-CMO: MS m / z 393.8[M-Na] + , the mass spectrometry data was consistent with the mol...

Embodiment 2

[0055] Embodiment 2, the preparation of gram rodent artificial antigen

[0056] Weigh 10 mg of gram murine hapten (CF-CMO) and dissolve it in 300 μL of DMF to obtain a hapten solution. Add 5mg EDC and 3mg NHS in the prepared hapten solution again (flow chart sees figure 1 ), placed on a magnetic stirrer, 300rpm room temperature reaction 5h. The reacted solution was centrifuged (5000 rpm), and the supernatant (280 μL) was collected. Get 40mg BSA and 50mg OVA afterward, be dissolved in the PBS damping fluid that contains 5% (volume percentage composition) DMF respectively in 7mL and 9mL contain in the PBS buffer fluid of 5% (volume percentage composition) DMF, obtain two kinds of albumen solutions. 200 μL and 180 μL of the above supernatant were added dropwise to the two protein solutions respectively, and placed in a magnetic stirrer for 5 hours of reaction to obtain a conjugate (artificial antigen) of the hapten and the carrier protein. The protein conjugate prepared above ...

Embodiment 3

[0057] Embodiment 3, the preparation and identification of the polyclonal antibody of Kemomurine

[0058] 1. Preparation of murine polyclonal antibody

[0059] A total of 6 mice were immunized with the CF-CMO-BSA prepared above as the immunogen, and CF-CMO-OVA was used as the coating agent for antiserum detection. The concentration of the complete antigen was determined by the Bradford method, and the concentrations of CF-CMO-BSA and CF-CMO-OVA were both 5 mg / mL.

[0060] For the first immunization, dilute the immunogen to 1 mg / mL (dilute with 0.01mol / L PBS), mix the diluted immunogen with Freund’s complete adjuvant in equal volume, and fully emulsify it, and inoculate it subcutaneously at multiple points on the back of the neck for 6-8 days. Week-old BALB / c mice (6 mice), the dose of inoculation immunogen was 100 μg / mouse, and the injection dose was 0.2 mL / mouse. Afterwards, booster immunization was performed every 3 weeks, and the immunogen was emulsified with an equal vol...

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Abstract

The invention provides a synthesis method and an application of a coumafuryl hapten. The structural formula of the coumafuryl hapten is shown in the description. The coumafuryl hapten is prepared by acondensation reaction of coumafuryl and carboxymethyl hydroxylamine semi-hydrochloride. The invention also provides a coumafuryl artificial antigen. The coumafuryl artificial antigen is prepared by coupling the coumafuryl hapten with a carrier protein. The complete antigen of the coumafuryl hapten prepared by the synthesis method of the coumafuryl hapten can expose the chemical structure of the coumafuryl as an antigen determinant, so a foundation is laid for the preparation of a high-sensitivity anti-coumafuryl antibody. Finally, an antibody CF-CMO-BSA-3 # with optimal properties is obtainedthrough screening, the highest titer can reach 3.2 * 10<4>, and the sensitivity is 0.32 ng / mL. A coumafuryl polyclonal antibody prepared from the coumafuryl hapten has the unique advantages of high sensitivity, high practical value and the like, and has a good application prospect in public health safety detection.

Description

technical field [0001] The invention relates to the field of public health, in particular to a method for preparing a murine hapten and an artificial antigen and an application thereof. Background technique [0002] Coumafuryl (CF) is a first-generation coumarin anticoagulant, which is highly toxic and effective in rodent control. It is a widely used rodenticide. Because its structure is similar to vitamin K, it can inhibit the synthesis of coagulation factors II, VII, IX, and X in the liver that vitamin K participates in. When rats eat a large amount of drugs, they will die from visceral hemorrhage, so as to achieve the effect of highly effective rodent control. However, internal hemorrhages such as skin congestion, gastric bleeding, intestinal bleeding, etc. will occur after the human body ingests an excessive amount of the rodent, and the consequences are more serious. Due to the better rodent control effect and wide application, some accidental ingestion, malicious poi...

Claims

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Application Information

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IPC IPC(8): C07D407/04C07K14/765C07K14/77C07K1/113C07K16/44G01N33/531G01N33/68
CPCC07D407/04C07K14/765C07K14/77C07K16/44G01N33/531G01N33/6854G01N2430/00
Inventor 温凯沈建忠王战辉江海洋于雪芝张素霞李红芳史为民
Owner CHINA AGRI UNIV
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