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Tutin hapten, and preparation method and application thereof

A technology of hydroxymasangolactone and hydroxymasanolactone, which is applied in the field of hydroxymasanolactone hapten and its preparation, can solve the problems of immunoassay and analysis that have not been reported yet, and achieve good application prospects, high titer, and preparation The effect of simple process

Active Publication Date: 2020-08-11
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The small molecule detection method developed based on immunoassay has the advantages of high sensitivity, high accuracy, fast detection speed, low cost, and simple operation. Many rapid detection methods of phytotoxins based on immunoassay have been reported. Immunoassay assays for esters and massanolactones have not been reported

Method used

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  • Tutin hapten, and preparation method and application thereof
  • Tutin hapten, and preparation method and application thereof
  • Tutin hapten, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation and Identification of Hydroxymasangolactone Hapten

[0049] (1) Hydroxyrizanolactone (20 mg) and succinic anhydride (6.8 mg) were dissolved in 3.5 mL of anhydrous pyridine, respectively, and the two solutions were mixed and placed in an oil bath magnetic stirrer to react in the dark for 8 h (60 o C, 240 rpm), to obtain the reaction product.

[0050] (2) The reaction system was quenched with water, extracted with ethyl acetate, washed with 0.1M dilute hydrochloric acid, and the organic phase was dried and concentrated.

[0051] (3) Column chromatographic separation to obtain the white target compound hydroxymasangolactone hapten.

[0052] Using liquid phase tandem high-resolution mass spectrometry to identify hydroxymasangolactone and the purified hapten, the results are shown in figure 1 , where, A is the precisely extracted ion current chromatogram, and B is the full-scan mass spectrogram in negative ion mode. According to its synthetic route, ...

Embodiment 2

[0053] Example 2 Preparation of artificial antigen of hydroxymasangolactone

[0054] (1) Weigh the hydroxymasangolactone hapten (8 mg) prepared in Example 1, N - Hydroxysuccinimide (4.1 mg) and dicyclohexylcarbodiimide (8.2 mg) were placed in a glass reaction vial, and 1 mL of DMF was added. The glass bottle containing the reaction solution was placed on a magnetic stirrer, and reacted at room temperature for 5 h at 400 rpm in the dark.

[0055] (2) Dissolve 30 mg of BSA in 9.5 mL of PBS buffer containing 10% (volume percent) DMF to obtain a protein solution.

[0056] (3) Add the liquid phase that has completed step (1) dropwise to the protein solution prepared in step (2), place the reaction solution on a magnetic stirrer, 4 o C for 5 h.

[0057] (4) Transfer the protein activation solution in step (3) to a dialysis bag with a molecular weight cut-off of 7 KDa, and then place the dialysis bag in PBS buffer and dialyze at 4 °C for 3 days (change the medium every 12 h).

[...

Embodiment 3

[0061] Example 3 Preparation and Identification of Polyclonal Antibody to Hydroxyrizanolactone

[0062] 1. Preparation of Polyclonal Antibody to Hydroxyrizanolactone

[0063] The hydroxybemoglide-BSA prepared in Example 2 was used as the immunogen to immunize mice, and the hydroxybemoglide-OVA was used as the coating source to detect the mouse antiserum. The concentration of the complete antigen was determined by the Bradford method, and the concentrations of the immunogen and the coating agent were both 2.9 mg / mL.

[0064] For the first immunization, the immunogen was diluted to 1 mg / mL (diluted with 0.01 M PBS, pH 7.4), and the diluted immunogen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified. Six-week-old Balb / c mice (6 mice were immunized) were spot inoculated, the dose of immunogen was 100 μg / mouse, and the injection dose was 0.2 mL / mouse. Immunization was boosted every 4 weeks, and the immunogen was emulsified with an equal volume of Fre...

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Abstract

The invention relates to the technical field of biochemical engineering, in particular to a tutin hapten, and a preparation method and application thereof. The tutin hapten has a structure as described in the specification. According to the invention, a conjugate of the tutin hapten provided by the invention and a carrier protein is utilized, and an anti-tutin antibody can be obtained by immunizing mice; preparation process is simple and economic; the valence of antibody can reach 135000 at most, and detection sensitivity is 7.7 ng / mL, so practical value is high, and the antibody has good application prospects in public health safety detection.

Description

technical field [0001] The invention relates to the technical field of biochemical industry, in particular to a hapten of hydroxymasangolactone and its preparation method and application. Background technique [0002] Coriaria Sinica Maxim is a poisonous plant in the genus Coriaria Sinica, also known as Buddleia. Ma Sang plant is rich in sesquiterpene ester toxic substances, such as Ma Sang Lactone, Hydroxy Sang Lactone, Ma Sang Ning and Ma Sang Ting, etc. Therefore, the whole plant of Ma Sang is poisonous, especially the immature fruit is the most toxic. Poisoning incidents caused by Ma Sang are not uncommon, mainly because children eat Ma Sang fruit by mistake. Patients with Masang poisoning initially manifested symptoms such as nausea, vomiting, dizziness, and headache, and those who were severely poisoned would experience frequent convulsions, persistent epilepsy, and coma. If patients with severe poisoning cannot receive timely and effective medical treatment, they wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/20C07K14/765C07K14/77C07K14/795C07K14/47C07K16/46G01N33/577G01N33/53
CPCC07D493/20C07K14/47C07K14/765C07K14/77C07K14/795C07K16/46G01N33/5308G01N33/577
Inventor 杨术鹏李熠傅怡周金慧张金震杨宇晖
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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