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14-hydroxygelsenicine hapten and artificial antigen and preparation method and application thereof

An artificial antigen and hapten technology, which is applied in the fields of peptide preparation, chemical instruments and methods, animal/human peptides, etc., can solve the problems that have not been reported on the immunoassay method of 14-Hydroxyglucan, and achieve good application. Prospect, high practical value, high valence effect

Inactive Publication Date: 2020-04-24
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

As the main alkaloid in Gelkins plants, so far, there is no report on the immunoassay method of 14-Hydroxykinesin

Method used

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  • 14-hydroxygelsenicine hapten and artificial antigen and preparation method and application thereof
  • 14-hydroxygelsenicine hapten and artificial antigen and preparation method and application thereof
  • 14-hydroxygelsenicine hapten and artificial antigen and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 114

[0042] Example 1 Preparation and Identification of 14-Hydroxykekinin Hexahapten (HGE-HS)

[0043] (1) 14-Hydroxykarpezine (20 mg) and succinic anhydride (10 mg) were dissolved in 6.0 mL of anhydrous pyridine respectively, the two solutions were mixed, and placed in an oil bath magnetic stirrer to react in the dark for 9 h ( 50°C, 240 rpm) to obtain the reaction product. Synthetic route see figure 1 .

[0044] (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.

[0045] (3) Column chromatographic separation yielded the white target compound 14-hydroxykaskinin hapten (21.3 mg, yield 83%).

[0046] The purified hapten was identified by high-resolution mass spectrometry, and the results were as follows figure 2 shown. 14-Hydroxygalesin: MS m / z 343.16523 [M+H] + ; HGE-HS: MS m / z 443.181283 [M+H] + . The m / z of HGE-HS increased by exactly 100 Da com...

Embodiment 2

[0047] Example 2 Preparation of 14-Hydroxy-Kelkinin-1 Artificial Antigen

[0048] (1) Weigh the 14-hydroxy kelecine hapten (10 mg), N - Hydroxysuccinimide (4.5 mg) and dicyclohexylcarbodiimide (9 mg) in a glass reaction vial, add 1 mL of DMF. 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.

[0049] (2) Dissolve 60 mg of BSA (OVA) in 10 mL of PBS buffer containing 10% (volume percent) DMF to obtain a protein solution.

[0050] (3) Add the liquid phase completed in step (1) dropwise to the protein solution prepared in step (2), place the reaction solution on a magnetic stirrer, and react at 4°C for 5 h.

[0051] (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 for dialysis at 4°C for 3 days (change the medium every 12 hours).

[0052] (5) After completing step (4), ...

Embodiment 314

[0054] Example 3 Preparation and Identification of 14-Hydroxy-Keskinin-1 Polyclonal Antibody

[0055] 1. Preparation of polyclonal antibody against 14-hydroxykelesine

[0056] HGE-HS-BSA prepared in Example 2 was used as an immunogen to immunize mice, and HGE-HS-OVA was used as a coating source to detect mouse antiserum. The concentration of the complete antigen was determined by the Bradford method, and the concentrations of HGE-HS-BSA and HGE-HS-OVA were both 5.5 mg / mL.

[0057] For the first immunization, dilute the immunogen to 1 mg / mL (dilute with 0.01 M PBS, pH 7.4), mix the diluted immunogen with Freund’s complete adjuvant in equal volume, and fully emulsify it, and subcutaneously place it on the back of the neck Inoculate 6-8 week-old Balb / c mice (5 mice were immunized), the dose of immunogen was 100 μg / mouse, and the injection dose was 0.2 mL / mouse. Then, a booster immunization was performed every 4 weeks, and the immunogen was emulsified with an equal volume of Fre...

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Abstract

The invention provides a 14-hydroxygelsenicine hapten and an artificial antigen and a preparation method and application thereof. The structure of the 14-hydroxygelsenicine hapten is shown as a formula (I) as described in the specifications, the 14-hydroxygelsenicine artificial antigen shown as the formula (I) is obtained by coupling the hapten shown as the formula (I) with carrier protein. Animals are immunized by using the 14-hydroxygelsenicine artificial antigen, so that a specific antibody with high titer and high sensitivity can be obtained. The 14-hydroxygelsenicine hapten and the prepared antibody provided by the invention provide a new means for establishing a rapid, simple, cheap, sensitive and specific 14-hydroxygelsenicine detection method, and also provide a powerful means forrapid detection of gelsemium elegans toxic honey and food poisoning.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a 14-hydroxy kelecine hapten and an artificial antigen and a preparation method and application thereof. Background technique [0002] hook kiss ( Gelsemium elegans ) is a highly poisonous plant, also known as big tea medicine, heartbroken grass, etc. It is widely distributed in the mountainous areas of South China in my country. In addition, it is also distributed in Southeast Asia and South Asia. Due to the bright yellow color of the flowers of Ginkgo, people often mistake it for honeysuckle and other traditional Chinese medicines and take it by mistake, which will lead to poisoning reactions and even death. Due to the wide distribution of glyphs in the mountainous areas of South China, there are reports of poisoning due to ingestion of glyphs every year in my country. In addition, ginkgo is one of the main poisonous nectar plants in my country. Durin...

Claims

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Application Information

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IPC IPC(8): C07D491/22C07K14/47C07K14/765C07K14/77C07K14/795C07K1/107C07K1/10C07K16/44G01N33/53
CPCC07D491/22C07K14/47C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53
Inventor 杨术鹏李熠周金慧傅怡张金震金钥杨宇辉黄京平袁媛赵文
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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