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Half antigen and antigen of tenuazonic acid and preparation method and application thereof

A technology of alternarinic acid and hapten, applied in chemical instruments and methods, antifungal/algae/lichen immunoglobulin, animal/human protein, etc. Large-scale screening and other issues, to achieve the effect of high accuracy, high sensitivity, and broad application prospects

Active Publication Date: 2012-06-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the limit standard of this toxin in crops has not been established at home and abroad, several articles at home and abroad have reported the detection of this toxin by liquid chromatography-mass spectrometry and other instrument methods, but the instrument is not suitable for large-scale screening due to the shortcomings of high price and need for special operation.

Method used

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  • Half antigen and antigen of tenuazonic acid and preparation method and application thereof
  • Half antigen and antigen of tenuazonic acid and preparation method and application thereof
  • Half antigen and antigen of tenuazonic acid and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation and Identification of Alternatus Acetic Acid Hapten

[0051] 1. Synthesis of Alternaria ketoacid haptens TeAH and TeAHGA

[0052] Add 30ml of absolute ethanol that has been dehydrated and dried into the three-neck flask, add 10mmol of isoleucine, and add 15ml of thionyl chloride. Reflux at 80°C for 3-4h, cool to room temperature, and concentrate in vacuo at 60°C to remove excess thionyl chloride. Dissolve it in dehydrated ethanol, add 12 mmol of newly prepared sodium ethoxide dropwise under stirring, and react for 30 minutes. Under stirring at 0-4°C, 12 mmol of diketene was added dropwise, and the drop was completed within 30 minutes, and then stirred and reacted at room temperature for 4 hours. 20ml of benzene was added, and 12mmol of newly prepared sodium ethoxide was added dropwise, and the mixture was stirred and refluxed for 3h. After cooling to room temperature, concentrate in vacuo at 60°C to remove the solvent, add dilute hydrochloric acid to adju...

Embodiment 2

[0066] Preparation and Identification of Alternarinic Acid Antigen

[0067] 1. Preparation of alternarinic acid antigen

[0068] Glutaraldehyde-linked protein: Weigh 1mmol of the hapten TeAH and 1 / 80mmol of OVA and dissolve them in a PBS solution with a pH value of 8.0, stir well at 25°C, and mix 50-200ul of 25% (volume ratio) ammonium The dialdehyde was added dropwise into the above mixed solution, and after the dropwise addition was completed, the mixture was stirred and reacted at 25° C. for 3 h. After the reaction, put the reaction solution in a dialysis bag and dialyze with normal saline at 4°C for 72 hours, during which the dialysate was changed 6 times to obtain the target product alternarinic acid antigen, which was stored at -20°C after aliquoting.

[0069] Active ester fanexin: Dissolve 1 mmol of TeAHGA in DMF, add 3 mmol of DCC and 4 mmol of N-hydroxysuccinimide (NHS) with stirring. Magnetic stirring was carried out overnight at 4°C. After centrifugation, the supe...

Embodiment 3

[0074] Detecting the effect of the antigen of the present invention by the alternarinic acid antibody

[0075] 1. Preparation of Alternarinic Acid Antiserum

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Abstract

The invention discloses half antigen and antigen of tenuazonic acid and a preparation method and application thereof. The antigen can serve as a compound of the tenuazonic acid half antigen and is provided with the structure as the formula I. The antigen of the tenuazonic acid is obtained by mixing TeAH and carrier protein, dissolving carrier protein into phosphate buffer solution (PBS), droppingglutaraldehyde solution, conducting stirring and reaction and then conducting dialysis through physiological saline, or the ntigen of the tenuazonic acid is obtained by dissolving TeAHGA into N, N-dimethylformamide, adding bicyclocaproyl carbodiimide and N-hydroxy succinimide into the mixture, stirring the liquid, conducting centrifugation to obtain supernatant as A liquid, dissolving carrier protein in the PBS, stirring and dissolving the PBS to obtain B liquid, dropping the A liquid into the B liquid for reaction and conducting dialysis through physiological saline. The antiserum valence obtained by antigen immune animals can reach 1: 3.2*104, the linear range of TeAH ranges from 0.130ng / mL to 8.789ng / mL(IC20-IC80), the half inhibition concentration is 1.095ng / mL, and the produced antigen is high in specificity, high in sensitivity and high in accuracy. Formula I.

Description

technical field [0001] The invention relates to a hapten and an antigen of alternarinic acid as well as a preparation method and application thereof. Background technique [0002] Tenuazonic acid (TeA, chemical name: 3-acetyl-4-hydroxy-5-sec-butylpyrrolin-2-one), is a secondary metabolite produced by Alternaria. One of the highest toxins. After the toxin was isolated and identified in the 1960s, it was found that the toxin has antitumor, weak antiviral activity, broad-spectrum, rapid and efficient herbicidal activity and other biological activities. The toxin can now be synthesized artificially, and its synthesis pathway is as follows: [0003] [0004] But along with the further biological and toxicological research of this toxin in various countries in recent years, the situation that this toxin contaminates crops (such as tomato, orange, grain, etc.) is quite common, because people's average daily intake is very low, cannot draw enough attention. Although eating fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D207/38C07K14/765C07K14/77C07K14/795C07K16/14G01N33/53
Inventor 王弘刘细霞杨星星沈玉栋徐振林孙远明杨金易
Owner SOUTH CHINA AGRI UNIV
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