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Solanine hapten, artificial antigen and antibody as well as preparation methods and applications thereof

A technology of artificial antigen and solanine, which is applied in the preparation methods of peptides, chemical instruments and methods, animal/human proteins, etc., can solve the problems of high sensitivity, cumbersome processing, expensive instruments, etc., and achieves easy control of reaction conditions and reaction. Simple operation and enhanced immunogenicity

Active Publication Date: 2022-01-07
ZHEJIANG INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colorimetry and thin-layer chromatography have low accuracy and are difficult to apply to high-sensitivity quantitative analysis; high-performance liquid chromatography can detect high-concentration solanin samples, but cannot accurately detect low-concentration samples, and mainly use C 18 Column separation, the separation effect of α-solanine and α-chaconine is not ideal; liquid chromatography-mass spectrometry can detect trace amounts of solanine in the sample, the recovery rate of the sample is high, the sensitivity is high, and the repeatability Well, it can be used to determine the content of low-content α-solanine in samples, and it is the most commonly used detection method at present
However, these methods have the disadvantages of cumbersome sample pretreatment, long detection time, and expensive instruments, and cannot be widely used in my country, and do not meet the requirements of on-site detection "accurate detection and screening of a large number of samples in a short period of time and at low cost".

Method used

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  • Solanine hapten, artificial antigen and antibody as well as preparation methods and applications thereof
  • Solanine hapten, artificial antigen and antibody as well as preparation methods and applications thereof
  • Solanine hapten, artificial antigen and antibody as well as preparation methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A preparation method of solanine hapten, the steps are as follows:

[0032] Take 0.868g of α-solanine and add 200mL of dry cyclohexane to dissolve, add 0.318g of mercaptopropionic acid and stir thoroughly, then add 0.1g of dilauroyl peroxide, stir at room temperature for 4 hours, stop the reaction, add 50mL of cold water at 0-4°C , stirred, allowed to stand, the water phase was separated, and the organic phase was evaporated to dryness to obtain a yellow oil, which was dissolved by heating in 60 mL of absolute ethanol, and recrystallized to obtain the solanine hapten.

Embodiment 2

[0034] A preparation method of solanine artificial antigen, the steps are as follows:

[0035] Take 36 mg of the solanine hapten prepared in Example 1, add N, N-dimethylformamide (DMF) 2 mL to dissolve, add 1-ethyl-(3-dimethylaminopropyl) carbonyl disulfide Acetate (EDC) 16mg, N-hydroxysuccinimide (NHS) 9.6mg, react for 3 hours to obtain hapten solution A; take BSA 50mg, add 0.05mol / L PB buffer solution to dissolve, and obtain B solution; Liquid A was added dropwise to liquid B, reacted at 4°C for 12 hours, purified by dialysis with 0.02mol / L PBS for 3 days, and changed the liquid three times a day to obtain the solanin artificial antigen coupled with bovine serum albumin, which was stored at -20°C. spare.

Embodiment 3

[0037] A preparation method of solanine artificial antigen, the steps are as follows:

[0038] Take 21.6 mg of solanine hapten prepared in Example 1, add 2 mL of DMF to dissolve, add 6.1 mg of NHS and 9.2 mg of EDC, and react at room temperature for 3 hours to obtain hapten solution A; take 50 mg of OVA, add 0.05mol / L PB buffer Solution was dissolved to obtain solution B; solution A was added dropwise to solution B, reacted at 4°C for 12 hours, purified by dialysis and purification with 0.02mol / L PBS for 3 days, and the solution was changed three times a day to obtain artificial solanin conjugated with ovalbumin Antigens, stored at -20°C, for later use.

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Abstract

The invention discloses a solanine hapten, an artificial antigen and an antibody as well as preparation methods and applications thereof. The solanine hapten provided by the invention retains the characteristic structure of solanine to the greatest extent, and a three-carbon-chain carboxyl group is introduced, so that the immunogenicity of the solanine hapten is obviously enhanced; the solanine artificial antigen obtained by coupling the solanine hapten and the carrier protein is used for immunizing animals, so that the animal immune response is more favorably stimulated to generate an antibody with stronger specificity and higher sensitivity, the sensitivity of the solanine antibody to alpha-solanine is 1 mu g / L and the cross reaction rate with alpha-canthariine is 87% through indirect competitive enzyme-linked immunosorbent assay detection, and a basis is provided for subsequently establishing various immunoassay methods of solanine.

Description

technical field [0001] The invention belongs to the field of food safety detection. More specifically, the present invention relates to solanine haptens, artificial antigens and antibodies and their preparation methods and applications. Background technique [0002] Solanin is a toxic glycoside alkaloid, also known as solanine, which is a secondary metabolite in the process of plant growth. Solanin forms glycosyl ligands with glucose, xylose, rhamnose and galactose. Its structure can be divided into solanine and chaconine according to the connected groups, of which α-solanine and α-chaconine account for the largest proportion, up to 95%. Solanin is widely present in Solanaceae plants (tomato, eggplant, potato, etc.), and has certain toxicity. The toxicity mechanism mainly includes inhibiting the activity of cholinesterase in the body and combining with sterols on the biofilm to cause toxicity. reaction. The poisoned person will experience symptoms such as digestive system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J71/00C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53C07K1/107
CPCC07J71/0052C07K14/765C07K14/77C07K14/795C07K16/44G01N33/5308
Inventor 刘柱陈碧莲徐潇颖华颖王峰赵超群
Owner ZHEJIANG INST FOR FOOD & DRUG CONTROL
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