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Xylazine hapten and artificial antigen and preparation method and application thereof

A technology of artificial antigen and xylazine, which is applied in chemical instruments and methods, animal/human peptides, animal/human proteins, etc., can solve the problems of complicated operation, expensive large-scale instruments, complicated pretreatment process, etc., and achieve the goal of preparation process Simple, good application prospects, and high practical value

Pending Publication Date: 2022-02-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the large-scale instruments required by these methods are expensive, complicated to operate, and poor in portability, making it difficult to achieve production-line detection, and the pretreatment process of these methods is complicated and time-consuming, and is not suitable for rapid detection of a large number of samples

Method used

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  • Xylazine hapten and artificial antigen and preparation method and application thereof
  • Xylazine hapten and artificial antigen and preparation method and application thereof
  • Xylazine hapten and artificial antigen and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Preparation and characterization of embodiment 1 xylazine hapten

[0047] The xylazine hapten provided in this embodiment has a structure as shown in formula (I) or formula (II):

[0048]

[0049] One, the preparation of xylazine hapten

[0050] 1, the preparation of xylazine hapten shown in formula (I)

[0051]Step 1: Add 241.86mg of 6-bromohexanoic acid into 20mL of dichloromethane, then add 5mL of thionyl chloride, reflux at 95°C for 1 hour, spin dry directly, and dropwise add to 10mL of tert- In butanol solution, react overnight at room temperature, post-treatment, suction filtration, pour into 10mL 5% sodium bicarbonate aqueous solution, extract with 30mL ethyl acetate, and rotary evaporate to dryness.

[0052] Step 2: Using N,N-dimethylformamide as solvent, add 2.64g tert-butyl 6-bromohexanoate and 3.16g xylazine to 20mL N,N-dimethylformamide, then add 2.70g Potassium carbonate and 1.23g of potassium iodide were reacted at 100°C overnight, and the post-treat...

Embodiment 2

[0067] Preparation and characterization of embodiment 2 xylazine artificial antigen

[0068] It is obtained by coupling the xylazine hapten of formula (I) or formula (II) with carrier protein. Divided into immunogen, coating original. In the preparation method of the immunogen and the coating source, the difference lies in the type of carrier protein used, the immunogen carrier protein mainly adopts KLH, and the coating original carrier protein mainly adopts BSA, and the coupling method used is active Ester method. The artificial antigen structure prepared by xylazine hapten shown in formula (I) and formula (II) is as follows:

[0069]

[0070] 1. Synthesis and identification of xylazine coating agent

[0071] 1. Preparation of xylazine coating agent

[0072] (1) Dissolve 49 mg of the compound shown in (I) prepared in Example 1 in 5 mL of DMF, add 21 mg of NHS and 42 mg of EDC, and stir overnight at room temperature to obtain solution I.

[0073] (2) Add 50mg of BSA in...

Embodiment 3

[0086] Preparation of embodiment 3 xylazine antiserum

[0087] The XYL ①-KLH and XYL ②-KLH solutions prepared in Example 2 were used to immunize 2 groups of female New Zealand white rabbits aged 3-4 months and weighing 1.5-2.0 kg, 2 in each group. Dilute each immunogen to 1 mg / mL with physiological saline, and emulsify with an equal amount of Freund's adjuvant. Freund's complete adjuvant was used for the first immunization, intradermal injection at multiple points on the back of the neck, and the immunization dose was 1 mg / mouse. Booster immunization was carried out after 4 weeks, once every 4 weeks, for a total of 3 times, the adjuvant was changed to Freund's incomplete adjuvant, and the immune dose was changed to multi-point subcutaneous injection on the back of the neck. One week after the fourth immunization, a large amount of blood was collected by heart blood collection. After taking blood, let the blood stand at 37°C for 2 hours, then stand at 4°C overnight, then cent...

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Abstract

The invention discloses an xylazine hapten and artificial antigen and a preparation method and application thereof. A specific antibody with high titer and high sensitivity can be obtained by immunizing animals with the xylazine artificial antigen. The xylazine hapten and the antibody prepared from the xylazine hapten provide a new means for establishing a rapid, simple, cheap, sensitive and specific silazine detection method. When the conjugate of the hapten and the carrier protein is used for preparing the xylazine antibody, the preparation process is simple and economical, the detection sensitivity of the antibody to the xylazine can reach 0.25 ng / mL, the practical value is high, and the antibody has a good application prospect especially in veterinary drug residue detection.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a xylazine hapten and an artificial antigen and a preparation method and application thereof. Background technique [0002] Xylazine (Xylazine, XYL) is an analgesic chemical baoding drug, its mechanism of action is to bind to the receptors of the presynaptic membrane of the central nervous system, and stimulate the inhibitory autoreceptors of the presynaptic membrane to inhibit the demethylation The release of adrenaline, which plays a role in sedation, analgesia and central muscle relaxation. At present, the drug is mainly used for sedation and analgesia in horses, cattle, sheep and other animals. Existing studies have shown that the residues of xylazine in animal-derived foods will cause great harm to the health of consumers. Long-term consumption of animal-derived foods containing xylazine residues may cause nausea, vomiting, dizziness, weakness, e...

Claims

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Application Information

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IPC IPC(8): C07D279/06C07K14/765C07K14/77C07K14/795C07K14/435C07K16/44G01N33/577G01N33/58G01N33/53
CPCC07D279/06C07K14/765C07K14/77C07K14/795C07K14/435C07K16/44G01N33/577G01N33/9486G01N33/948G01N33/587Y02P20/55
Inventor 温凯王战辉沈建忠于雪芝余文博江海洋张素霞史为民罗亮
Owner CHINA AGRI UNIV
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