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Benzocaine hapten, artificial antigen and antibody, and preparation and application thereof

A technology of benzocaine and artificial antigen, applied in the field of artificial antigen, antibody and its preparation and application, benzocaine hapten, to achieve high accuracy, high antiserum titer and good sensitivity

Active Publication Date: 2020-03-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the defect and deficiency of existing benzocaine and its detection method, provide a kind of benzocaine hapten, artificial antigen, antibody and preparation and application thereof, utilize artificial antigen of the present invention Immunized animals, the obtained antibodies have significant advantages such as high specificity, good sensitivity, and high accuracy

Method used

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  • Benzocaine hapten, artificial antigen and antibody, and preparation and application thereof
  • Benzocaine hapten, artificial antigen and antibody, and preparation and application thereof
  • Benzocaine hapten, artificial antigen and antibody, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The preparation method of embodiment 1 hapten

[0070] In a 25mL round-bottomed flask, 825mg (5mmol) benzocaine was dissolved in 25mL of anhydrous methanol (solvent), and after dissolving, 1mL of triethylamine (catalyst) was added, and 670mg of phenylaldehyde was slowly added dropwise under magnetic stirring. After reflux reaction at 80°C for 3 hours, add 0.12g of sodium borohydride for reduction, condense and reflux reaction at 50°C for 1 hour, spin dry methanol at 40°C, dissolve in water, extract with ethyl acetate for 3 times, and dissolve ethyl acetate at 50°C The resulting reaction mixture was purified by silica gel column, eluted with petroleum ether:ethyl acetate=3:1 (volume ratio), and then spin-dried at 50°C to obtain the product, which was the target hapten (ie, benzocaine hapten), the chemical structure of the target hapten is as figure 1 as shown, figure 2 It is the identification map of benzocaine hapten mass spectrometry.

[0071] ESI-MS analysis m / z 2...

Embodiment 2

[0072] The preparation method of embodiment 2 immunogen / coating original

[0073] In the preparation methods of the immunogen and the coating source, the difference lies in the structural formula of the hapten, the type of carrier protein used and the coupling method. The immunogen carrier protein is mainly bovine serum albumin (BSA), and the original carrier protein of the coating is mainly Chicken ovalbumin (OVA) was used, and the coupling method used was active ester method or diazotization method. Bovine serum albumin was purchased from Beijing Boao Tuoda Technology Co., Ltd., and chicken ovalbumin was purchased from Sigma Company.

[0074] Immunogen (active ester method): Dissolve 140 mg of benzocaine hapten (BZKY-299) in 0.6 mL of DMF, dissolve 50 mg of BSA in 10 mL of PBS (phosphate buffer saline) with a pH of 7.4, and add half Antigen dissolving solution, magnetically stirred for 10 minutes, after mixing evenly, the protein-hapten mixture was obtained, 90 mg of EDC wa...

Embodiment 3

[0079] Example 3 Antibody Preparation and Identification

[0080] The immunogen prepared in Example 2 was evenly emulsified with an equivalent amount of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations), and then immunized animals. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles and ear veins respectively. The second immunization was done after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. The enzyme plate was purchased from Thermo Fisher Corporation of the United States. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the hea...

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Abstract

The invention provides benzocaine hapten, artificial antigen and antibody, and preparation and application thereof. The benzocaine hapten has the structure represented as the formula (I), and the benzocaine artificial antigen has the structure represented as the formula (II) or (III). Because there is less limit standard of the benzocaine, reporting of the synthesis of hapten and artificial antigen of the benzocaine is rare, and there is less related immunoassay method of the benzocaine, and the existing detection technology of benzocaine mainly includes instrument-based methods which are complex in operation and high in pretreatment demand and cost, the antiserum prepared from animals immunized by the artificial antigen has high titer, so that the antibody is high in specificity and sensitivity and is good in accuracy.

Description

technical field [0001] The invention relates to the technical field of food safety immunoassay, in particular to a benzocaine hapten, artificial antigen, antibody and its preparation and application. Background technique [0002] In fishery production, often due to long-distance and high-density transportation, fish bodies are prone to struggle, injury, fish disease infection or death due to lack of oxygen. In order to increase the survival rate of fish and reduce economic losses, benzocaine is also widely used as a circulation agent. Link fish anesthetics. It can forcibly change the physiological state of the fish during transportation, reduce the metabolic intensity of the fish, make the fish move sluggishly, and enter a state similar to dormancy, thereby preventing the fish from being injured due to violent movement in the container. [0003] With people's increasing concern and demands for food safety, residues of food and drugs have become a social hot issue. As an an...

Claims

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Application Information

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IPC IPC(8): C07C229/60C07K14/765C07K14/77C07K14/79C07K14/795C07K16/44G01N33/53
CPCC07C229/60C07K14/765C07K14/77C07K14/79C07K14/795C07K16/44G01N33/5308
Inventor 罗林徐振林罗双子贾宝珠何镇熹孙远明沈玉栋雷红涛王弘
Owner SOUTH CHINA AGRI UNIV
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