A kind of tyramide hapten, antigen and antibody and its preparation method and application

A tyramine hapten, tyramine technology, applied in the preparation of carboxylic acid amide, chemical instruments and methods, cyanide reaction preparation and other directions, can solve the problems of poor repeatability and unreasonable antibody preparation scheme, and achieve high accuracy and linearity. Broad and specific effects

Active Publication Date: 2022-02-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the defects of unreasonable and poor repeatability of tyramide artificial antigen and antibody preparation scheme in the prior art, and provides a tyramide hapten and its corresponding artificial antigen and antibody and its preparation method

Method used

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  • A kind of tyramide hapten, antigen and antibody and its preparation method and application
  • A kind of tyramide hapten, antigen and antibody and its preparation method and application
  • A kind of tyramide hapten, antigen and antibody and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0063] The preparation method of embodiment 1 tyramide hapten

[0064] S11. Put 1g of tyramine and 1.197g of methyl 4-formylbenzoate into a 25mL round bottom flask, mix well, add 25mL of anhydrous methanol to fully dissolve, condense and reflux at 60°C for 20h

[0065] S12. Add 0.55g of sodium borohydride for reduction, and condense and reflux for 1 hour at 50°C;

[0066] S13. Methanol is spin-dried, dissolved in water, extracted 3 times with ethyl acetate, and then ethyl acetate is spin-dried;

[0067] S14. Redissolve the product with 20mL of methanol, and add 0.22g of potassium hydroxide, and reflux at 70°C for 3h;

[0068] S15. Spin dry and dissolve in saturated saline, adjust the pH to about 5-6 with hydrochloric acid, extract with n-butanol to obtain the target tyramine hapten, and its structure is shown in formula I.

[0069] Formula I.

[0070] The ESI-MS identification results of the above tyramide haptens are as follows: figure 1 As shown, the synthesized compou...

Embodiment 2

[0071] Embodiment 2 The preparation method of immunogen / coating original

[0072] In the preparation method of the immunogen and the coating source, the difference lies in the structural formula of the hapten, the use type of the carrier protein and the coupling method. The immunogen carrier protein mainly adopts bovine serum albumin (BSA), and the coating Chicken ovalbumin (OVA) was mainly used as the original carrier protein, and the coupling method used was carbodiimide method or glutaraldehyde two-step method.

[0073] A preparation method (immunogen (carbodiimide method)) of a tyramide antigen, which comprises the following steps:

[0074] S21. 9mg BSA is dissolved in the PBS buffer of 5mL PH7.4;

[0075] S22. Dissolve 11 mg of tyramide hapten (Tyr-271) in 400 μL of DMF and 500 μL of first-grade water, add it to the protein solution obtained in S21, and stir for 10 minutes;

[0076] S23. Dissolve 12 mg of EDC in 200 μL of water, then add dropwise to the protein-hapten m...

Embodiment 3

[0090] Example 3 Antibody preparation and identification

[0091] Emulsify the prepared immunogen with an equal amount of immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) and immunize animals. BalB / C mice aged 6 to 7 weeks were immunized by subcutaneous injection in the back, subcutaneous injection in various parts, abdominal cavity and foot respectively. The second immunization was performed 2 weeks later, and the booster immunization was performed every 2 weeks thereafter. One week after the third booster immunization, blood was collected from the tail of the mice, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, intraperitoneal injection is used to boost the immunization. Three days later, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 4°C and 10,000 rpm for 15 minutes, and the supernatant was tak...

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Abstract

The invention provides a tyramide hapten, artificial antigen, antibody and preparation and application thereof. The tyramide hapten has the molecular structure shown in formula I (formula I), and the tyramide artificial antigen has the molecular structure shown in formula II (formula II). The invention solves the defects of unreasonable and poor repeatability in the tyramide artificial antigen and antibody preparation scheme in the prior art. The titer of the antiserum obtained by immunizing animals with the artificial antigen prepared by the hapten of the present invention can reach 1:1000, the minimum detection limit is 26.8 ng / mL, and the half-inhibitory concentration is as low as 391.0 ng / mL, and the antibody has specificity High, good sensitivity, high accuracy, wide linear range and other significant advantages.

Description

technical field [0001] The invention belongs to the technical field of food safety immunological detection, and more specifically relates to a hapten of tyramine, an artificial antigen and an antibody, and a preparation method and application thereof. Background technique [0002] Tyramine is a biogenic amine substance. According to the structural classification, it belongs to the aromatic biogenic amine; according to the composition classification, it belongs to the monoamine biogenic amine. Tyramine is widely found in various foods, especially in fermented foods and spoiled foods, where the content of tyramine is very high. [0003] Tyramine is an effective physiologically active substance that exists in biological organisms. It is a neuroactive substance that plays an irreplaceable physiological function in the normal metabolism of the body. However, when the intake of tyramine exceeds 100 mg, it can cause migraine; and when the intake exceeds 1080 mg, it can cause sever...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C229/38C07C227/18C07K14/765C07K14/77C07K16/44G01N33/53G01N33/543G01N33/58G01N33/68
CPCC07C229/38C07C227/18C07C227/16C07C231/10C07K14/765C07K14/77C07K16/44G01N33/5308G01N33/543G01N33/581G01N33/6854C07C233/73
Inventor 罗林罗双子徐振林孙远明杨金易蔡泽伟沈玉栋王弘雷红涛
Owner SOUTH CHINA AGRI UNIV
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