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Method for synthesizing artificial antigen of specific ractopamine

A technology of ractopamine and artificial antigen, applied in chemical instruments and methods, specific peptides, animal/human proteins, etc., to achieve high specificity, good specificity, and low immunogenicity

Active Publication Date: 2013-07-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a novel ractopamine hapten and complete antigen synthesis method for the existing ractopamine antigen synthesis technology and the deficiencies and defects of corresponding antibodies, making it possible to prepare highly specific ractopamine monoclonal antibodies

Method used

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  • Method for synthesizing artificial antigen of specific ractopamine
  • Method for synthesizing artificial antigen of specific ractopamine
  • Method for synthesizing artificial antigen of specific ractopamine

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the preparation of ractopamine hapten

[0040] 1) Octopamine 306mg (2mmol), add ethyl levulinate 288.3μL (2mmol), methanol 10mL, triethylamine 1mL, sodium cyanoborohydride 145mg (2.2mmol), stir at room temperature for 12h, rotary steaming, to obtain intermediate product.

[0041] 2) Dissolve the intermediate product in 15% KOH methanol solution (15mL), keep it at 80°C for 2h under reflux, then evaporate to dryness, and dissolve the solid residue in 5mL of distilled water, adjust the pH value to 2.0 with 6M HCl, and use ethyl acetate After extraction three times, the extract was collected and evaporated to dryness to obtain hapten compound I with a yield of about 30%.

Embodiment 2

[0042] Embodiment 2, the preparation of ractopamine artificial antigen

[0043] Take 25mg (0.1mmol) hapten, add 2mL N,N-dimethylformamide (DMF) to dissolve, then add N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-di Methylaminopropyl) carbodiimide

[0044](EDC) (hapten, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a molar ratio of 1:1.5 :2), at 4°C, mix well in the dark, stir for 60min, and then react for 12h at room temperature. Take 85mg (0.00125mmol) of bovine serum albumin (the molar ratio of hapten to bovine serum albumin is 80:1), and add 10mL of 0.1M pH9.6 carbonate buffer. The activated ractopamine hapten solution was slowly added dropwise to the bovine serum albumin solution, and reacted at room temperature for 24 hours. The ractopamine artificial antigen compound II was obtained by dialysis with PBS buffer solution for 2 days, during which the PBS buffer solution was changed 4 times.

Embodiment 3

[0045] Embodiment 3, the preparation of ractopamine artificial antigen

[0046] Take 25mg (0.1mmol) hapten, add 2mL N,N-dimethylformamide (DMF) to dissolve, and pre-cool at 0°C for 30min. At 0°C, add tri-n-butylamine and isobutyl chloroformate (the molar ratio of hapten:tri-n-butylamine:isobutyl chloroformate is 1:1.2:1.2), and react at 0°C for 1 hour. Take 85mg (0.00125mmol) of bovine serum albumin (the molar ratio of hapten: bovine serum albumin is 80:1), add 10mL of 0.1M pH9.6 carbonate buffer, and pre-cool at 0°C for 30min. At 0°C, the activated hapten solution was slowly added dropwise to the bovine serum albumin solution, reacted at 0°C for 1 hour, and then reacted at room temperature for 24 hours. The ractopamine artificial antigen compound II was obtained by dialysis with PBS buffer solution for 2 days, during which the PBS buffer solution was changed 4 times.

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Abstract

The invention relates to a method for synthesizing the artificial antigen of specific ractopamine, and belongs to the technical field of biochemical engineering. The method for synthesizing the artificial antigen of specific ractopamine disclosed by the invention comprises the following steps of: mixing octopamine and ethyl levulinate in methanol solution, reducing through sodium cyanoborohydride under catalysis of triethylamine, reacting to obtain an intermediate product, hydrolyzing the intermediate product under the alkaline condition to obtain molecule containing active carboxyl, wherein the homology of the molecule and ractopamine molecule is more than 70%, and the molecule can be used as hapten of ractopamine; and coupling the hapten to carrier protein to obtain the artificial antigen of specific ractopamine. Experimental results show that an antibody generated by immune animal by using the method disclosed by the invention is high in specificity and sensitivity, the titer of obtained antiserum can be up to 64000, the detection limit is 0.1 ng / mL, and the 50% inhibiting concentration IC50 is 1 ng / mL. The antigen or the antibody disclosed by the invention can be used for establishing an enzyme-linked immunosorbent analyzing method and a colloidal gold test paper rapid detecting method, used for rapidly detecting ractopamine residue in foods, and has wide application prospect.

Description

technical field [0001] The invention relates to a method for synthesizing a highly specific ractopamine artificial antigen, which belongs to the technical field of biochemical engineering. Background technique [0002] Ractopamine is a synthetic β-adrenergic receptor agonist that can be used to treat congestive heart failure, muscular dystrophy, increase muscle, reduce fat accumulation, and is beneficial to fetal and neonatal growth. Its molecular structural formula is: [0003] [0004] Ractopamine can be used in animal nutrition formulations, and has been used as an animal feed additive to promote pigs, cattle, and turkeys to grow muscle (commonly known as lean meat) and reduce body fat. Its meat residual toxicity is far lower than other clenbuterol additives with the same function, and is currently widely used in animal husbandry and breeding. At the same time, it can increase the daily weight gain of animals, improve the utilization rate of feed, and increase the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07C229/12C07K16/44G01N33/68
Inventor 胥传来刘丽强王利兵匡华徐利广马伟胡拥明
Owner JIANGNAN UNIV
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