Synthesis method of specific salbutamol artificial antigen

A technology of albuterol and artificial antigen, which is applied in chemical instruments and methods, specific peptides, animal/human proteins, etc., to achieve high specificity and high sensitivity

Inactive Publication Date: 2013-06-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Purpose of the present invention: aiming at the deficiencies and defects of the existing albuterol antigen synthesis technology and corresponding antibodies, a novel albuterol hapten and complete antigen synthesis method is provided, which makes it possible to prepare highly specific salbutamol monoclonal antibodies

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  • Synthesis method of specific salbutamol artificial antigen
  • Synthesis method of specific salbutamol artificial antigen
  • Synthesis method of specific salbutamol artificial antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Preparation of albuterol hapten

[0040] 200mg (0.836mmol) of albuterol was dissolved in 15mL of ethanol. At room temperature, 99μL of 37% formaldehyde solution was added dropwise, and the reaction was stirred for 30min. Then, 110mg (0.836mmol) of 6-aminocaproic acid was added, and the reaction was continued for 6h. 6M HCl was slowly added dropwise to the reaction solution to adjust the pH to 2-3, 10 mL of ethyl acetate was added to extract three times, the organic phase was collected, and dried by rotary evaporation at 37° C. to obtain the albuterol hapten.

Embodiment 2

[0041] Embodiment 2, the preparation of albuterol artificial antigen

[0042] Take 25mg (0.0678mmol) albuterol hapten, add 2mL N,N-dimethylformamide (DMF) to dissolve, then add N-hydroxysuccinimide (NHS), 1-ethyl-3-(3- Dimethylaminopropyl) carbodiimide (EDC) (the molar ratio of hapten, NHS, EDC) is 1:1.5:2), at 4°C, mix well in the dark, stir for 60min, and then at room temperature Reaction 12h. Take 56mg (0.00084mmol) of bovine serum albumin (the molar ratio of hapten to bovine serum albumin is 80:1), add 10mL of 0.1M pH9.6 carbonate buffer. The activated hapten solution was slowly added dropwise to the bovine serum albumin solution, and reacted for 24 hours at room temperature. Dialyze with PBS buffer solution for 2 days, during which the PBS buffer solution was changed 4 times to obtain salbutamol artificial antigen.

Embodiment 3

[0043] Embodiment 3, the preparation of albuterol artificial antigen

[0044]Take 25 mg (0.0678 mmol) of albuterol hapten, add 2 mL of N,N-dimethylformamide (DMF) to dissolve, and pre-cool at 0°C for 30 minutes. At 0°C, add tri-n-butylamine and isobutyl chloroformate (the molar ratio of hapten, tri-n-butylamine, and isobutyl chloroformate is 1:1.2:1.2), and react at 0°C for 1 hour. Take 56mg (0.00084mmol) of bovine serum albumin (the molar ratio of hapten to 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. Dialyze with PBS buffer solution for 2 days, during which the PBS buffer solution was changed 4 times to obtain salbutamol artificial antigen.

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Abstract

The invention discloses a synthesis method of a specific salbutamol artificial antigen, belonging to the technical field of biological chemical engineering. The synthesis method disclosed by the invention comprises the following steps of: activating salbutamol through a formaldehyde solution, and connecting 6-aminocaproic acid in the ortho-position of a phenolic hydroxyl group to obtain a salbutamol hapten; and coupling a carboxyl group on the salbutamol hapten with an amino group on a carrier protein to obtain the salbutamol artificial antigen. The synthesis method disclosed by the invention can make up for the insufficiencies and defects of the existing salbutamol antigen synthesis technologies, the salbutamol artificial antigen with high specificity is obtained, the specificity of a produced antibody is high, and the sensitivity is high; and experimental results show that the antiserum titre of an animal immunized by using the salbutamol artificial antigen disclosed by the invention can achieve 80000, the detection limit is 0.5ng / mL, and the half-inhibitory concentration IC50 is 5ng / mL. The antigen or the antibody disclosed by the invention can be used for establishing an enzyme-linked immunosorbent analytical method and a colloidal gold test strip rapid assay method so as to rapidly detect the residues of the salbutamol in a food and further realize broad application prospects.

Description

technical field [0001] The invention discloses a method for synthesizing a highly specific salbutamol artificial antigen, which belongs to the technical field of biochemical engineering. Background technique [0002] Salbutamol (salbutamol), is a short-acting β2 adrenoceptor agonist, used as an asthma drug. Adding a small amount of salbutamol to livestock feed can increase the lean meat mass and meat replacement rate of livestock, and reduce fat, but its toxicity is much higher than that of ractopamine, which has the same function. From September 10, 2002, the use of albuterol in feed and animal drinking water was prohibited in China. The structural formula of salbutamol is as formula (1). [0003] [0004] Formula 1) [0005] At present, the detection methods of albuterol in my country mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass chromatography (LC / MS), enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, EL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K16/44G01N33/68G01N33/53
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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