Synthesis method of structurally specific salbutamol complete antigen

A technology of salbutamol and complete antigen, applied in the field of biochemical industry, can solve the problems of high detection cost, long cycle, complicated preparation, etc., and achieve the effect of high sensitivity and high specificity

Active Publication Date: 2014-04-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The instrumental analysis method has the disadvantages that the sample must be diluted, filtered, and extracted in multiple steps, and the preparation is complicated and cumbersome.
Although the instrument method is the confirmatory method for the detection of albuterol, due to its cumbersome operation and long sample pretreatment process, the detection cost is high and the cycle is long, which cannot meet the requirements of rapid screening of large batches of samples and rapid on-site detection

Method used

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  • Synthesis method of structurally specific salbutamol complete antigen
  • Synthesis method of structurally specific salbutamol complete antigen
  • Synthesis method of structurally specific salbutamol complete antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of albuterol hapten

[0033] 2-Bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethan-1-one 500mg, dissolved in 20mL acetone, add 560mg potassium carbonate, mix well, then add 430μL dibenzylamine 60℃ Reflux for 12 hours to generate compound B; add 230 mg lithium aluminum hydride to react for 12 hours at -20°C to generate compound C; under the catalysis of Pd / C, react with hydrogen gas to generate compound D; add 0.3 mL of ethyl levulinate, 0.6 mL Triethylamine, 430mg of sodium acetoxyborohydride reacted for 12h to generate compound E; add 6mL of 1mol / L NaOH solution for hydrolysis for 4h, rotary steam, add 10mL of water to dissolve, adjust the pH of the reaction solution to 3.0, stand at 4°C Recrystallize to obtain a white precipitate, which is the salbutamol hapten after centrifugal drying.

Embodiment 2

[0034] Embodiment 2, the preparation of albuterol complete antigen

[0035] Take 25mg albuterol hapten, add 2mL DMF to dissolve, then add NHS and EDC respectively (the molar ratio of hapten: NHS:EDC is 1: 1.5: 2), mix well at 4°C in the dark, stir the reaction for 60min, and then Reaction at room temperature 25 ℃ for 12h. Take 75mg bovine serum albumin (the molar ratio of hapten and bovine serum albumin is 80:1), add 10mL 0.1M pH9.6 carbonate buffer. The activated hapten solution was slowly added dropwise to the protein solution, and reacted at room temperature for 24 hours. Dialyze with PBS buffer solution for 2 days, during which the medium was changed 4 times to obtain the complete antigen of albuterol.

Embodiment 3

[0036] Embodiment 3, the preparation of albuterol complete antigen

[0037] Take 25mg albuterol hapten, add 2mL 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 1h. Take 75mg 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 30 minutes. At 0°C, the activated hapten solution was slowly added dropwise to the protein 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 medium was changed 4 times to obtain the complete antigen of albuterol.

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Abstract

The invention discloses a synthesis method of a structurally specific salbutamol complete antigen and belongs to the technical field of biochemical engineering. The synthesis method comprises the following steps: dissolving 2-bromo-1-[4-hydroxy-3-(hydroxymethyl)phenyl]aceto-1-ketone in acetone; after adding potassium carbonate, reacting with dibenzylamine to generate a compound B; generating a compound C under catalysis of lithium aluminium hydride; reacting with hydrogen under catalysis of Pd/C to generate a compound D; performing a reductive amination reaction with ethyl levulinate to generate a compound E; and hydrolyzing under alkaline condition, and recrystallizing to generate salbutamol hapten named as 4-((2-hydroxy-2-(4-hydroxy-3-(hydroxymethyl)phenyl)ethyl) amino) valeric acid. A complete antigen is obtained by coupling the carboxyl on the hapten and amidogen on carrier proteins. Experimental results show that the antiserum titer of animals immune to the complete antigen achieves 81,000, the limit of detection is 0.3ng/mL, and half inhibiting concentration is 3.2ng/mL; a generated antibody is high in specificity and sensitivity; the antigen or the antibody has a broad application prospect.

Description

technical field [0001] The invention discloses a method for synthesizing a structure-specific complete antigen of albuterol, which belongs to the technical field of biochemical engineering. Background technique [0002] Salbutamol (salbutamol), is a short-acting β2 adrenergic receptor 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 salbutamol analogues with the same function. From September 10, 2002, the use of albuterol in feed and animal drinking water was prohibited in China. [0003] [0004] [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, ELISA), colloidal gold test strip method, etc. . The ins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/77C07K1/107C07K16/44G01N33/53
CPCC07K14/765C07K14/77C07K14/795C07K16/44C07K19/00
Inventor 胥传来刘丽强王利兵
Owner JIANGNAN UNIV
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