Abstract
A novel enzyme-linked receptor assay (ELRA) based on β2-adrenergic receptor (β2-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.
Highlights
Multiresidue Detection Based on β2 Adrenergic Receptor enormous economic benefits, the illegal abuse of such agents never stopped, which caused many incidents of poisoning
The results showed that the aniline-type β-agonists clenbuterol had the highest affinity, were the phenolic-type β-agonists, such as ractopamine and salbutamol, and yet carbuterol and terbutaline showed the lowest affinity
To obtain abundant active β2-adrenergic receptor (β2-AR) is the premise for the development of receptor assay used for the multianalyte detection of β-agonists
Summary
Multiresidue Detection Based on β2 Adrenergic Receptor enormous economic benefits, the illegal abuse of such agents never stopped, which caused many incidents of poisoning. In addition to the abuse of some known β-agonists such as clenbuterol (CBL) and salbutamol (SAL), a series of novel β-agonist derivatives with similar structure and function have been synthetized to evade detection by routine screening methods [7,8]. It is urgently needed to establish a high-throughput screening approach for multiresidue determination of β-agonists
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