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Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

Wang J, She Y, Wang M, Jin M, Li Y, Wang J, Liu Y - PLoS ONE (2015)

Bottom Line: After activity identification, the recombinant receptor was used in the development of direct competitive ELRA.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Quality Standards and Testing Technology for Agro-products of CAAS, Key Laboratory of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing, 100081, P. R. China; Department of Food Science, Hebei North University, Zhangjiakou, 075000, P. R. China.

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.

No MeSH data available.


Related in: MedlinePlus

The calibration curves constructed by plotting the B/B0 ratio against the concentration of β-agonists.Each value represents the mean ± SD from 3 independent measurements.
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pone.0139176.g005: The calibration curves constructed by plotting the B/B0 ratio against the concentration of β-agonists.Each value represents the mean ± SD from 3 independent measurements.

Mentions: A typical calibration curve was constructed and illustrated in Fig 5 by plotting [B/B0] × 100% against the β-agonist concentration, where B and B0 were the absorbance of the β-agonist at the standard point and at zero concentration of the β-agonist, respectively (Table D in S1 Document). The IC50 values of CBL, SAL, and RAC calculated from the fitting equations were 34, 53, and 63 μg/L, respectively. The IC50 values in this study were much lower than that of the reported radiolabeled receptor assay (higher than 110 μg/L) [37], and in the same order of magnitude with the previous ELRA method [38]. Besides, among the above 3 studies, the lowest IC50 value of each study originated from CBL, SAL, and RAC, respectively. The difference may be ascribed to several possible reasons. However, the main reason was the different structure and binding affinity caused by the various sources of β2-AR. Moreover, different expressions and detection systems might also contribute to it.


Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

Wang J, She Y, Wang M, Jin M, Li Y, Wang J, Liu Y - PLoS ONE (2015)

The calibration curves constructed by plotting the B/B0 ratio against the concentration of β-agonists.Each value represents the mean ± SD from 3 independent measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589316&req=5

pone.0139176.g005: The calibration curves constructed by plotting the B/B0 ratio against the concentration of β-agonists.Each value represents the mean ± SD from 3 independent measurements.
Mentions: A typical calibration curve was constructed and illustrated in Fig 5 by plotting [B/B0] × 100% against the β-agonist concentration, where B and B0 were the absorbance of the β-agonist at the standard point and at zero concentration of the β-agonist, respectively (Table D in S1 Document). The IC50 values of CBL, SAL, and RAC calculated from the fitting equations were 34, 53, and 63 μg/L, respectively. The IC50 values in this study were much lower than that of the reported radiolabeled receptor assay (higher than 110 μg/L) [37], and in the same order of magnitude with the previous ELRA method [38]. Besides, among the above 3 studies, the lowest IC50 value of each study originated from CBL, SAL, and RAC, respectively. The difference may be ascribed to several possible reasons. However, the main reason was the different structure and binding affinity caused by the various sources of β2-AR. Moreover, different expressions and detection systems might also contribute to it.

Bottom Line: After activity identification, the recombinant receptor was used in the development of direct competitive ELRA.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Quality Standards and Testing Technology for Agro-products of CAAS, Key Laboratory of Agro-Product Quality and Safety, Ministry of Agriculture, Beijing, 100081, P. R. China; Department of Food Science, Hebei North University, Zhangjiakou, 075000, P. R. China.

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.

No MeSH data available.


Related in: MedlinePlus