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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

SDS-PAGE of the purified β2-AR protein from cells transfected with the plasmid of pTriEx-1.1 Hygro-β2-AR.(Lanes 1-3) 10 μL of Ni-NTA-purified protein, (lane M) molecular weight standards. The receptor expressed in HEK293 was glycosylated and migrated as 2 bands, with the minor band showing an apparent molecular mass of around 47 kDa and the major band around 52 kDa.
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pone.0139176.g004: SDS-PAGE of the purified β2-AR protein from cells transfected with the plasmid of pTriEx-1.1 Hygro-β2-AR.(Lanes 1-3) 10 μL of Ni-NTA-purified protein, (lane M) molecular weight standards. The receptor expressed in HEK293 was glycosylated and migrated as 2 bands, with the minor band showing an apparent molecular mass of around 47 kDa and the major band around 52 kDa.

Mentions: Based on the principle of combination between His-tag protein and Ni2+, the recombinant receptor was purified by Ni-affinity chromatography adopted with Ni-NTA produced by Qiagen. Unlike Ni-iminodiacetic acid chelated by Ni2+ with triple bond, the purification filler was chelated by Ni2+ with quadrivalent bond free from abscission in the maximal degree, so as to guarantee the protein of high purity. To realize the best eluting effect, the imidazole concentration in the elution buffer was optimized. In Fig 3, it can be observed that β2-AR was eluted completely when the imidazole concentration reached 250 mmol/L in the elution buffer. The purified β2-AR appeared in electrophoresis as 2 predominant forms of 52 kDa and 47 kDa, respectively (Fig 4). It was stored at -80°C at a concentration of 80,000 μg/L.


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)

SDS-PAGE of the purified β2-AR protein from cells transfected with the plasmid of pTriEx-1.1 Hygro-β2-AR.(Lanes 1-3) 10 μL of Ni-NTA-purified protein, (lane M) molecular weight standards. The receptor expressed in HEK293 was glycosylated and migrated as 2 bands, with the minor band showing an apparent molecular mass of around 47 kDa and the major band around 52 kDa.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139176.g004: SDS-PAGE of the purified β2-AR protein from cells transfected with the plasmid of pTriEx-1.1 Hygro-β2-AR.(Lanes 1-3) 10 μL of Ni-NTA-purified protein, (lane M) molecular weight standards. The receptor expressed in HEK293 was glycosylated and migrated as 2 bands, with the minor band showing an apparent molecular mass of around 47 kDa and the major band around 52 kDa.
Mentions: Based on the principle of combination between His-tag protein and Ni2+, the recombinant receptor was purified by Ni-affinity chromatography adopted with Ni-NTA produced by Qiagen. Unlike Ni-iminodiacetic acid chelated by Ni2+ with triple bond, the purification filler was chelated by Ni2+ with quadrivalent bond free from abscission in the maximal degree, so as to guarantee the protein of high purity. To realize the best eluting effect, the imidazole concentration in the elution buffer was optimized. In Fig 3, it can be observed that β2-AR was eluted completely when the imidazole concentration reached 250 mmol/L in the elution buffer. The purified β2-AR appeared in electrophoresis as 2 predominant forms of 52 kDa and 47 kDa, respectively (Fig 4). It was stored at -80°C at a concentration of 80,000 μg/L.

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