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Development of an enzyme‐linked immunosorbent assay and a beta‐1 adrenergic receptor–based assay for monitoring the drug atenolol
Author(s) -
Sapir A.,
Shalev A. Hariton,
Skalka N.,
Bronshtein A.,
Altstein M.
Publication year - 2013
Publication title -
environmental toxicology and chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.1
H-Index - 171
eISSN - 1552-8618
pISSN - 0730-7268
DOI - 10.1002/etc.2078
Subject(s) - ligand binding assay , polyclonal antibodies , receptor , atenolol , recombinant dna , chemistry , sf9 , microbiology and biotechnology , bioassay , radioligand assay , enzyme , transfection , immunoassay , g protein coupled receptor , adrenergic receptor , antibody , biology , biochemistry , spodoptera , immunology , endocrinology , gene , genetics , blood pressure
Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive‐binding assay, based on the interaction between ATL and its target receptor, β1 adrenergic receptor ( β1AR ). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme‐linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross‐react with any of the tested beta‐blocker drugs. Furthermore, a human β1AR ( h‐β1AR ) was stably expressed in Spodoptera frugiperda cells ( Sf9 ). The receptor was employed to develop a competitive‐binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h‐β1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose‐dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost‐effective large‐scale, high‐throughput monitoring of ATL in environmental, agricultural, and biological samples. Environ. Toxicol. Chem. 2013;32:585–593. © 2012 SETAC