title: Determination of receptor protein binding site specificity and relative binding strength using a time-resolved competition assay.
abstract: BACKGROUND
  Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand.
  
  
  METHODS
  To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain.
  
  
  RESULTS
  Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement.
  
  
  CONCLUSIONS
  The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.
authors.affiliation: Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Charles University in Prague, Hyerovskeho 1203, 50005 Hradec Kralove, Czech Republic. Electronic address: pavel.barta@faf.cuni.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Barta
authors.affiliation: Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Charles University in Prague, Hyerovskeho 1203, 50005 Hradec Kralove, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Volkova
authors.affiliation: Rudbeck Laboratory/BMS, Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Dag Hammarskjölds väg 20, 75185, Uppsala, Sweden.
authors.forename: Adrian
authors.initials: A
authors.lastname: Dascalu
authors.affiliation: Rudbeck Laboratory/BMS, Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Dag Hammarskjölds väg 20, 75185, Uppsala, Sweden.
authors.forename: Diana
authors.initials: D
authors.lastname: Spiegelberg
authors.affiliation: Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Charles University in Prague, Hyerovskeho 1203, 50005 Hradec Kralove, Czech Republic.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Trejtnar
authors.affiliation: Rudbeck Laboratory/BMS, Department of Radiology, Oncology and Radiation Sciences, Uppsala University, Dag Hammarskjölds väg 20, 75185, Uppsala, Sweden; Ridgeview Instruments AB, Skillsta 4, 74020 Vänge, Sweden.
authors.forename: Karl
authors.initials: K
authors.lastname: Andersson
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000494:pharmacology*; D001667:Binding, Competitive / Q000187:drug effects*; D000068818:Cetuximab; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D000077544:Panitumumab; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D008024:Ligands; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D000077544:Panitumumab; D066246:ErbB Receptors; D000068818:Cetuximab
keywords: Binding assay; Cetuximab; Competition assay; Competitor; EGF receptor; Methods; Panitumumab; TGF-α; Tracer