title: A comparison of in vitro methods for determining the membrane receptor expression in cell lines.
abstract: BACKGROUND
  Determining the number of expressed receptors per cell (NRPC) in cell lines is an important prerequisite for many experimental procedures in biomedical research. This paper focuses on the comparison of a newly developed method of determining NRPC - the Kinetic extrapolation method (KEX) - with the standard saturation method. These two methods, both based on radiolabeled ligand-receptor binding, were compared with the data on receptor expression found using quantified western blotting.
  
  
  METHODS
  Four cell lines with different expressions of epidermal growth factor receptor (EGFR) were chosen for the experiment: A431, HaCaT, HCT116 and HepG2. Two radiolabeled monoclonal antibodies specific for EGFR were used as ligands: [(131)I]-cetuximab and [(131)I]-panitumumab. The classic manual technique based on the saturation of cell receptors was performed on cells seeded in 24-well plates. The KEX method uses the LigandTracer, a special instrument which detects ligand retention in real time from seeded cells onto a rotating Petri dish. The western blot analysis was performed according to the routinely used procedure.
  
  
  RESULTS
  A very close accordance between the manual saturation technique and the KEX method was found in all four cell lines used. The NRPC in the cell lines follows the same order using both ligands: A431>HaCaT>HCT116≈HepG2. Similarly, consistent data on EGFR expression in the studied cell lines were obtained using western blot analysis and the radiolabeled ligand binding assays.
  
  
  CONCLUSIONS
  The KEX method could be as similarly useful for determining receptor expression as is the classic saturation method and western blotting.
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic.
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Novy
authors.forename: Pavel
authors.initials: P
authors.lastname: Barta
authors.forename: Jana
authors.initials: J
authors.lastname: Mandikova
authors.forename: Milan
authors.initials: M
authors.lastname: Laznicek
authors.forename: Frantisek
authors.initials: F
authors.lastname: Trejtnar
mesh_terms: D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D013223:Statistics as Topic / Q000379:methods*
chemical_list: D008024:Ligands; D066246:ErbB Receptors