title: Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors.
abstract: Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.
authors.affiliation: Department of Discovery Biology, 3-Dimensional Pharmaceuticals Inc., 665 Stockton Drive, Exton, PA 19341, USA. cbauman4@prdus.jnj.com
authors.forename: Christian A
authors.initials: CA
authors.lastname: Baumann
authors.forename: Lee
authors.initials: L
authors.lastname: Zeng
authors.forename: Robert R
authors.initials: RR
authors.lastname: Donatelli
authors.forename: Anna C
authors.initials: AC
authors.lastname: Maroney
mesh_terms: D001331:Automation; D001671:Biochemistry / Q000379:methods; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation; D020128:Inhibitory Concentration 50; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D016184:Receptors, Colony-Stimulating Factor / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D003902:Detergents; D000939:Epitopes; D016184:Receptors, Colony-Stimulating Factor; D019000:Phosphotyrosine; D006639:Histidine; D017830:Octoxynol