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J Biochem Biophys Methods. 2004 Jul 30;60(1):69-79.

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors.

Author information

1
Department of Discovery Biology, 3-Dimensional Pharmaceuticals Inc., 665 Stockton Drive, Exton, PA 19341, USA. cbauman4@prdus.jnj.com

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.

PMID:
15236912
DOI:
10.1016/j.jbbm.2004.05.003
[Indexed for MEDLINE]

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