Establishment of homogeneous time-resolved fluorescence immunoassay for high throughput screening of protein tyrosine kinase inhibitors / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors.</p><p><b>METHODS</b>Specific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-2) kinase activity was investigated.</p><p><b>RESULTS</b>A homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/microl, 100 micromol/L and 1 micromol/L, respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC50 value of 86.7 nmol/L, which was close to the values tested using other methods.</p><p><b>CONCLUSION</b>The homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.</p>