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Duplexed On‐Microbead Binding Assay for Competitive Inhibitor of Epidermal Growth Factor Receptor by Quantitative Flow Cytometry
Author(s) -
Lan WenJun,
Hao GaoKe,
Wang Jin,
Zhang RuiHua,
Lan Wei,
Wang RuiMing,
Sun Rui,
Wang TengFei
Publication year - 2010
Publication title -
basic and clinical pharmacology and toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.805
H-Index - 90
eISSN - 1742-7843
pISSN - 1742-7835
DOI - 10.1111/j.1742-7843.2010.00538.x
Subject(s) - microbead (research) , competitive binding , epidermal growth factor receptor , flow cytometry , epidermal growth factor , fluorescein isothiocyanate , tyrosine kinase , chemistry , microbiology and biotechnology , receptor , ligand binding assay , autophosphorylation , fluorescence , biology , kinase , biochemistry , protein kinase a , physics , quantum mechanics
Upon binding agonist, the epidermal growth factor receptor (EGFR) is dimerized and auto‐phosphorylated to activate downstream pathway that induces diverse physiology and pathology processes. Conventional methods for evaluation of EGFR inhibitors are limited. This study describes a duplexed on‐microbead binding assay allowing competitive EGFR inhibitors to be quantificationally evaluated in vitro . Polystyrene microbeads barcoded by fluoresceine isothiocyanate fluorescence as high brightness and low brightness microspheres were coated with receptor tyrosine kinase (RTK) ligand‐epidermal growth factor (EGF)/stem cell factor (SCF) and ATP/GTP, respectively. High and low brightness microbeads were mixed and incubated with EGFR and its competitive inhibitor in binding assay buffer. Phycoerythrin (PE) fluorescence‐labelled antibody was employed to report the level of EGFR binding to EGF/SCF and ATP/GTP. Values were numbered via PE molecules assessed by quantitative flow cytometry. Results from this study demonstrated that incubation with EGFR identified by PE‐labelled antibody can make EGF‐ and ATP‐coated microbeads luminous. And EGF or ATP‐competitive EGFR inhibitors, respectively, alleviated this in a concentration‐dependent manner. Coating microbeads with SCF or GTP as a negative control cannot capture EGFR. The duplexed on‐microbead binding assay in this study might be useful for discovering ligand‐ and ATP‐competitive EGFR inhibitors in a rapid and quantificational approach.