Duplexed on-microbead binding assay for competitive inhibitor of epidermal growth factor receptor by quantitative flow cytometry.
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.
Laboratory for Biomedical Engineering & Drug Discovery, Shandong Institute of Light Industry, Jinan, China. lanwenjun0522@sina.com
Wen-Jun
WJ
Lan
Gao-Ke
GK
Hao
Jin
J
Wang
Rui-Hua
RH
Zhang
Wei
W
Lan
Rui-Ming
RM
Wang
Rui
R
Sun
Teng-Fei
TF
Wang
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology; D000068818:Cetuximab; D003164:Competitive Bidding; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000069347:Erlotinib Hydrochloride; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008863:Microspheres; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D004815:Epidermal Growth Factor; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors; D000068818:Cetuximab