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SPR显微镜用于表皮生长因子受体表达水平及细胞表面结合动力学定量检测

发布时间: 2018-07-19 16:02 来源: 上海纳腾仪器有限公司
领域: 分子生物学,蛋白/抗体/蛋白质组,生物制药/仿制药,药物代谢
样品:A431cell,anti-EGFR项目:A431cell,anti-EGFR
参考:Anal. Chem., 2015, 87 (19), pp 9960–9965

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SPR显微镜用于表皮生长因子受体表达水平及细胞表面结合动力学定量检测

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Epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) is a membrane bound protein that has been associated with a variety of solid tumors and the control of cell survival, proliferation, and metabolism. Quantification of the EGFR expression level in cell membranes and the interaction kinetics with drugs are thus important for cancer diagnosis and treatment. Here we report mapping of the distribution and interaction kinetics of EGFR in their native environment with the surface plasmon resonance imaging (SPRi) technique. The monoclonal anti-EGFR antibody was used as a model drug in this study. The binding of the antibody to EGFR overexpressed A431 cells was monitored in real time, which was found to follow the first-order kinetics with an association rate constant (ka) and dissociation rate constant (kd) of (2.7 ± 0.6) × 105 M–1 s–1 and (1.4 ± 0.5) × 10–4 s–1, respectively. The dissociation constant (KD) was determined to be 0.53 ± 0.26 nM with up to seven-fold variation among different individual A431 cells. In addition, the averaged A431 cell surface EGFR density was found to be 636/μm2 with an estimation of 5 × 105 EGFR per cell. Additional measurement also revealed that different EGFR positive cell lines (A431, HeLa, and A549) show receptor density dependent anti-EGFR binding kinetics. The results demonstrate that SPRi is a valuable tool for direct quantification of membrane protein expression level and ligand binding kinetics at single cell resolution. Our findings show that the local environment affects the drug-receptor interactions, and in situ measurement of membrane protein binding kinetics is important.

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