Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic.
Nucl Med Biol. 2012 Oct;39(7):893-6. doi: 10.1016/j.nucmedbio.2012.02.007. Epub 2012 Apr 10.
Determining the number of expressed receptors per cell (NRPC) in cell lines is an important prerequisite for many experimental procedures in biomedical research. This paper focuses on the comparison of a newly developed method of determining NRPC - the Kinetic extrapolation method (KEX) - with the standard saturation method. These two methods, both based on radiolabeled ligand-receptor binding, were compared with the data on receptor expression found using quantified western blotting.
Four cell lines with different expressions of epidermal growth factor receptor (EGFR) were chosen for the experiment: A431, HaCaT, HCT116 and HepG2. Two radiolabeled monoclonal antibodies specific for EGFR were used as ligands: [(131)I]-cetuximab and [(131)I]-panitumumab. The classic manual technique based on the saturation of cell receptors was performed on cells seeded in 24-well plates. The KEX method uses the LigandTracer, a special instrument which detects ligand retention in real time from seeded cells onto a rotating Petri dish. The western blot analysis was performed according to the routinely used procedure.
A very close accordance between the manual saturation technique and the KEX method was found in all four cell lines used. The NRPC in the cell lines follows the same order using both ligands: A431>HaCaT>HCT116≈HepG2. Similarly, consistent data on EGFR expression in the studied cell lines were obtained using western blot analysis and the radiolabeled ligand binding assays.
The KEX method could be as similarly useful for determining receptor expression as is the classic saturation method and western blotting.
确定细胞系中表达的受体数量（NRPC）是许多生物医学研究实验程序的重要前提。本文重点比较了一种新开发的确定 NRPC 的方法 - 动力学外推法（KEX）与标准饱和法。这两种方法均基于放射性标记的配体-受体结合，并用使用定量 Western blot 发现的受体表达数据进行了比较。
选择了四个具有不同表皮生长因子受体（EGFR）表达的细胞系进行实验：A431、HaCaT、HCT116 和 HepG2。使用两种针对 EGFR 的放射性标记单克隆抗体作为配体：[（131）I]-西妥昔单抗和[（131）I]-帕尼单抗。经典的手动技术基于细胞受体的饱和，在接种于 24 孔板的细胞上进行。KEX 方法使用 LigandTracer，这是一种特殊的仪器，可以实时从接种的细胞检测到在旋转培养皿中的配体保留。Western blot 分析按照常规程序进行。
在使用的所有四个细胞系中，手动饱和技术和 KEX 方法之间发现非常紧密的一致性。使用两种配体，细胞系中的 NRPC 遵循相同的顺序：A431>HaCaT>HCT116≈HepG2。同样，使用 Western blot 分析和放射性标记的配体结合测定法，在研究的细胞系中获得了一致的 EGFR 表达数据。
KEX 方法在确定受体表达方面与经典饱和法和 Western blot 同样有用。
