Department of Clinical Laboratory and Molecular Pathology, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, Japan.
PLoS One. 2013 Oct 4;8(10):e75579. doi: 10.1371/journal.pone.0075579. eCollection 2013.
Transcription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites.
Non-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured.
The percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes.
This assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.
转录因子核因子-κB（NF-κB）在调节免疫炎症反应中发挥关键作用。然而，临床实验室中并没有方便的检测系统可以及时定量 NF-κB 活性水平。因此，我们开发了一种基于荧光相关光谱（FCS）的新型高通量定量检测方法，用于检测细胞核提取物中的 NF-κB 活性水平，并评估该方法的性能。该方法的基本原理是计算 NF-κB 与荧光标记 DNA 探针的结合分数，这些探针含有 NF-κB 结合位点。
采用非荧光竞争探针来归一化样品之间核提取物的粘度的影响，并消除荧光探针的非特异性结合的影响。为了准确定量，将人重组 NF-κB p50 混入 U937 细胞核提取物中，并计算混合物中荧光探针与 NF-κB 的结合分数进行定量。为了评估该方法是否可用于测量人淋巴细胞中的 NF-κB 活性，测量了围手术期全身炎症反应综合征患者的 NF-κB 活性水平。
回收率为 88.9%。批内变异系数约为 10%。成功测量了围手术期的 NF-κB 活性水平。FCS 测量的检测时间在 20 分钟内。
该检测系统可用于在医院实验室中及时定量 NF-κB 活性水平。
