title: A rapid and high-throughput quantitation assay of the nuclear factor κB activity using fluorescence correlation spectroscopy in the setting of clinical laboratories.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  This assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.
authors.affiliation: Department of Clinical Laboratory and Molecular Pathology, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Toyama, Japan.
authors.forename: Kenu
authors.initials: K
authors.lastname: Harada
authors.forename: Shintaro
authors.initials: S
authors.lastname: Mikuni
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Beppu
authors.forename: Hideki
authors.initials: H
authors.lastname: Niimi
authors.forename: Shigeki
authors.initials: S
authors.lastname: Abe
authors.forename: Nobuko
authors.initials: N
authors.lastname: Hano
authors.forename: Koichi
authors.initials: K
authors.lastname: Yamagata
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.forename: Isao
authors.initials: I
authors.lastname: Kitajima
mesh_terms: D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000032:analysis*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D016328:NF-kappa B