title: Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy.
abstract: Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques to measure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrP(Sc)) was comparable to that of enzyme-linked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis.
authors.affiliation: Laboratory of Supramolecular Biophysics, Research Institute of Electronic Science, Hokkaido University, Sapporo 060-0818, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Fujii
authors.forename: Motohiro
authors.initials: M
authors.lastname: Horiuchi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Ueno
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakata
authors.forename: Issei
authors.initials: I
authors.lastname: Nagao
authors.forename: Mamoru
authors.initials: M
authors.lastname: Tamura
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay; D011328:Prions / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods