Detection of recombinant bovine prion protein by fluorescence correlation spectroscopy
Fumihiko Fujii^'^, Hiroshi Sakata^'^, Masayoshi Ueno^, Takayuki Ya- nagiya^, Mamoru Tamura^ and Masataka Kinjo^
Inovation Plaza Hokkaido, JST, Nishi 11-chome, Kita 19-jyo, Kita-ku, Japporo 060-0819 Japan ^Fujirebio
<e-mail> ffiijii@sapporo.jst-plaza.jp
Sapporo 060-0819 Japan ^Fujirebio Inc. ^RIES, Hokkaido Univ.
Abstract
Fluorescence correlation spectroscopy (FCS) is a detection technique for molecular parameters, such as concentration, diffusion constant and inter- action in solution with single molecule level. FCS consists of a confocal optics w^ith a well focused laser beam and detects fluorescent fluctuation that is caused by the diffusion of fluorescently labeled molecules through a tiny open volume element (< one femtoliter). Measured fluctuation can be used to calculate the lateral diffusion coefficient and thus the size of the molecule. We propose FCS as a newly method for the detection of prion diseases.
Although FCS is well suited for development of homogeneous assays, an obstacle lies on the detection of antibody-antigen systems. The dif- ference in molecular weight has to be at least a factor of four between un- bound fluorescently-labeled antibodies and the immune complexes, be- cause FCS sensitivity is based on the diffusion coefficients. Therefore, it was difficult to discriminate the immune complexes of prion (180 kDa) and labeled antibodies (150 kDa). To overcome this limitation, we have applied the fluorescent-labeled Fab' fragments (50 kDa) and IgG for the detection of monomeric prion proteins (30 kDa).
By using FCS, recombinant bovine prion proteins could be detected at about 5 nM concentrations. The value was about one order less sensitive than ELISA analysis. However, this study provides the basis for a rapid and specific assay method test for prion diseases using FCS.
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