Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

摘要

Prion diseases or transmissible spongiform encephalopathies(TSEs)are characterized by the accumulation in the brain of PrP~(Sc),an abnormal isoform of the host-encoded glycoprotein PrP~C.PrP~(Sc)is a reliable marker for the post mortem diagnosis of TSEs but its use as a marker for a pre-clinical blood test has been hampered by the low levels of PrP~(Sc)in blood.We have evaluated confocal fluorescence spectroscopy(CFS)as an ultrasensitive method for the immunological detection of PrP in brain homogenates and blood serum.Using recombinant PrP,we determined the detection limit of our CFS assay to be in the picomolar range which is in the same order of magnitude as the corresponding luminescence immunoassay(Prionics~R-Check LIA).The analytical sensitivity was further investigated using brain homogenates from BSE infected cattle that showed low levels of PrP~(Sc)by Western blot analysis.In these brain homogenates,PrP~(Sc)was detected by confocal fluorescence spectroscopy down to a dilution of 64-fold whereas the luminescence immunoassay was able to detect PrP~(Sc)in brain homogenates diluted down to at least 128-fold suggesting that in this antibody sandwich assay the luminescence immunoassay has a higher analytical sensitivity than confocal fluorescence spectroscopy.We also demonstrated that blood serum is a suitable matrix for a confocal fluorescence spectroscopy based TSE test,and found that serum did not interfere with the detection of spiked recombinant PrP.