FORPRION

BAVARIAN RESEARCH ASSOCIATION PRIONS

LMU 8 Development of a Diagnostic Test for Prion Diseases in Blood

For epidemiological surveillance, for prevention of transmission, and for early diagnosis a prerequisite for any potential treatment of infected persons, there is an urgent need for a highly sensitive diagnostic test for prion disease suited for diagnostic specimens available for routine analysis such as body fluids, especially CSF and blood. As blood has been shown to contain infectivity (albeit at low titres) in animal models of prion disease (Brown et al., 2001) and as PrPSc has been shown to be the main component of the infectious particle, the principal aim of this project was to develop new methods that allow the development of a rapid and highly sensitive diagnostic test for PrPSc in the blood/CSF of humans and animals. Due to the low titre of infectivity that can be expected to be present in CSF and blood, direct detection of PrPSc by “classical” methods such as Western blot or ELISA appears to be impossible. Therefore, our approach was to • develop methods to enrich and amplify the PrPSc present in diagnostic specimens. • combine these methods with a novel highly sensitive single particle detection ethod termed SIFT (“scanning for intensely fluorescent targets”) derived from ual-color fluorescence correlation spectroscopy (Bieschke et al., 2000, Giese et al., 2000) The first phase of the project was focussed on optimisation of the SIFT detection technology and on sample processing in model systems such as brain tissue and bood/CSF spiked with purified PrPSc. To optimise sample processing, we evaluated a number of protocols for biochemical purification of PrPSc in regard to speed, yield, enrichment factor, and compatibility with SIFT detection of PrPSc-aggregates. Special emphasis was put on i) the optimisation of homogenisation parameters and lysis buffer in regard to yield and reliability, ii) comprehensive comparison of different tissues and body fluids, animal species, and prion strains in regard to purification properties of PrPSc, iii) automation of sample processing, iv) comprehensive analysis of a range of antibodies (including new antibodies resulting from an ongoing collaboration with J. Grassi, CEA, France) in regard to detection of PrPSc from different species and prion strains, and v) the establishment of automated software for analysis of SIFT data. Importantly, we also used a new method termed PMCA (“protein misfolding cyclic amplification”) that allows amplification of PrPSc in a cyclic process analogous to the amplification of nucleic acids in the PCR reaction. By inventing the method of serial PMCA, we could provide a technique that – for the first time – allows amplification of PrPSc by several orders of magnitude. In the second project period we focussed on the refinement of individual test components with special regard to automation in the context of a rapid diagnostic test and in regard to optimised combination of the sequential processing steps, PrPSc amplification, biochemical enrichment of PrPSc and SIFT detection, respectively. Tasks for the second funding period included i) improvement of amplification factor by optimisation of PMCA parameters, by addition of potential co-factors (e.g. various metal ions), and by addition of additional PrP substrate for conversion (e.g. recombinant PrP derived from E. coli and purified PrPC that is glycosylated and contains a GPI anchor), ii) automation of PMCA and use of an indirect sonication system in sealed multi-well plates instead of "open" sonication using a microtip, iii) evaluation of the effect of blood/CSF or blood components on PMCA efficiency, iv) further analysis of the biochemical, biophysical, and biological properties of PrPres generated in vitro. Finally, we wanted to make the newly developed methods widely available for diagnostic and scientific use by dissemination of the necessary technology and knowhow.