|dc.description.abstracteng||In the present work, we were interested in applying different recombinant prion
proteins (rec PrP) as substrates in the real-time quaking-induced conversion (RT-QuIC)
assay to improve the diagnostic accuracy in different prion diseases. We were also
interested in adapting the RT-QuIC for detecting infectious isoform of prion protein
(PrPSc) in a less invasive biological fluid (tear fluid) samples from prion disease patients.
Finally, we intended to study the morphology of fibrils generated during the RT-QuIC by
different substrates and the characteristics of the competitive seeding of different rec PrP
To this aim, hamster-sheep, full-length human (FL Hu), FL Hu with E200K, and fatal
familial insomnia (FFI) mutations were produced and purified through fast protein liquid
chromatography (FPLC). Subsequently, different rec PrP substrates were tested in the RTQuIC using CSF and tear fluid from patients with prion and non-prion disease controls.
In the CSF RT-QuIC, we observed an increase of the sensitivity in prion diseases 95%
sCJD patients (with different codon PRNP 129 MV geno- and subtypes), 100 % in E200K,
and 88 % in FFI) when using FL Hu E200K substrate compared to hamster-sheep (84.6
% in sCJD, 88 % in E200K, and 23 % in FFI) and FL Hu (76 % in sCJD, 88 % in E200K,
and 36 % in FFI) substrates (retrospective study). We also observed a reduced lag phase
duration and higher area under the curve (AUC) values when using FL Hu E200K
substrate (compared to the other substrate) in different prion diseases.
Consequently, a reduction of the duration of the assay to 60 h (instead of 80 h) can be
considered. Another prospective study comparing the agreement between the rec Hu
E200K substrate with the hamster-sheep (in routine use) revealed a substantial agreement
between both substrates.
Transmission electron microscope studies indicated different subtle morphologies of
PrP-fibrils generated during the RT-QuIC with different rec substrates. A mixture of
different rec PrP substrates (e.g., Hu E200K substrate with hamster-sheep) provoked a
mean signal response in the RT-QuIC, which indicates that two rec PrP substrates with
different PRNP sequences do not inhibit each other; they were able to convert and
aggregate independently from each other.
In tear fluid samples from prion disease patients, we used the higher sensitive FL Hu
E200K substrate in the RT-QuIC. Here, we observed a sensitivity of 75 %, while the
specificity remained at 100 %.
Our study demonstrated that using the FL Hu E200K substrate in the CSF RT-QuIC
improved the sensitivity of the assay, in particular for FFI diagnosis, and it enables the
detection of PrPSc in less-invasive tear fluid samples, which may be quite useful not only
for diagnostic but also for follow-up studies (e.g., after therapeutic intervention).||de