Improved PMCA technology for diagnosis, strain typing, risk assessment and transconformation studies of prion diseases
2007: 2 years / Grant: 75 000 euros
Project: The first objective of the project “VolePMCA” was to assess whether vole brain homogenate could be used as substrate in PMCA (protein misfolded cyclic amplification) for the efficient amplification of prionsfrom differentsources. Another part of the project wasto investigate the possible occurrence of the de novo generation of prions in PMCA and to decipher the origin of this phenomenon.
Main results and related published data: Vole brain substrates were shown to be a powerful
substrate for both homologous and heterologous PMCA. Heterologous amplification wassuccessfully
obtained with different prion strains from humans and various animal species. This property of vole
substrate has been exploited in recent years by different authors and with different amplification
settings and techniques. The second part of the project tookthe cue from the observation that, after
a few serial automated PMCA (saPMCA) rounds, zputative de novo PK-resistant PrPTSE were
sometimes obse rved in non-prion seeded negative controls. By testing several PMCA conditions
(analysis in different laboratories, rigorous prion-free conditions, different PMCA settings, number
of consecutive rounds of PMCA and the age of the vole used as brain subtrate) the team proposed
that the most likely explanation for the appearance of de novo PrPTSE was the occurrence of crosscontamination
during saPMCA. The equipment used (pipettes, sonicator and thermal bath) and the
working environment (laminar flow hood, bench)seemed not to be critical forcontamination events.
However, the presence of high numbers of seeded tubes in the sonicator and the resulting aerosols following violent shaking was shown to be a determinant for cross contamination. This could be circumvented by the use of MultiplySafecup Biosphere tubes (screw cap and volume limitation system), but, in particular, by limiting the amplification rounds to one or two. In fact, ultra-efficient amplification conditions dramatically increase the risk of spurious amplifications deriving from accidental, minimal contaminations.