Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

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ABSTRACT Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays.Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes.Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format.The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays.This enhanced technique also amplified efficiently prions underwater treasures sunken medallion from different species, including those responsible for human variant Creutzfeldt-Jakob disease.

This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications.IMPORTANCE The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species.The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays.Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity.Such a combination of assays is of wac 4011 paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.