Open AccessQuaking-induced conversion of prion protein on a thermal mixer accelerates detection in brains infected with transmissible spongiform encephalopathy agents
Author(s) -
Nadine Kaelber,
Cyrus Bett,
David M. Asher,
Luisa Gregori
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0225904
Subject(s) - chronic wasting disease , virology , transmissible spongiform encephalopathy , recombinant dna , scrapie , titer , hamster , bovine spongiform encephalopathy , microbiology and biotechnology , incubation period , proteinase k , biology , incubation , prion protein , medicine , virus , pathology , enzyme , biochemistry , disease , gene
Detection of misfolded prion protein, PrP TSE , in biological samples is important to develop antemortem tests for transmissible spongiform encephalopathies (TSEs). The real-time quaking-induced conversion (RT-QuIC) assay detects PrP TSE but requires dedicated equipment and relatively long incubation times when applied to samples containing extremely low levels of PrP TSE . It was shown that a microplate shaker with heated top (Thermomixer-C) accelerated amplification of PrP TSE in brain suspensions of 263K scrapie and sporadic Creutzfeldt-Jakob disease (sCJD). We expanded the investigation to include TSE agents previously untested, including chronic wasting disease (CWD), macaque-adapted variant CJD (vCJD) and human vCJD, and we further characterized the assays conducted at 42°C and 55°C. PrP TSE from all brains containing the TSE agents were successfully amplified using a truncated hamster recombinant protein except for human vCJD which required truncated bank vole recombinant protein. We compared assays conducted at 42°C on Thermomixer-C, Thermomixer-R (without heated top) and on a fluorimeter used for RT-QuIC. QuIC on Thermomixer-R achieved in only 18 hours assay sensitivity similar to that of RT-QuIC read at 60 hours (or 48 hours with sCJD). QuIC on Thermomixer-C required 24 hours to complete and the endpoint titers of some TSEs were 10-fold lower than those obtained with RT-QuIC and Thermomixer-R. Conversely, at 55°C, the reactions with sCJD and CWD on Thermomixer-C achieved the same sensitivity as with RT-QuIC but in shorter times. Human vCJD samples tested at higher temperatures gave rise to high reactivity in wells containing normal control samples. Similarly, reactions on Thermomixer-R were unsuitable at 55°C. The main disadvantage of Thermomixers is that they cannot track formation of PrP fibrils in real time, a feature useful in some applications. The main advantages of Thermomixers are that they need shorter reaction times to detect PrP TSE , are easier to use, involve more robust equipment, and are relatively affordable. Improvements to QuIC using thermal mixers may help develop accessible antemortem TSE tests.