title: Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve.
abstract: In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.
authors: Department of Neurological Science, Tohoku University Graduate School of Medicine, Japan.
authors: Atsuko
authors: A
authors: Takeuchi
authors: Mayumi
authors: M
authors: Komiya
authors: Tetsuyuki
authors: T
authors: Kitamoto
authors: Masanori
authors: M
authors: Morita
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002623:Chemistry Techniques, Analytical; D006224:Cricetinae; D004202:Disinfectants / Q000494:pharmacology*; D019286:Endopeptidase K / Q000378:metabolism; D011328:Prions / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017510:Protein Folding; D014773:Virology / Q000379:methods*
chemical_list: D004202:Disinfectants; D011328:Prions; D019286:Endopeptidase K