Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve

Atsuko Takeuchi, Mayumi Komiya, Tetsuyuki Kitamoto, Masanori Morita

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)502-509
Number of pages8
JournalMICROBIOLOGY and IMMUNOLOGY
Volume55
Issue number7
DOIs
Publication statusPublished - 2011 Jul

Keywords

  • Disinfectant
  • Evaluation
  • Prion
  • Protein misfolding cyclic amplification

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Virology

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