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Critical factors influencing prion inactivation by sodium hydroxide
Author(s) -
Bauman P. A.,
Lawrence L. A.,
Biesert L.,
Dichtelmüller H.,
Fabbrizzi F.,
Gajardo R.,
Gröner A.,
Jorquera J. I.,
Kempf C.,
Kreil T. R.,
Von Hoegen I.,
Pifat D. Y.,
Petteway S. R.,
Cai K.
Publication year - 2006
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/j.1423-0410.2006.00790.x
Subject(s) - proteinase k , scrapie , infectivity , chemistry , western blot , immunoprecipitation , in vitro , antibody , prion protein , epitope , microbiology and biotechnology , incubation , incubation period , virology , hamster , biochemistry , biology , enzyme , virus , immunology , medicine , gene , disease , pathology
Background and Objectives Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP Sc ; prions) that are generally resistant to conventional pathogen‐inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. Materials and Methods A decrease in prion infectivity correlates with the disappearance of the protease‐resistant core of PrP Sc (PrP RES ) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0·1 m NaOH at 4 or 18 °C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. Results Treatment of SBH with 0·1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 °C caused a reduction in the PrP RES signal of 3·5 and 4·0 log 10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60‐min incubation in NaOH further enhanced PrP RES reduction to ≥ 4·5 log 10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. Conclusions The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrP RES signal. Detergent may liberate lipid membrane‐protected PrP Sc to improve access to NaOH, which can then inactivate PrP Sc by altering its structure. In cases of unidentified exposure to PrP Sc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.