Critical factors influencing prion inactivation by sodium hydroxide.
OBJECTIVE
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.


METHODS
A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) 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 degrees 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 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 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 PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.
Bauman
P A
PA
Talecris Biotherapeutics, Research Triangle Park, NC, USA.
Lawrence
L A
LA
Biesert
L
L
Dichtelmüller
H
H
Fabbrizzi
F
F
Gajardo
R
R
Gröner
A
A
Jorquera
J I
JI
Kempf
C
C
Kreil
T R
TR
von Hoegen
I
I
Pifat
D Y
DY
Petteway
S R
SR
Cai
K
K
D000818:Animals; D001681:Biological Assay; D006224:Cricetinae; D003666:Decontamination; D019286:Endopeptidase K / Q000737:chemistry*; D018620:PrPSc Proteins / Q000737:chemistry* / Q000472:pathogenicity; D017096:Prion Diseases / Q000517:prevention & control*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry; D012972:Sodium Hydroxide / Q000737:chemistry*
D018620:PrPSc Proteins; D012972:Sodium Hydroxide; C025231:sarkosyl; D019286:Endopeptidase K; D012521:Sarcosine