1 Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland
2 ZLB Bioplasma AG, Bern, Switzerland
Correspondence
Fabian Käsermann
kaesermann{at}ibc.unibe.ch
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ABSTRACT |
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MAIN TEXT |
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An important aspect in the prevention of prion disease transmission is the disinfection of potentially contaminated fluids, equipment and medical instruments. Sodium hydroxide (NaOH) solutions are commonly used as a disinfectant for surgical instruments or for the cleaning of production equipment. It is known that NaOH has the potential to lower the infectivity of different TSE agents (Brown et al., 1984, 1986
; Di Martino et al., 1993
; Prusiner et al., 1981
). Unfortunately, these data are limited and contradictory (Tateishi et al., 1991
; Taylor, 2000
) and no inactivation kinetics of TSE agents by NaOH have been published so far. Therefore, the goal of this study was to investigate the effect of NaOH on PrPSc and to measure reduction kinetics. We have chosen an immunological approach: the measurement of PrPres by Western blot analysis.
In a first set of experiments, hamster brain homogenate was exposed to different concentrations of NaOH. Stock brain homogenates (10 % in Tris-buffered saline) from hamsters, which had been inoculated with the 263K hamster-adapted scrapie agent, were obtained from Bayer or BioReliance. The stock hamster brain homogenate was diluted with water to 0·125 % and re-homogenized for 45 s with a Miccra D-8 drive and a DS-8/P dispersing tool at approximately 23 000 r.p.m. (ART-Labortechnik). NaOH concentrations were adjusted to the desired molarity by addition of 1/20 of the total sample volume using NaOH stock solution (0·22 M). pH values did not deviate by more than 0·3 units of pH from theoretical values, as measured by indicator sticks, and did not change significantly over the period of incubation. The suspension was neutralized after different incubation periods at room temperature by adding the same amount of HCl in 1 M HEPES (pH 7·0). Subsequently, the PK-sensitive forms of prion protein were digested as follows: the samples were incubated with PK (Roche Diagnostics) at a final concentration of 150 µg ml-1 for 1 h at 37 °C. PK-resistant prion proteins (PrPres) were detected by Western blot analysis, as described by Lee et al. (2000). The prion proteins were then sedimented by centrifugation for 1 h at 20 200 g and pellets were suspended in protein loading buffer, heated and serially diluted in 0·5 log10 steps. Aliquots of the dilutions (15 µl) were separated on a 10 % Bis/Tris NuPAGE gel (Invitrogen) and transferred onto a Nitrocellulose membrane (Invitrogen). Detection of prion proteins was performed using the 3F4 antibody (Signet) as the primary antibody and alkaline phosphatase-conjugated goat anti-mouse IgG2a (Southern Biotech Associates) as the secondary antibody. Alkaline phosphatase activity was visualized using CDP-Star substrate (Applied Biosystems) and subsequent exposure to X-ray film.
The results demonstrate that treatment of the 0·125 % scrapie-infected brain homogenate with 0·1 M NaOH eliminated the PrPres signal on Western blots within seconds. Incubation with 10 mM NaOH only had a minor effect on the PrP signal. Whereas with 25 mM NaOH, an intermediate reduction rate of the PrPres signal was observed; PrPres was below the detection limit after 15 min of incubation in NaOH (Fig. 1a). In control experiments, serial dilutions (0·5 log10 steps) of NaOH-treated brain homogenates (0·1 M for 15 min) from infected and non-infected hamsters were analysed on Western blots without PK digestion. Prion proteins were observed in both scrapie-infected and non-infected samples, showing that NaOH treatment does not interfere with the immunological detection system (Fig. 1b
). The PrPres signal was absent in samples containing non-infected brain homogenate, whether the samples were exposed to NaOH or not (data not shown).
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Logarithmic reduction factors were calculated for each NaOH concentration and at each time point. Treatment with 0·1 M NaOH resulted in a rapid reduction of at least 3·54 log10 PrPres. Treatment with 25 and 50 mM NaOH also showed a reduction of 3·54 log10 after a prolonged incubation time. With 10 mM NaOH, a minor reduction in PrPres was observed (Fig. 2).
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Samples were either digested with PK (150 µg ml-1 for 1 h at 37 °C in PBS) (+PK) or were kept untreated (in PBS) (-PK) followed by washing in PBS. The remaining proteins were dissociated from the iron particles by heating the iron powder samples in protein loading buffer. Proteins were then separated by SDS-PAGE and analysed by Western blotting. An aliquot representing the re-dissolved material from a surface of approximately 50 cm2 was loaded per lane on the gel. Prion protein was detectable in all samples that were not treated with PK (-PK). In samples kept at neutral pH and digested with PK, a signal corresponding to PrPres was detected (+PK). In samples incubated with NaOH, the PrPres signal was seen only at the starting point (0 min); in samples incubated for 15 min in NaOH, no PrPres signal was observed (Fig. 3).
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The results of the present study show that if traces of infectious prion proteins would contaminate metal surfaces, such as surgical instruments or production equipment in the blood industry, the PK resistance of these prion proteins would be eliminated efficiently by treatment with NaOH. Our results, which showed clearance of PrPSc (4 log10 with 0·1 M NaOH at room temperature), are in good agreement with previously published single-point measurements that demonstrated loss of infectivity (
4·8 log10 of hamster scrapie and CJD in guinea pigs upon exposure to 0·1 M NaOH) (Brown et al., 1986
). In addition, it seems that other groups may have obtained inactivation at high pH (>11): Somerville (2002)
described inactivation at elevated temperature and stated, as unpublished results, that similar effects are found with increasing pH (>pH 11)...
Compared to earlier studies that reported contradictory results, we used a much lower concentration of prion protein, which might serve as an explanation for the differences observed. However, the final concentration of 0·125 % brain homogenate is much closer to the expected real-life situation. The results of this study, obtained in biochemical in vitro experiments, form a good basis for further investigations. Further studies, especially the direct determination of infectivity in bioassays, need to be done to confirm the in vitro results.
If the infectivity of TSE agents is obligatorily linked to PrPres, our results imply that a commonly used cleaning procedure for the decontamination of metal surfaces (NaOH treatment) can substantially lower the risk of TSE cross-contamination. This inactivation of TSE occurs much faster and more readily than currently assumed. Furthermore, based on this study, a method for the disinfection of metal surfaces, such as surgical instruments potentially contaminated with TSE, can be proposed: equipment should be soaked for a short period of time in NaOH and the protease-sensitive prion proteins can then be removed with PK treatment.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, P., Rohwer, R. G. & Gajdusek, D. C. (1986). Newer data on the inactivation of scrapie virus or CreutzfeldtJakob disease virus in brain tissue. J Infect Dis 153, 11451148.[Medline]
Caughey, B., Raymond, G. J., Kocisko, D. A. & Lansbury, P. T., Jr (1997). Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies. J Virol 71, 41074110.[Abstract]
Di Martino, A., Safar, J., Ceroni, M. & Gibbs, C. J., Jr (1993). Inactivation of the scrapie agent in a scaled-down procedure for the purification of gangliosides from brain tissue. Dev Biol Stand 80, 187194.[Medline]
Flechsig, E., Hegyi, I., Enari, M., Schwarz, P., Collinge, J. & Weissmann, C. (2001). Transmission of scrapie by steel-surface-bound prions. Mol Med 7, 679684.[Medline]
Foster, P. R., Welch, A. G., McLean, C., Griffin, B. D., Hardy, J. C., Bartley, A., MacDonald, S. & Bailey, A. C. (2000). Studies on the removal of abnormal prion protein by processes used in the manufacture of human plasma products. Vox Sang 78, 8695.[CrossRef][Medline]
Lee, D. C., Stenland, C. J., Hartwell, R. C. & 7 other authors (2000). Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein. J Virol Methods 84, 7789.[CrossRef][Medline]
Lee, D. C., Stenland, C. J., Miller, J. L. & 8 other authors (2001). A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins. Transfusion 41, 449455; erratum 41, 1079.
McKinley, M. P., Bolton, D. C. & Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 5762.[Medline]
Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A. & Prusiner, S. B. (1986). Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci U S A 83, 23102314.[Abstract]
Miller, J. L., Petteway, S. R., Jr & Lee, D. C. (2001). Ensuring the pathogen safety of intravenous immunoglobulin and other human plasma-derived therapeutic proteins. J Allergy Clin Immunol 108 (Suppl. 4), S91S94.[CrossRef][Medline]
Oesch, B., Doherr, M., Heim, D. & 7 other authors (2000). Application of Prionics Western blotting procedure to screen for BSE in cattle regularly slaughtered at Swiss abattoirs. Arch Virol 16, 189195.
Prusiner, S. B. (1998). Prions. Proc Natl Acad Sci U S A 95, 1336313383.
Prusiner, S. B., Groth, D. F., McKinley, M. P., Cochran, S. P., Bowman, K. A. & Kasper, K. C. (1981). Thiocyanate and hydroxyl ions inactivate the scrapie agent. Proc Natl Acad Sci U S A 78, 46064610.[Abstract]
Race, R., Jenny, A. & Sutton, D. (1998). Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis. J Infect Dis 178, 949953.[Medline]
Safar, J., Wang, W., Padgett, M. P., Ceroni, M., Piccardo, P., Zopf, D., Gajdusek, D. C. & Gibbs, C. J., Jr (1990). Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. Proc Natl Acad Sci U S A 87, 63736377.[Abstract]
Safar, J., Roller, P. P., Gajdusek, D. C. & Gibbs, C. J., Jr (1993). Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J Biol Chem 268, 2027620284.
Schaller, O., Fatzer, R., Stack, M. & 9 other authors (1999). Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol 98, 437443.[CrossRef][Medline]
Shaked, G. M., Fridlander, G., Meiner, Z., Taraboulos, A. & Gabizon, R. (1999). Protease-resistant and detergent-insoluble prion protein is not necessarily associated with prion infectivity. J Biol Chem 274, 1798117986.
Somerville, R. A. (2002). TSE agent strains and PrP: reconciling structure and function. Trends Biochem Sci 27, 606612.[CrossRef][Medline]
Somerville, R. A., Oberthur, R. C., Havekost, U., MacDonald, F., Taylor, D. M. & Dickinson, A. G. (2002). Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications. J Biol Chem 277, 1108411089.
Tateishi, J., Tashima, T. & Kitamoto, T. (1991). Practical methods for chemical inactivation of CreutzfeldtJakob disease pathogen. Microbiol Immunol 35, 163166.[Medline]
Taylor, D. M. (2000). Inactivation of transmissible degenerative encephalopathy agents. Vet J 159, 1017.[CrossRef][Medline]
Wille, H., Prusiner, S. B. & Cohen, F. E. (2000). Scrapie infectivity is independent of amyloid staining properties of the N-terminally truncated prion protein. J Struct Biol 130, 323338.[CrossRef][Medline]
Received 15 May 2003;
accepted 16 July 2003.