Ablation of prion protein immunoreactivity by heating in saturated calcium hydroxide.

Abstract

Prions, the infectious agents that cause transmissible spongiform encephalopathies (TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments. Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to digest protein components of offal. The purpose of this work was to determine if incubating brain material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution (Ca(OH)2) would abolish immunoreactivity of the infectious prion (PrPSc) as determined by western blot.
After incubating for as few as 10 minutes in saturated calcium hydroxide at 99 degrees C, immunoreactivity of protease resistant bands by western blot analysis is completely lost.
Boiling in limewater may offer an alternative for disposal of carcasses and enable alternative uses for rendered products from potentially infected carcasses.

Full text

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BMC Research Notes
Open Access
Short Report
Ablation of prion protein immunoreactivity by heating in saturated
calcium hydroxide
Justin J Greenlee*1, Eric M Nicholson*1, Amir N Hamir1, Gary P Noyes2,
Mark T Holtzapple3 and Marcus E Kehrli Jr1
Address: 1Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 2300 Dayton
Avenue, Ames, IA, USA, 50010, 2Oceaneering Space Systems, Houston, TX, USA 77058 and 3Department of Chemical Engineering, Texas A&M
University, College Station, TX, USA 77843
Email: Justin J Greenlee* - justin.greenlee@ars.usda.gov; Eric M Nicholson* - eric.nicholson@ars.usda.gov;
Amir N Hamir - amir.hamir@ars.usda.gov; Gary P Noyes - gnoyes@oceaneering.com; Mark T Holtzapple - mth4500@chemail.tamu.edu;
Marcus E Kehrli - marcus.kehrli@ars.usda.gov
* Corresponding authors
Abstract
Background: Prions, the infectious agents that cause transmissible spongiform encephalopathies
(TSEs), are relatively resistant to destruction by physical, enzymatic, and chemical treatments.
Hydrolysis in boiling saturated calcium hydroxide (limewater) utilizes inexpensive chemicals to
digest protein components of offal. The purpose of this work was to determine if incubating brain
material from scrapie-infected sheep in near-boiling saturated calcium hydroxide solution
(Ca(OH)2) would abolish immunoreactivity of the infectious prion (PrPSc) as determined by
western blot.
Findings: After incubating for as few as 10 minutes in saturated calcium hydroxide at 99°C,
immunoreactivity of protease resistant bands by western blot analysis is completely lost.
Conclusion: Boiling in limewater may offer an alternative for disposal of carcasses and enable
alternative uses for rendered products from potentially infected carcasses.
Background
Scrapie in sheep, bovine spongiform encephalopathy
(BSE), chronic wasting disease (CWD) of deer and elk,
and Creutzfeldt-Jakob disease in humans are chronic neu-
rodegenerative diseases associated with the accumulation
of the protease-resistant, disease-associated isoform of the
prion protein (PrPSc) in selected regions of the central
nervous system. PrPSc is relatively resistant to inactivation
by standard decontamination procedures [1] and can
remain infectious after undergoing sterilization proce-
dures under high pressure, treatment with disinfectants,
or exposure to dry heat.[2]
The host of any particular prion disease determines the
potential distribution of PrPSc in the tissues. Brain, spinal
cord, and distal ileum and in some species, peripheral
lymphoid tissues contain infectious material (PrPSc).
Removal, handling, and disposal of tissue with the poten-
tial to harbor infectious PrPSc represent a challenge to the
meatpacking and by-product industries. Efficient meth-
ods of inactivating potentially infectious material are
needed.
A number of procedures have been described to decon-
taminate surfaces or reusable medical devices, [3-7] but
Published: 28 October 2008
BMC Research Notes 2008, 1:99doi:10.1186/1756-0500-1-99
Received: 23 April 2008
Accepted: 28 October 2008
This article is available from: http://www.biomedcentral.com/1756-0500/1/99
© 2008 Greenlee et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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few of these methods are appropriate for decontamina-
tion of large volumes of animal tissue from TSE-infected
animals. Chemical means of inactivation such as concen-
trated hypochlorite (bleach) solutions or sodium hydrox-
ide solution can damage equipment, cause respiratory
irritation in workers exposed to fumes, pose problems
with disposal, or may not be appropriate for processing
large amounts of material. Disposal of large volumes of
chemically treated material may present problems for
some municipal sewage systems. High-pressure and tem-
perature methods of inactivation [3] or reducing infectiv-
ity[8] of tissues and proteolytic digestion by enzymes
secreted by thermophylic bacteria[9] have not been dem-
onstrated to be effective on a large scale.
Methods for detecting abnormal prion in meat and bone
meal (MBM) are under development,[10] but methods
that inactivate abnormal prions and leave material with
by-product value (concentrated amino acids, feed addi-
tive, fertilizer, etc.) may be more desirable. Incineration
can inactivate the agent,[2] but precludes any recovery or
reuse of raw material.
Inexpensive, effective means of inactivating prions in offal
are needed for safe disposal of carcasses and to ensure the
safety of animal by-products if used as a feed source. Ulti-
mately, the success of any potential method of inactiva-
tion must be measured by comparing infectivity in cell
cultures[11] or animal models of prion disease, but west-
ern blot can be used to screen for unfolding or denatura-
tion of PrPSc, which would result in a loss of reactivity
with prion-specific antibodies. The purpose of this study
was to determine if incubating brain material from
scrapie-infected sheep in near-boiling saturated calcium
hydroxide solution would abolish immunoreactivity of
the infectious prion (PrPSc) as determined by western
blot.
Methods
Preparation of brain material
Brain immunoreactive for PrPSc by immunohistochemis-
try and western blot from intracerebrally inoculated sheep
or brain from scrapie-free control animals[12] was
homogenized at a final concentration of 40% (w/v) in 10
mM Tris 5 mM MgCl2, pH 7.5 using a tissue homogenizer
(Powergen 125 homogenizer, Fisher Scientific) with dis-
posable probe. Secondly, ultrasonic dismembranation
(Fisher Scientific Model 500 Ultrasonic Dismembrator,
Fisher Scientific) was performed for four 30-second inter-
vals in an ice bath with brief vortex mixing between soni-
cation. The samples were then centrifuged at 2,000 × g for
2 minutes at room temperature. The supernatant was then
treated with 100 U/mL benzonase (Novagen) at 37°C for
1 hour. The material was used immediately for the inacti-
vation experiments or frozen at -80°C for later use.
Calcium Hydroxide (Ca(OH)2 treatment
The 40% brain homogenates were diluted to a final con-
centration of 10% (w/v) brain in saturated Ca(OH)2 con-
taining excess insoluble Ca(OH)2. Samples of 10% (w/v)
brain homogenate in 10 mM Tris 5 mM MgCl2, pH 7.5
were processed in parallel to those homogenized in satu-
rated Ca(OH)2 to serve as a heat-only control. All samples
except from the zero timepoint were placed at 99°C for
incubation. The zero timepoint sample and samples
measured at subsequent timepoints were neutralized by a
1:6.9 dilution into 1 M Tris-HCl to stop the reaction. Ice-
cold acetone (4 volumes) was added to each sample, and
samples were then placed at -20°C for at least 1 hour.
Preparation of samples and analysis by western blot
Following incubation at -20°C for 1 hour, the calcium
hydroxide treated samples were centrifuged at 13,000 × g
for 10 minutes at room temperature. The supernatant was
discarded and the protein pellet was allowed to air dry.
Once dry, the pellet was dissolved in SDS-PAGE sample
buffer such that 1.2 mg tissue equivalent (assuming 100%
yield) was loaded on the gel. Positive and negative scrapie
control samples were homogenized in 10 mM Tris-HCl 5
mM MgCl2 pH 7.5 and loaded on a gel at ~1 mg tissues
equivalent in SDS-PAGE sample buffer without treatment
or after 240 minutes at 99°C. For the control samples,
proteinase K (USB) was added to a final concentration of
80 μg/ml; digestion was conducted at 37°C for 1 hour and
stopped by the addition of Pefabloc (Roche Diagnostics)
to a final concentration of 0.1 mg/mL.
For SDS-PAGE and western blot, a 4–20% commercially
prepared SDS-PAGE gel was loaded and run according to
the manufacturer's instructions, blotted to a polyvinyli-
dene difluoride (PVDF) membrane (GE Healthcare) and
blocked with 3% bovine serum albumin. Western blot
detection was conducted using mouse anti-PrP mono-
clonal antibody P4 (R-Biopharm AG), which targets to
amino acids 89–104 of the ovine prion protein
sequence,[13] at a 1:10,000 dilution (0.1 μg/mL) as the
primary antibody. Western blots were repeated using
monoclonal antibody F99/97.6.1, which targets residues
220–225 of ovine prion protein sequence.[14] A bioti-
nylated sheep anti-mouse secondary antibody at 0.05 μg/
ml and a streptavidin-horseradish peroxidase (HRP) con-
jugate (GE Healthcare) were used according to the manu-
facturer's instructions in conjunction with the ECL Plus
detection system (GE Healthcare) and imaged using a
Kodak Image Station In-Vivo F. Primary antibody incuba-
tions were conducted with the membrane at either room
temperature for 1 hour or 4°C overnight (≥ 12 hours).
Secondary antibody and streptavidin-HRP conjugate
incubations were conducted at room temperature for 1
hour. Band intensity was determined using Kodak Molec-
ular Imaging software.

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Results and discussion
Scrapie-positive and scrapie-negative samples were con-
firmed based upon the presence or absence, respectively,
of proteinase K-resistant PrP (Figure 1). For both scrapie-
positive and scrapie-negative samples, PrP is clearly visi-
ble in the first sample taken after dilution into saturated
Ca(OH)2, however, after only 10 minutes there is virtually
no detectable PrP in either the scrapie-positive or scrapie-
negative samples (Figure 2) on a western blot. Analysis of
the western blot indicates that greater than 95% of all
detectable PrP is hydrolyzed in the first 10 minutes. Sam-
ples homogenized in 10 mM Tris-HCl 5 mM MgCl2 pH
7.5 remained immunoreactive after 240 minutes at 99°C
(results not shown).
As the infectious agent, reduction in PrPSc levels is strongly
suggestive of reduced infectivity. However, PrPSc is
detected indirectly via western blot and disruption of anti-
body-PrPScinteractions for which there would be no corre-
sponding reduction in infectivity could be one potential
explanation for lack of immunoreactivity. Western blots
were performed with multiple antibodies with different
binding epitopes to assure that lack of immunoreactivity
was not due to loss of an epitope at a single site. Failure to
reduce PrPSc levels while reducing infectivity is much less
likely.[15]
Base-catalyzed hydrolysis using sodium hydroxide at con-
centrations ≥ 1 N is an accepted means of decontamina-
tion of PrPSc-contaminated material.[16] Reports indicate
that even this fails to completely inactivate some PrPSc iso-
lates.[17] Failures may be more a result of the consump-
tion of hydroxide ion in the hydrolysis than an inability
of 2N NaOH to hydrolyze PrPSc. The pH of 2 N NaOH is
greater than 14. In contrast, saturated Ca(OH)2 is nearer
pH 12.5, which is still sufficient to rapidly hydrolyze pep-
tide bonds. As the hydroxide ions are consumed in the
case of NaOH, the pH of the solution would drop; how-
ever, using a slurry of saturated Ca(OH)2 solution and
insoluble Ca(OH)2 the hydroxide ion concentration and
thus the pH is readily maintained. Additionally, Na+ ions
are poorly tolerated in feed, whereas Ca2+ ions are much
more readily tolerated. Thus, although Ca(OH)2, is a
weaker base than the often-used NaOH it offers some
practical advantages to prion inactivation on an industrial
scale for the purposes of producing a safe, alternative by-
product.
New methods of prion inactivation for application to
offal would reduce complications with carcass disposal
and assure safety of rendered materials used as an animal
feed source. Immunoassay alone is not sufficient to eval-
uate the efficacy of a particular method of inactivation
[11] as removal of all immunoreactive PrPSc correlates
poorly with levels of infectivity measured by bioassay.
Animal inoculations appear to be a standardizable
method for the evaluation of prion inactivation, but a 15-
month incubation period is required to guarantee total
absence of infectivity.[3] Results of this study demonstrate
the potential for heating in near-boiling saturated
Western blot of non-treated brain homogenates
Figure 1
Western blot of non-treated brain homogenates.
Representative western blots of scrapie-positive and scrapie-
negative sheep brain homogenates. Lane 1-Molecular weight
marker; lane 2-Scrapie-positive brain without proteinase K
digestion; lane 3-Scrapie-positive brain treated with protein-
ase K; lane 4-Blank; lane 5-Scrapie-negative brain without
proteinase K digestion; lane 6-scrapie-negative brain with
proteinase K digestion. Only the scrapie-positive brain
homogenate exhibits proteinase K resistance.
Western blot after calcium hydroxide treatment
Figure 2
Western blot after calcium hydroxide treatment.
Western blot analysis of calcium hydroxide treatment of
scrapie-negative (A) and scrapie-positive (B) sheep brain
homogenate. For both, Lane 1-molecular weight marker; lane
2–0 minutes; lane 3–10 minutes; lane 4–20 minutes; lane 5–
30 minutes; lane 6–60 minutes; lane 7–120 minutes; lane 8–
240 minutes. PrP immunoreactivity is abolished in scrapie-
positive samples after 10 minutes treatment with calcium
hydroxide.

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Ca(OH)2 as a method of PrPSc inactivation with practical
and safety advantages to the handling of the hydrolysate.
However, confirmation of loss of infectivity requires bio-
assay or cell culture infectivity studies as loss of immuno-
reactivity in WB is not equivalent to loss of infectivity.
Bioassay using inoculum treated with heating in saturated
calcium hydroxide is currently underway. Because strain
differences to some methods of inactivation have been
demonstrated,[6] it may be necessary to pursue inactiva-
tion studies using material derived from cattle with BSE
and cervids with CWD.
Competing interests
Gary Noyes and Mark Holtzapple are named as co-inven-
tors on a U.S. Patent Application of the boiling-saturated-
calcium-hydroxide protein-depolymerization process.
Neither has any stock or ownership in the company that
has licensed the patent from Texas A&M University.
Authors' contributions
JG, EN, MH, GN, and MK conceived of the idea for this
research. JG and EN designed the experiments, analyzed
and interpreted data, and wrote the manuscript. AH pro-
vided tissues and reagents and critically reviewed the man-
uscript. MK supervised data collection and reviewed the
manuscript. All the authors have read and approved the
final manuscript.
Acknowledgements
The authors thank Leisa Mandell and Semakaleng Lebepe-Mazur for provid-
ing technical support to this project. Mention of trade names or commer-
cial products in this article is solely for the purpose of providing specific
information and does not imply recommendation or endorsement by the
U.S. Department of Agriculture.
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