Journal of Virology, November 1999, p. 9386-9392, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection of Bovine Spongiform
Encephalopathy-Specific PrPSc by Treatment with Heat and
Guanidine Thiocyanate
Rudolf K.
Meyer,1,*
Bruno
Oesch,2
Rosmarie
Fatzer,1
Andreas
Zurbriggen,1 and
Marc
Vandevelde1
BSE Reference Center, Institute of Animal
Neurology, University of Bern, CH-3012 Bern,1
and Prionics Ltd., University of Zürich, CH-8057
Zürich,2 Switzerland
Received 10 May 1999/Accepted 2 August 1999
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ABSTRACT |
The conversion of a ubiquitous cellular protein (PrPC),
an isoform of the prion protein (PrP), to the pathology-associated isoform PrPSc is one of the hallmarks of transmissible
spongiform encephalopathies such as bovine spongiform encephalopathy
(BSE). Accumulation of PrPSc has been used to diagnose BSE.
Here we describe a quantitative enzyme-linked immunosorbent assay
(ELISA) that involves antibodies against epitopes within the
protease-resistant core of the PrP molecule to measure the amount of
PrP in brain tissues from animals with BSE and normal controls. In
native tissue preparations, little difference was found between the two
groups. However, following treatment of the tissue with heat and
guanidine thiocyanate (Gh treatment), the ELISA discriminated
BSE-specific PrPSc from PrPC in bovine brain
homogenates. PrPSc was identified by Western blot,
centrifugation, and protease digestion experiments. It was thought that
folding or complexing of PrPSc is most probably reversed by
the Gh treatment, making hidden antigenic sites accessible. The
digestion experiments also showed that protease-resistant PrP in BSE is
more difficult to detect than that in hamster scrapie. While the
concentration of PrPC in cattle is similar to that in
hamsters, PrPSc sparse in comparison. The detection of
PrPSc by a simple physicochemical treatment without the
need for protease digestion, as described in this study, could be
applied to develop a diagnostic assay to screen large numbers of samples.
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INTRODUCTION |
Extensive research on the molecular
mechanism of scrapie in the Syrian golden hamster showed that
accumulation of a partially protease-resistant prion protein
(PrPSc) is the hallmark of transmissible spongiform
encephalopathies (TSE) (9). PrPSc is an isoform
of the prion protein (PrP) (1, 25). It originates from
PrPC, a ubiquitous protease-sensitive cellular protein.
PrPSc has been defined as being partly resistant to
digestion by proteinase K under standardized conditions (1).
All isoforms are translational products of the same Prnp gene
(18). The TSE-specific PrP isoforms accumulate by conversion
of PrPC.
Demonstration of PrPSc has been used for the diagnosis of
TSE, for example in dot-blot immunoassays (23), in
enzyme-linked immunosorbent assays (ELISA) (22), by
immunocytochemistry on histologic sections (17), or in
Western blot analyses of brain homogenates (11). The last
two techniques have also been applied in bovine spongiform
encephalopathy (BSE) (3, 6). Since most antibodies will not
discriminate between the two isoforms in immunocytochemistry or Western
blot analyses (15), antibody detection of the
protease-resistant PrPSc requires removal of the
PrPC by protease digestion. The ratio of protease-sensitive
to protease-resistant pathological PrP may vary and is characteristic
for a particular TSE strain (22). In addition, since in some
infectious fractions of BSE-infected mice no protease-resistant PrP
could be found, the value of protease digestion in TSE diagnosis has
been questioned (8). To avoid protease digestion, a
monoclonal antibody that specifically recognizes PrPSc was
developed (7), but its successful use in ELISA procedures has not yet been reported.
A PrPSc-specific sandwich ELISA has been described for the
Syrian golden hamster. The assay was based on the comparison of the affinity of an antibody to the native and denatured samples. The monoclonal antibody used (3F4) was specific for epitopes of the PrP
protein, which were buried by the transformation of PrPC
into PrPSc. In native samples it bound to PrPC
only, but after the protein was unfolded by treatment with 4 M
guanidine hydrochloride, the antibody bound to both PrPC
and its pathological isoform (22). However, to avoid
denaturation of the antibodies, the guanidine hydrochloride had to be
diluted, with a concomitant reduction in assay sensitivity. Compared to scrapie in hamsters, in BSE the amount of PrPSc appears to
be rather limited and more difficult to detect. For its detection,
strongly denaturing conditions, such as detergent, acid, and heat
treatment, are included in most immunocytochemistry protocols,
presumably to uncover antigenic epitopes (5, 12).
In an attempt to develop a BSE-specific immunocapturing ELISA and to
improve antibody binding to bovine PrP, we tested a variety of physical
and chemical treatments by exposing BSE brain homogenates to defined
heat levels and guanidine thiocyanate (GdnSCN) concentrations. As a
result, we found a variable amount of PrPSc, which was not
present in control animals.
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MATERIALS AND METHODS |
Animals and brain material.
Brain material was derived from
15 BSE-affected Swiss cattle. The diagnosis was based on the finding of
spongiform change and neuronal vacuolation in susceptible areas and on
immunocytochemical demonstration of PrPSc accumulation in
the brain stem. Control samples included samples derived from 20 age-matched normal cattle from a BSE-free country. Brain tissue from
the fish Salmo truta was used in some experiments as a
negative control and for dilutions.
Preparation of brain homogenates.
Fragments of brain tissue
(
0.5 g) were homogenized in 320 mM sucrose solution (10 ml/g [wet
weight] of brain tissue) with an Ultra-Turrax T25 apparatus (Janke and
Kungel, Staufen, Germany). The homogenate was cleared by a short
(5-min) centrifugation at 7,000 × g. For some
experiments, the pellets were resuspended and homogenized again in the
same sucrose solution at a volume corresponding to that of the
supernatant. The total amount of protein in most samples was measured
by the bicinchoninic acid reaction (Pierce, Rockford, Ill.).
Anti-PrP antibodies.
For detection of PrP in Western blots
and for ELISA, three different anti-PrP antibodies were used, one
monoclonal antibody and two rabbit antisera. Monoclonal antibody 6H4
was derived from PrP-null mice immunized with recombinant PrP of the
bovine sequence (Prionics Ltd., Zürich, Switzerland). It binds to
an epitope in the center of the protease-resistant core (7).
A rabbit antiserum was raised against full-length recombinant PrP
(R#26; supplied by Prionics Ltd.). A second rabbit antiserum, C15S, was raised against a peptide of the bovine PrP sequence (2),
GQGGTHGQWNKPS, located near the N terminus of the putative
protease-resistant core of the bovine PrP molecule. All antibodies used
in this study could detect PrPSc in immunocytochemistry
(data not shown) and Western blot analysis (Fig.
1).
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FIG. 1.
Western blot of samples from BSE-affected (lanes a to h)
and control (lanes i to m) animals. The medulla oblongata was
homogenized, cleared by a short centrifugation and analyzed either
undigested (lanes a, c, e, and i) or after digestion with 20 (lanes h
and m), 50 (lanes g and l) or 100 (lanes b, d, f, and k) µg of
proteinase K (PK) per ml. The first antibody used was either C15S
(lanes a and b), R#26 (lanes c and d), or 6H4 (lanes e to m).
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Preparation and testing of the peptide antibody.
The peptide
was coupled to ovalbumin or keyhole limpet hemocyanin by using the
Imject activated immunogen conjugation kit (Pierce) and purified by gel
filtration as specified by the supplier. Several rabbits were immunized
by subcutaneous injection of coupled keyhole limpet hemocyanin. For the
first immunization, the protein concentration was adjusted to 1 mg/ml
and the protein was emulsified in a 1:2 ratio with complete Freund
adjuvant. For the following four boosters, the protein was mixed 1:2
with incomplete Freund adjuvant.
The antisera were assessed by testing various dilutions in ELISA
plates. The plates (Nunc-Immuno Plate MaxiSorp F96; Nunc, Roskilde,
Denmark) were coated by overnight incubation (4°C) with 200 ng of
peptide-coupled ovalbumin per well in carbonate buffer (15 mM
Na2CO3, 35 mM NaHCO3, 0.02%
NaN3 [pH 9.6]). The plates were then blocked with 0.2 ml
of 10% dry milk in RPB buffer (13.7 mM NaCl, 2.7 mM KCl, 1.4 mM
KH2PO4, 8.1 mM Na2HPO4
[pH 7.3]) plus 0.01% Tween 20 (RPB-Tween) per well for 1 h at
37°C. The antisera were serially diluted in RPB-Tween containing
3.3% dry milk and added to the plates (100 µl/well). After
incubation for 1 h at 37°C, the plates were washed with
RPB-Tween and the antibodies bound were detected by incubation with a
horseradish peroxidase (HRP)-conjugated swine anti-rabbit antibody
(Dako, Glostrup, Denmark) diluted 1:300 in phosphate-buffered
saline-Tween (137 mM NaCl, 2.7 mM KCl, 1.4 mM
KH2PO4, 8.1 mM Na2HPO4
[pH 7.3]) containing 0.01% Tween 20 and 3.3% dry milk. After
incubation for 1 h at 37°C, the plates were washed again with
phosphate-buffered saline-Tween and filled with 0.2 ml of
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) solution
(Boehringer, Mannheim, Germany) per well for reaction with HRP.
Oxidation of the ABTS substrate resulted in a change in the optical
density, which was measured with an ELISA plate reader at 405 nm. Only
antisera which had a half-maximal binding at dilutions of at least
1:16,000 were used.
Western blotting.
Western blotting was carried out as
specified for the Prionics BSE/scrapie test kit. Briefly, samples were
first separated on sodium dodecyl sulfate-12.5% polyacrylamide gels
and then blotted on polyvinylidene difluoride membranes (Millipore).
The membranes were then blocked for 1 h in blocking buffer
(Tropix, Bedford, Mass.). The first and second antibodies were diluted
1:1,000 to 1:5,000 in blocking buffer and successively incubated with
the membranes after thorough washing with TBS-Tween (25 mM Tris base, 137 mM NaCl, and 27 mM KCl [pH 7.6], containing 0.05% Tween). Second
antibodies were labeled with either HRP or alkaline phosphatase. Detection was carried out with a chemiluminescent substrate, either ECL
(Amersham) or CDP-Star (Tropix), according to the provider.
ELISA for PrP.
ELISA plates were coated by overnight
incubation with 0.1 ml of monoclonal antibody 6H4 diluted 1:100 in
carbonate buffer at 4°C per well. The plates were blocked with 0.2 ml
of RPB-Tween containing 10% dry milk per well for 1 h at 37°C
and then incubated with 0.1 ml of bovine brain homogenate diluted 1:2
in RPB buffer per well in triplicate for 1 h at 37°C. After
being washed with RPB-Tween, the bound PrP was quantified by successive
incubation with two other antibodies, a rabbit PrP antiserum (C15S or
R#26) and an HRP-conjugated swine anti-rabbit antibody (each for 1 h at 37°C). The rabbit PrP antiserum was diluted 1:500 in RPB-Tween containing 3% dry milk, the swine anti-rabbit antibody was diluted 1:300 in PBS-Tween containing 3% dry milk, and 0.1 ml/well was applied. After incubation, the plates were washed with PBS-Tween and
filled with 0.2 ml of ABTS solution per well for reaction with HRP.
Quantification of the ELISA results.
To test linearity,
samples of bovine brain homogenates from healthy control animals were
diluted with brain homogenates from the fish Salmo truta,
which has no proteins cross-reacting with the antibodies mentioned
above. To calibrate the assay, different concentrations of recombinant
PrP (Prionics) were mixed with fish brain homogenate and measured again
in the ELISA with R#26 as a second antibody. The concentration of
purified recombinant PrP was 3.07 ± 0.16 mg/ml (n = 3) as measured by the BCA protein assay (Pierce). It was diluted
to 172 ng/ml. Appropriate volumes were further diluted with fish brain
homogenate and pipetted into the wells of a coated immunoplate to an
end concentration of 0.5 to 4 ng of recombinant PrP in 100 µl. A
standard of bovine brain was equally diluted, and samples of 100 µl
each containing 5 to 50 µl of the homogenate were distributed to
other wells of the same plate (see Table 1). The PrP content of the
bovine brain standard was calculated in terms of recombinant PrP from
the slope of the two linear curves obtained from the optical density
(OD)-concentration relationship. The values obtained for the
concentration of PrPC in this specific brain homogenate
were 79 ng/ml in a first experiment (slope for recombinant PrP, 0.6686 OD unit/ng, and slope for the brain homogenate, 0.05267 OD unit/µl)
and 78 ng/ml in a second (0.7484 and 0.0585 OD unit/µl). The
concentrations of all other brain homogenates were obtained by
calibration with this standard.
To create a standard, the bovine brain homogenate was diluted with fish
brain homogenate to a concentration corresponding to 75 ng/ml. A
100-µl/well concentration of a 1:2 dilution of this standard in RPB
buffer was included in each plate to determine the length of the HRP
reaction and to allow quantification of unknown samples. The OD of the
plates was read when the standard reached an OD between 0.900 and
1.100. To allow comparison of the results of different plates, zero
values were subtracted first and then all results were divided by the
value of the standard. Its OD (75 ng of PrP/ml) became 1.000 at this step.
A quadratic equation was fitted to the nonlinear dose-response curve of
the peptide antibody C15S. The formula finally used for calculation in
Microsoft Excel was OD = 1.887*POWER((SQRT(0.068*x+1)
1)/2.04;2), where x is the concentration of PrP in nanograms per milliliter.
Validation of the ELISA.
To calculate the detection limit,
the average standard deviation of the blank (0.003 ± 0.001, n = 24) was used. Accordingly, an OD of 0.006 ± 0.003 (average of the three wells of a plate) was found to be
significantly different from zero, which corresponded to a detection
limit of 4 ng/ml. The upper limit was given by the capacity of PrP
binding of 6H4 bound to the plates (a 1:100 dilution) and was found to
be about 6 ng/well, corresponding to 120 ng/ml. The interassay
variation coefficient (reproducibility, 100 × standard
deviation/average) was 5% (maximum, 11%) as judged from evaluation of
24 standard deviations obtained from the standard, which was included
in each assay.
Western blotting of samples bound to the ELISA plates.
To
identify proteins bound to the wells of 6H4-coated ELISA plates, the
wells were washed with sample buffer and the released proteins were
analyzed by Western blotting.
GdnSCN and heat treatment.
Before being examined in the
ELISA, brain homogenates were treated with either several
concentrations of GdnSCN, different high temperatures, or a combination
of the two. The final method used for processing the brain tissue
homogenates was as follows. Brain homogenates were diluted 1:2 with 0.2 M GdnSCN in RPB buffer. Then 0.4 ml of the diluted samples was
transferred to 2-ml glass ampoules. The ampoules were closed and heated
in a laboratory oven set at 150°C (actual temperature, 150 to
160°C) for 10 minutes (Gh treatment). After the ampoules were cooled
to room temperature, their contents were assayed for PrP in parallel
with unheated samples.
Ultracentrifugation and proteinase K digestion.
For
sedimentation studies, 1-ml samples of cleared brain homogenate were
centrifuged in a Beckman ultracentrifuge at 200,000 × g for 1 h. The supernatant was separated from the pellet. The pellet was resuspended in 1 ml of RPB buffer with the help of the
Ultra-Turrax. The resuspended pellet and supernatant were analyzed
separately by ELISA.
For protease digestion, proteinase K (Boehringer, Mannheim, Germany)
was diluted to 0.04 to 0.2 mg/ml in RPB buffer containing 0.2 M GdnSCN
from a 14-mg/ml stock solution. The mixture was added to an equal
volume of brain homogenate and incubated for 20 min at 37°C. The
reaction was stopped by incubation of the samples for 10 min at 150°C
(ELISA) or by addition of Pefabloc SC (Western blots) as specified by
the supplier (Boehringer).
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RESULTS |
Development of a PrP-specific ELISA.
A quantitative
PrP-specific immunocapturing ELISA was developed. The plates were
coated with a PrP-specific monoclonal antibody (6H4) and incubated with
cleared bovine brain homogenate. In a first step, the proteins bound to
the plates were removed for analysis. PrP could be identified in
Western blots.
For detection of PrP bound to the plates, one of two PrP-specific
rabbit antisera was used as a second antibody. One (R#26) was raised
against recombinant PrP, and the other (C15S) was raised against a
synthetic peptide of the bovine sequence (see Materials and Methods). A
PrP fraction could be detected in samples from both BSE-affected
animals and in controls, independent of the second antibody used. The
PrP fraction was identified as PrPC by protease
digestion and ultracentrifugation (see below). Animals with BSE could
not be distinguished from controls by ELISA of untreated preparations.
Heat treatment in the presence of GdnSCN exposed epitopes of
BSE-specific PrP.
The effect of GdnSCN on PrP detection was
explored by adding GdnSCN to a standard of bovine brain homogenate.
When C15S was used as the second antibody, little change in OD was
observed for up to 0.1 M GdnSCN in comparison to a control. However, at 0.25 M GdnSCN, the OD was reduced by about 50%, and it was further decreased with increasing GdnSCN concentrations. No detection of a
BSE-specific PrP fraction was achieved by adding GdnSCN alone (without
subsequent dilution) (Fig. 2A).
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FIG. 2.
Effect of GdnSCN and heat on PrPSc
detection. (A) Increasing amounts of GdnSCN were added to samples of
brain homogenate derived from either BSE-affected animals or unaffected
controls. All samples were measured in the ELISA, and the resulting OD,
given as a percentage of the untreated control, is plotted against the
GdnSCN concentration. (B) Samples of brain homogenate from BSE-affected
animals or controls were incubated for the indicated time (in minutes)
at 150°C. (C) The experiment as described in panel B was repeated
with samples containing 0.1 M GdnSCN. The data with standard error bars
represent the mean of two independent experiments.
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To explore the effect of heat, brain homogenates were incubated for
various times at 150°C (temperatures below 100°C had no effect) and
the resulting OD was measured in the ELISA. The result was compared to
the optical density of the corresponding unheated sample. Only
reduction but no increase in the optical density could be observed by
heat treatment alone (Fig. 2B).
The heat experiments were repeated in the presence of 0.1 M GdnSCN (Gh
treatment). In heat-exposed samples from BSE-affected animals, a large
increase in OD was detected, which reached a maximum at an incubation
time of about 10 min (Fig. 2C). In some experiments, the signal was up
to three times stronger than the OD of the unheated control.
The BSE-specific OD signal (i.e., PrPSc; see below) could
be detected only by using the peptide antiserum C15S as second
antibody. R#26, raised against the recombinant protein, caused no
difference in OD. In addition, no equivalent increase was obtained when
other denaturing agents were present in addition to GdnSCN, e.g.,
Pefabloc or 0.05% sarcosine, as used for scrapie-associated filament
(SAF) preparations (4). For most control animals, 10-min
incubations in the presence of 0.1 M GdnSCN resulted in some (<30%)
reduction in the OD. Most other proteins were denatured by the Gh
treatment and could be removed by a short centrifugation
(7,000 × g for 5 min). The PrP signal remained
in the supernatant and was not significantly different from the signal
measured without centrifugation.
The physicochemical mechanism for diminished OD observed by heat or
addition of GdnSCN was not further investigated.
Quantification of PrP.
When serial dilutions were measured,
R#26 showed a linear dose-response curve (Table
1) and the peptide antiserum showed a
nonlinear one. The nonlinear dose-response curve was obtained both with
and without heat treatment (Fig. 3). It
was not influenced by varying the antiserum concentration. The cause of
this nonlinearity is under investigation.
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FIG. 3.
Dose-response relationship for Gh-treated and unheated
samples derived from a BSE-affected animal. Samples of brain homogenate
from a BSE-affected animal were diluted as indicated with fish brain
homogenate to 50 µl/well and tested in the ELISA in triplicate either
with or without heating. C15S was used as the second antibody. By
calibration, the OD of the standard was 1.000, which corresponded to a
PrP concentration of 75 ng/ml.
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The assay was calibrated by comparing the ODs of different dilutions of
a standard brain homogenate with that of recombinant PrP of known
concentration (Table 1). The standard was included in all plates and
used for calculation of the concentration of unknown samples. Compared
to recombinant PrP, PrPC was less susceptible to
spontaneous degradation and therefore was preferred as a standard. A
quadratic equation was fitted to the nonlinear dose-response curve
observed with C15S (see Materials and Methods). Thus, calculation of
the concentration of both PrPC and PrPSc was
based on recombinant PrP.
Characterization of PrPSc.
PrPSc
became obvious by a dramatic increase in OD following Gh treatment. For
characterization of this PrP, samples of brain stem homogenates from
BSE-affected animals were ultracentrifuged to sediment protein
aggregates and cellular organelles. Samples of pellet and supernatant
were Gh treated and analyzed by ELISA. All of PrPSc,
together with about half of the total PrPC, was found in
the pellet fraction; i.e., PrPSc either had been aggregated
or was associated with cellular organelles before treatment (Table
2). In some experiments, the homogenates were digested with proteinase K before being subjected to Gh treatment. In samples from control animals, all of the protease-sensitive PrP
measured by the ELISA disappeared with 20 µg of proteinase K per ml.
In samples from BSE-affected animals, less than half of the initial PrP
was digested when using 20 µg of proteinase K per ml, but the PrP
disappeared proportionally when more proteinase K was added (Fig.
4). The samples were also analyzed by
Western blotting. Protease-resistant PrP could be observed in
proteinase K-digested fractions of all samples from BSE-affected
animals (Fig. 1, lanes f to h) but not in control samples (lanes k to m).
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TABLE 2.
Average PrP protein concentration of medulla oblongata
homogenate from BSE-affected animals before and
after ultracentrifugation
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FIG. 4.
Proteinase K digestion of samples from bovine medulla
oblongata. Samples from four different BSE-affected animals and samples
from four different control animals were incubated with increasing
concentrations (20, 50, and 100 µg/ml) of proteinase K (PK). The
samples were digested for 30 min at 37°C, Gh treated, and measured in
the ELISA at the end of the incubation period. Averaged results and
standard deviations are plotted together with the results from
untreated and Gh-treated but undigested samples.
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PrPC and PrPSc in different brain areas of
BSE-affected and control animals.
The PrP content of the medulla
oblongata, thalamus, and cerebral cortex of several BSE-affected
animals and controls was measured (Table
3). High animal-to-animal variation in
both PrPC and PrPSc was found. These variations
were scarcely reduced when the PrP content was based on total protein,
as measured by the BCA reaction.
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TABLE 3.
Average concentration of PrP protein in cleared bovine
brain homogenates as measured by ELISA using C15S as the
second antibody
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A significant amount of PrPSc was detected in the medulla
oblongata of all BSE-affected animals used in this study. Its use as a
BSE assay was investigated. For each animal, the OD difference between
treated and untreated samples was calculated and results from 15 BSE-affected animals and the 20 negative controls obtained from a
BSE-free country were compared. The range was 0.102 to 0.668 for BSE
and 0.214 to 0.077 for controls. We found that 83% of the control
animals had either a negative difference or a value not significantly
different from zero. Four control animals (17%) had values between
0.051 and 0.077. All of the BSE-affected animals had differences larger
than 0.1, with no values overlapping (Fig.
5). Accordingly, all BSE-affected animals
used in this study could be identified by the OD difference. However,
the sample size was small and samples were not blinded.
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FIG. 5.
Box and whisker plot of the difference in OD of
Gh-treated and unheated samples derived from control (n = 20) and BSE-affected (n = 15) animals. For each
animal, the difference in OD was calculated by subtracting the value of
the unheated sample from that of the Gh-treated sample. The boxes
enclose the middle half of the data. Whiskers indicate the range of
"typical" data values. Medians ( ) and possible (*) and probable
( ) outliers are displayed.
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A different result was obtained for the thalamus and cortex (Table 3).
PrPSc could be detected in only 20% of the samples
obtained from the thalamus of BSE-affected animals. All other samples
could not be distinguished from controls (Table 3). No
PrPSc was found in the cortex.
To improve sample handling and to reduce standard deviations, samples
had been cleared by a short centrifugation step prior to the ELISA;
i.e., some of the PrP was lost by this procedure. To estimate the
extent of this fraction, pellets were resuspended and analyzed for PrP.
On average, 49% ± 10% (n = 55) of total PrP could be
recovered by this procedure. When corrected for this loss, the amount
of PrPC detected in bovine brain was about the same as
reported for hamster brain (1 to 5 µg/ml) (22) but the
amount of PrPSc was much smaller (0.5 to 1 µg/ml compared
to 10 to 40 µg/ml in hamster brain).
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DISCUSSION |
For the development of an ELISA to detect BSE, we used three
different antibodies against bovine PrP in a variety of binding studies
on normal and BSE-affected bovine brain tissues. All three antibodies
were found to bind to PrP and its protease-resistant isoform
PrPSc following denaturing procedures as required for
Western blotting (Fig. 1). They also bound to native PrP in fresh brain
tissue, but we did not detect a significant quantitative difference
between normal controls and BSE-affected animals in cleared untreated tissue homogenates. Very limited binding of PrPSc to the
first or second antibody used in the ELISA could explain this lack of
difference between normal and BSE-affected tissue in native untreated
preparations. Similar observations have been described before for other
PrP antibodies (22, 26).
Following heat and GdnSCN treatment of the fresh tissue homogenate, a
much stronger ELISA signal was obtained in the animals with BSE than in
the controls. Thus, the treatment revealed a PrP fraction specific for
BSE. To investigate whether this PrP fraction was related or identical
to PrPSc, ultracentrifugation and protease digestion
studies with BSE-affected samples were performed. The PrP fraction
detected by our ELISA did sediment by ultracentrifugation. This
indicated that PrP was present either as large aggregates or complexed
to cellular organelles. Both possibilities are characteristic of
PrPSc, which is either aggregated to SAFs or associated
with the microsomal membranes (11). SAF formation is most
effective in the presence of detergent (10), which in our
ELISA interfered with PrP detection. In this respect, our brain
homogenates were different from the detergent-treated brain fractions
used for SAF isolation and were likely to contain little SAF. Digestion
with 20 µg of proteinase K per ml destroyed much of the
PrPC but not the PrP revealed by Gh treatment (Fig. 4).
Protease-resistant PrPSc could be demonstrated by Western
blots in this fraction (Fig. 1, lane h). Both protease resistance and
the relative amount of PrPSc in BSE are limited compared to
scrapie in hamsters (22). In BSE-affected tissue, 23% ± 13% (n = 4) of PrPSc disappeared when the
proteinase K concentration was increased from 20 to 100 µg/ml (Fig.
4). Immunoprecipitation studies with a PrPSc-specific
antibody revealing multiple forms of disease-specific PrP with
different sensitivities to proteinase K had been described (7). The ratio of protease-sensitive to protease-resistant pathological PrP has been used recently for characterization of a
particular TSE strain (22).
Our results suggest that the epitopes of PrPSc, which are
recognized by one or several of the antibodies used in our assay, become accessible only following Gh treatment. This could perhaps be
related to complexing of PrPSc with other fractions
(14) or to a different conformation from PrPC
(13). Significant conformational transitions and unfolding of PrPSc by concentrated GdnHCl or GdnSCN with simultaneous
loss of infectivity are well documented (19, 20). Hidden
epitopes of PrPSc could be exposed by treatment with GdnSCN
(16). Treatment with GdnSCN had the additional advantage of
PrPSc solubilization (21). We compensated for
the much lower concentration of GdnSCN (0.1 versus 4 M) used in our
experiments by heating. Thus, the treatment increased the solubility of
PrPSc and reversed, at least partially, its folding and
complexing, making hidden antigenic sites accessible (7).
The antibody R#26, which was raised against full-length PrP, did not
reveal an increased signal following treatment. This indicates either
that the chosen treatment was not sufficient to fully expose epitopes
recognized by R#26, implying that Gh-treated PrPSc is not
completely reversed to PrPC, or that R#26 could
immunodetect most if not all of the PrP present in the untreated BSE
brain homogenates and further denaturation exposed no additional epitope(s).
The detection of protease-resistant bovine PrPSc is
difficult without extensive extraction and centrifugation
(4). For diagnostic purposes, optimal incubation conditions
have to be elaborated to minimize unintentional digestion of
PrPSc while eliminating PrPC. In contrast to
Western blotting, in which both isoforms can be distinguished by size,
any remaining PrPC could interfere with the diagnosis by
ELISA. The Gh treatment approach had the advantage of not depending on
protease digestion for discrimination of PrPSc from
PrPC. All BSE-affected animals used in this study could be
distinguished from controls solely by the amount of their
PrPSc. The amounts of both PrPSc and
PrPC vary depending on the anatomic location of the sample,
as described for experimentally infected Syrian golden hamsters
(24). The former is restricted to well-confined
neuroanatomic areas; the latter depends on the amount of gray matter in
a sample, since PrP is expressed mostly in neurons. Our experiments
show little effect of Gh treatment on samples of cortex or thalamic
nuclei. Thus, the sample size and sample location must be optimized to reduce individual variations and to increase sensitivity.
We believe that the detection of PrPSc by a simple
physicochemical treatment, as used in this study, could be applied to
develop a diagnostic assay for TSE. Trials to test large numbers of
samples to validate the diagnostic usefulness of the technique are in progress. The detection of BSE and perhaps other TSE syndromes by ELISA
may simplify mass screening by allowing automation.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Swiss Federal
Veterinary Office and by the Swiss Federal Office for Education and
Science (Fair5-CT97-3311).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Animal Neurology, University of Bern, Bremgartenstrasse 109a, CH-3012 Bern, Switzerland. Phone: (41) 31 631 2206. Fax: (41) 31 631 2538. E-mail: meyer{at}itn.unibe.ch.
| |
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Journal of Virology, November 1999, p. 9386-9392, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
