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From: TSS ()
Subject: Diagnosis of human prion disease PRUSINER ET AL FULL TEXT PNAS | March 1, 2005 | vol. 102 | no. 9 | 3501-3506
Date: March 4, 2005 at 4:39 pm PST

PNAS | March 1, 2005 | vol. 102 | no. 9 | 3501-3506


Diagnosis of human prion disease

Jiri G. Safar *, , Michael D. Geschwind , , Camille Deering
*, Svetlana Didorenko *, Mamta Sattavat ¶, Henry Sanchez ¶,
Ana Serban * , Martin Vey ||, Henry Baron **, Kurt Giles *,
, Bruce L. Miller , , Stephen J. DeArmond * , ¶ and Stanley
B. Prusiner *, , ,

*Institute for Neurodegenerative Diseases, Memory and Aging
Center, and Departments of Neurology, ¶Pathology, and
Biochemistry and Biophysics, University of California, San
Francisco, CA 94143; ||ZLB Behring, 35041 Marburg, Germany;
and **ZLB Behring, 75601 Paris, France

Contributed by Stanley B. Prusiner, December 22, 2004


With the discovery of the prion protein (PrP),
immunodiagnostic procedures were applied to diagnose
Creutzfeldt–Jakob disease (CJD). Before development of the
conformation-dependent immunoassay (CDI), all immunoassays
for the disease-causing PrP isoform (PrPSc) used limited
proteolysis to digest the precursor cellular PrP (PrPC).
Because the CDI is the only immunoassay that measures both
the protease-resistant and protease-sensitive forms of
PrPSc, we used the CDI to diagnose human prion disease. The
CDI gave a positive signal for PrPSc in all 10–24 brain
regions (100%) examined from 28 CJD patients. A subset of 18
brain regions from 8 patients with sporadic CJD (sCJD) was
examined by histology, immunohistochemistry (IHC), and the
CDI. Three of the 18 regions (17%) were consistently
positive by histology and 4 of 18 (22%) by IHC for the 8
sCJD patients. In contrast, the CDI was positive in all 18
regions (100%) for all 8 sCJD patients. In both gray and
white matter, 90% of the total PrPSc was protease-sensitive
and, thus, would have been degraded by procedures using
proteases to eliminate PrPC. Our findings argue that the CDI
should be used to establish or rule out the diagnosis of
prion disease when a small number of samples is available as
is the case with brain biopsy. Moreover, IHC should not be
used as the standard against which all other
immunodiagnostic techniques are compared because an
immunoassay, such as the CDI, is substantially more

Human prion diseases include Creutzfeldt–Jakob disease
(CJD), kuru, and Gerstmann–Sträussler–Scheinker disease.
Sporadic CJD (sCJD) accounts for 85% of all cases of human
prion disease, familial CJD (fCJD) for 10–15%, and infection
from exogenous, frequently iatrogenic CJD (iCJD) prions, for
<1% (1). Prions consist solely of a disease-causing prion
protein (PrPSc) that is derived from the cellular isoform
(PrPC) (2). During prion replication, PrPSc stimulates
conversion of PrPC into nascent PrPSc.
Human prions from many cases of sCJD, fCJD, and iCJD were
transmitted to apes and monkeys, but few titrations were
performed, so there is little quantitative data on the
levels of prions from these investigations (3). The
development of mice expressing human prion protein (HuPrP)
and chimeric mouse–human transgenes (MHu2M) (4–7) allowed us
to measure the levels of prions in human brains as reported
here. The incubation times of these mice were sufficiently
abbreviated to allow endpoint titrations. Based on these
endpoint titrations, we surmise that each of three cases of
sCJD harbors a different strain of prion. We also used the
titrations to calibrate PrPSc measurements that were
determined by the conformation-dependent immunoassay (CDI).
Full-length, protease-resistant PrPSc (rPrPSc) and
previously unrecognized protease-sensitive forms of PrPSc
(sPrPSc) can be detected by the CDI (8). Most PrPSc
accumulating in the frontal cortex and white matter of sCJD
cases was sPrPSc. Other immunoassays for PrPSc detect the
N-terminally truncated protein PrP 27–30 derived from PrPSc;
these include Western blotting, ELISA, and histoblotting. It
is unclear what forms of PrPSc are detected by
immunohistochemistry (IHC) after hydrolytic autoclaving in
the presence of formic acid.

Because the CDI can readily detect PrPSc molecules
comprising one ID50 unit, we examined the diagnostic
sensitivity of the test by measuring PrPSc in many different
brain regions. We performed these measurements on brains of
28 people who died of either sCJD, fCJD(E200K), or iCJD.
Whereas the CDI registered a positive signal in every brain
region examined in all of the cases, standard histopathology
and IHC were much less effective in diagnosing CJD. Indeed,
the poor performance of these histological techniques
indicates that they should no longer be used to rule out
prion disease in a brain biopsy from a single cortical site
and must be applied to multiple cortical and subcortical
brain samples at autopsy.

Materials and Methods

Preparation of Brain Homogenates. For biochemical analysis
only, slices from 24 different anatomical areas of human
brains weighing 250–350 mg were homogenized to a final 15%
(wt/vol) in 4% (wt/vol) Sarkosyl in PBS, pH 7.4, by three
75-s cycles in a reciprocal homogenizer MiniBeadBeater-8
(BioSpec Products, Bartlesville, OH) as described in refs.
8–10. The resulting homogenate was diluted to a final 5%
(wt/vol) by using PBS containing 4% (wt/vol) Sarkosyl. The
diluted samples were either treated with a proteinase
inhibitor mixture for measurements of PrPSc or digested with
2.5 or 10 µg/ml proteinase K (PK) for 60 min at 37°C on the
shaker. After a clarification spin at 500 x g for 5 min at
room temperature in a drum rotor (Jouan, Winchester, VA),
the samples were mixed with stock solution containing 10%
sodium phosphotungstate (NaPTA) and 85 mM MgCl2, pH 7.4, to
obtain a final concentration of 0.32% NaPTA. After a 1-h
incubation at 37°C on a rocking platform, the samples were
centrifuged at 14,000 x g in a Jouan MR23i centrifuge for 30
min at room temperature. The resulting pellets were
resuspended in H2O containing protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2
µg/ml leupeptin) and assayed by the CDI.

Sandwich CDI for PrPSc. For capture of HuPrP, the mAb MAR1
was used (11), and detection was accomplished with mAb 3F4
(12) labeled with Eu-chelate of
traacetic acid at pH 9.6 for 16 h at room temperature
according to the manufacturer's protocols (Wallac, Turku,
Finland), as described in ref. 8. The principle,
development, calibration, and calculation of PrPSc
concentration from CDI data have been described in refs.
8–10. The results were expressed as the difference in
Ab-binding between native and denatured samples [(D - N)] of
the time-resolved fluorescence of aliquots measured in cpm.
In some cases, the concentration of PrPSc is directly
proportional to (D - N) value and was calculated from the
formula described in refs. 8–10.

Histopathologic Procedures. Autopsies were performed shortly
after death, and brain tissue was either immediately frozen
or immersion-fixed in 10% buffered formalin for embedding in
paraffin. We stained 8-µm-thick sections with hematoxylin
and eosin (H&E) to evaluate vacuolation. Vacuolation scores,
visual estimates of the percentage of gray matter area in a
slide occupied by vacuoles, were determined by a single
observer (S.J.D.). Reactive astrocytic gliosis was evaluated
by glial fibrillary acidic protein immunostaining by using a
rabbit antiserum (DAKO). Hydrolytic autoclaving pretreatment
of the formalin-fixed tissue sections was used to detect
PrPSc, as described in ref. 13. The Bielschowsky silver
stain and IHC for -synuclein, tau, and ubiquitin were used
as needed to test for Alzheimer's disease,
synucleinopathies, tauopathies, and other neurodegenerative

Additional methods describing the diagnosis of prion
disease, human samples acquisition, construction of
transgenic (Tg) mice, endpoint titrations in Tg mice, sample
tracking and data processing, and sandwich CDI for PrPSc are
described in Supporting Text, which is published as
supporting information on the PNAS web site.


Patient Groups, Clinical Diagnosis, and Codon 129 Polymorphi
sm. Human brain specimens were obtained from 46 patients who
underwent pathologic evaluation. Of the 28 cases in the
prion disease group, 24 were diagnosed with sCJD, three with
fCJD(E200K), and one with iCJD (see Table 3, which is
published as supporting information on the PNAS web site).
The PrP polymorphism at codon 129 [methionine (M) or valine
(V)] was determined by DNA sequencing for all 28 patients in
the prion disease group. As shown for sCJD, 13 patients were
MM, 7 were MV, and 4 were VV (Table 3).

Analytical Sensitivity of the CDI for Detection of Human
Prions. We used Eu-labeled 3F4 mAb (12) for detection and
MAR1 mAb (11) to capture HuPrPSc in a sandwich CDI format
(10). For a normal human brain homogenate that contains only
PrPC, the (D - N) was 1,789 cpm.

Brain homogenates from three cases of sCJD and one case of
fCJD were serially diluted in 3-fold increments into normal
human plasma and assayed by the CDI (Fig. 1). The
sensitivity of the CDI in detecting both sCJD and fCJD
prions was equal to or greater than that for the detection
of human prions by bioassay in Tg(MHu2M)5378/Prnp0/0 mice
(Fig. 1; see also Fig. 5 and Table 4, which are published as
supporting information on the PNAS web site). Within the
linear range, there was a good correlation between PrPSc
concentration measured by CDI and prion titer measured by

Fig. 1. Correlation between CDI and bioassay in Tg mice.
Sandwich CDI protocol for the detection of PrPSc was
compared to titration bioassays in Tg(MHu2M)5378/Prnp0/0
mice for three sCJD brains (a) and one fCJD brain (b).
Samples were precipitated with PTA and digested with 2.5
µg/ml PK for 1 h at 37°C. The MAR1 mAb was used (11) for
capture, and Eu-labeled 3F4 mAb was used for detection. The
(D - N) values measured in cpm are directly proportional to
the concentration of PrPSc (8, 10). Data points and bars
represent the average ± SD obtained from three to four
independent measurements. The cutoff (D - N) value of 1,789
cpm for this sandwich CDI protocol was calculated by [mean +
3(SD)] and determined from 100 brain samples obtained from
patients who died from nonneurologic disease (n = 6),
Alzheimer's disease (n = 7), and other neurologic diseases
(n = 5).

Diagnostic Sensitivity of the CDI for Detection of Human
Prions. To evaluate the diagnostic sensitivity of the CDI
for the detection of different CJD prions, we performed
multiple blind tests on brain tissue from 24 sCJD cases, 3
fCJD cases, 1 iCJD case, and 18 controls (Table 3). Data
from the controls exhibited a Gaussian distribution, with
the median (D - N) value oscillating around zero, as
expected for samples containing only residual PrPC after
phosphotungstate (PTA) precipitation (Fig. 2 and Table 5,
which is published as supporting information on the PNAS web
site). In contrast, median (D - N) values for sCJD and fCJD
cases are 106, which is six orders of magnitude higher than
the median of the control group (Fig. 2). The dynamic range
of the CJD data approaches 104, and all values are above
threshold. After performing 493 tests, the CDI identified
all CJD cases with 100% accuracy, and no false positives
occurred in the control group (Table 6, which is published
as supporting information on the PNAS web site).

Fig. 2. Statistical distribution of the CDI data for the
detection of PrPSc. Multiple samples from control (n = 18)
(a) and CJD brains (n = 27) (b) were tested. The control
group included cases of Alzheimer's disease (AD), patients
who died from other neurological diseases (OND), and cases
with no specific brain pathology at autopsy (NSPA). Each
brain sample was tested two to four times. Results are
expressed as (D - N) in cpm.

Codon 129 and the Anatomical Distribution of PrPSc in sCJD
Brains. By using the CDI, we determined the levels of PrPSc
in 24 brain regions for some patients and as few as 10 for
others because frozen samples for all regions were not
available (Fig. 6, which is published as supporting
information on the PNAS web site). Generally, the highest
concentrations of HuPrPSc were detected in the primary
visual cortex, thalamus, and cerebellum. All other areas of
the cortex and subcortical gray matter displayed substantial
accumulation of PrPSc. From these quantitative studies, we
conclude that, although the codon 129 genotype may influence
the levels of PrPSc deposition, the differences among the
three codon 129 genotypes (MM, MV, and VV) are less
prominent than expected from qualitative lesion profiles and
Biopsies of Human Brains. A 52-year-old female experienced
memory problems, difficulty with concentration, anxiety,
confabulation, and visual hallucinations. A left parietal
lobe biopsy was performed 3 months after symptoms began to
rule out CJD. The biopsy contained a sample of cortex
extending from the pial surface to the underlying white
matter. No characteristic vacuolation or PrPSc deposits were
identified in sections of the biopsy (Fig. 3 b and f).

Fig. 3. Routine histology and PrPSc IHC on brain sections of
two CJD patients. (a and e) Patient with CJD (MV2 subtype)
in which vacuolation and PrPSc deposits were identified in
all brain regions sampled, and kuru-type plaques were
identified in the cerebellar cortex. (b and f and c and g)
Patient with CJD (MM1 subtype) in which neither vacuolation
nor PrPSc deposits were found in a brain biopsy (b and f) or
at routine autopsy (c and g). (d and h) By using
high-intensity MRI signals as a guide, a second set of brain
sections were prepared from this patient; vacuolation of the
neuropil as well as coarse PrPSc deposits were found. (a–d)
H&E stain. (Scale bar, 50 µm.) (e–h) IHC for PrPSc. (Scale
bar, 30 µm.)

The patient died 3.5 months after the biopsy. Routine
sampling of multiple brain regions again failed to reveal
sufficient degrees of gray matter vacuolation (Fig. 3c) to
make the diagnosis of human prion disease. IHC for PrPSc
show rare punctate deposits in the neocortical regions
sampled (Fig. 3g). At the time, one of us (S.J.D.) believed
such infrequent deposits might be an artifact, and,
therefore, the inconclusive IHC and routine histology
prevented a definitive diagnosis of CJD. By using the MRI
scan performed a week before death as a guide, new samples
were obtained from cortical regions with high signal
intensities. In these regions, clusters of vacuoles
measuring 40–60 µm in diameter were found in cortical layers
2 and 3 (Fig. 3d) and coarse PrPSc deposits were associated
with the clusters of vacuoles (Fig. 3h). Small amounts of
PrPSc deposits were also found away from the vacuoles. The
clusters of vacuolation and deposits of PrPSc tended to be
small and highly focal, occupying <1% of the cortical
cross-sectional area.
Two years later, when unfixed frozen samples from the right
hemisphere corresponding to neuropathologically positive and
negative contralateral brain regions were analyzed by the
CDI, all 13 of the regions examined were strongly positive
for PrPSc. The data from histology with H&E staining, IHC,
and the CDI are summarized in Table 7, which is published as
supporting information on the PNAS web site. (D - N) values
varied from almost 300,000 cpm in the medulla to >3,000,000
cpm in the thalamus, globus pallidus, frontal cortex,
occipital cortex, as well as the parietal cortex, the region
where the initial biopsy was taken on the contralateral

Diagnostic Sensitivity of the CDI and IHC. When brain
sections from 10 CJD cases [8 sCJD and 2 fCJD(E200K)] were
analyzed by H&E staining, IHC with -PrP mAbs, and the CDI
(Tables 1 and 2), we discovered that, like the biopsied
patient reported above, the CDI was vastly superior to both
histologic examination for vacuolation of the neuropil and
IHC for PrP immunostaining. The microscopic studies were
performed on formalin-fixed, paraffin-embedded tissue
sections with knowledge that the patients were clinically
diagnosed with CJD, but without knowledge of the CDI

Table 1. Comparison of the diagnostic sensitivity of the
vacuolation profile, IHC, and sandwich CDI in the detection
of PrPSc in different anatomical areas of sCJD brains

Table 2. Comparison of the diagnostic sensitivity of the
vacuolation profile, IHC, and sandwich CDI for the detection
of PrPSc in different anatomical areas of fCJD brains

From 18 brain regions of the 8 sCJD cases, we compared the
results of histology, IHC, and CDI. Only the CDI gave
consistently positive (100%) PrPSc signals for all 18 brain
regions in all 8 patients. By routine histology, only 3 of
18 regions were found positive in the 8 sCJD cases (Table
1); this represents a diagnostic sensitivity of 17%. The
entorhinal cortex, temporal lobe cortex, and the caudate
nucleus were 100% positive from the brains of all eight
patients analyzed by histology. The remaining 15 regions
varied from 0% to 87% positive for the 8 sCJD brains
The results with IHC were similar to those obtained by
histological examination, which was unexpected because IHC
is generally thought to be more sensitive than histology. By
IHC, only 4 of 18 regions were found positive in all 8 sCJD
patients (Table 1); these results represent a diagnostic
sensitivity of 22%. The frontal cortex, parietal cortex,
temporal lobe cortex, and the insula were 100% positive from
the brains of all 8 patients. The remaining 14 regions
varied from 13% to 87% positive for the 8 sCJD brains
examined. Comparing histology, IHC, and the CDI, only the
temporal lobe region gave consistently positive results
(100%) for the 8 sCJD patients (Table 1).

Next, we compared histology, IHC, and CDI analysis on the
brains of two patients who died of fCJD(E200K). Of the 18
regions examined by histology or IHC, 9 were found positive
in the 2 patients (Table 2); these results represent a
diagnostic sensitivity of 50%. In contrast, the CDI was
positive in all 18 regions for both patients.

Asymmetric Lesions and PrPSc Deposits in the Brain. To
address the possibility of sampling bias, we examined the
brain of a 79-year-old female in whom rapidly progressive
motor and language decline were associated with myoclonic
jerks and an electroencephalogram with periodic spikes
characteristic of CJD. The patient died 14 days after a
diffusion-weighted MRI scan showed high intensity signals in
the left cerebral cortex but few or no such signals in the

Histologic analysis showed moderately severe vacuolation
scores in multiple cortical regions of the left cerebral
hemisphere with little or none in analogous regions on the
right (Table 8, which is published as supporting information
on the PNAS web site). Unbiased stereological counts of
neurons in different cerebral cortical layers showed marked
loss from all layers of the left frontal cortex (Brodmann
areas 44 and 45), but no loss was found on the right (data
not shown). Morphometric quantification of IHC for glial
fibrillary acidic protein showed marked astrocytic gliosis
in the left cerebral cortex and only focal, mild gliosis on
the right. We found more PrPSc deposits in the left cortex
than in the right but these were not quantified (Table 8).
PrPSc was found in all locations of analogous right and left
cortical samples by the CDI; levels of PrPSc in the right
cortex were 5–50% lower than those from the left. Much lower
levels of PrPSc in the right cortex might have been expected
based the minimal microscopic changes, reflecting again the
incongruity between microscopic and CDI analyses.

Levels of sPrPSc and rPrPSc. To determine the relationship
between sPrPSc and rPrPSc in the brains of sCJD patients,
samples were either PTA-precipitated to measure the
concentration of total PrPSc or PK-digested then
PTA-precipitated to obtain the concentration of rPrPSc, as
described in refs. 8 and 9. These treated samples were then
subjected to analysis by the CDI. Surprisingly, >80% of
total PrPSc was susceptible to proteolytic degradation (Fig.
4). Despite a 20-fold lower concentration of PrPSc in white
matter, the ratio between sPrPSc and rPrPSc remained
constant. In conclusion, sPrPSc constitutes a major fraction
of total PrPSc in the frontal cortex and white matter of the
sCJD-infected brains.

Fig. 4. Most PrPSc in the frontal cortex and white matter of
sCJD brains is protease-sensitive [gray bars, calculated
from measurements of total PrPSc (white bars) and rPrPSc
(black bars)]. Before measurement by the CDI, undigested
samples were PTA-precipitated to measure total PrPSc or
digested with 50 µg/ml PK at 37°C for 1 h, followed by PTA
precipitation to determine rPrPSc (8, 9). The graph shows
the means ± SEM obtained from duplicate measurements of
samples from the frontal cortex (n = 19) and white matter (n
= 12) of sCJD-infected brains.

It is noteworthy that IHC of formalin-fixed,
paraffin-embedded tissue sections occasionally showed PrPSc
deposits in white matter; however, histoblot analysis, which
is our most sensitive and specific tissue-based method,
routinely failed to identify rPrPSc in white matter (data
not shown). In contrast, the CDI found PrPSc in white matter
in all cases of sCJD and fCJD (Fig. 4 and Tables 1 and 2).


The clinical diagnosis of human prion disease is often
difficult until the patient shows profound signs of
neurologic dysfunction. It is widely accepted that the
clinical diagnosis must be provisional until a tissue
diagnosis either confirms or rules out the clinical
assessment. Before the availability of Abs to PrP, a tissue
diagnosis was generally made by histologic evaluation of
neuropil vacuolation. IHC with
anti-glial-fibrillary-acidic-protein Abs in combination with
H&E staining preceded the use of anti-PrP Ab staining.

Recently, the role of IHC in the diagnosis of scrapie in the
brains of eight clinically affected goats inoculated with
the SSBP1 prion isolate has been challenged (14). Thalamic
samples taken from seven of eight goats with scrapie were
positive for PrPSc by Western blotting but negative by IHC.
The eighth goat was negative by Western blotting and IHC.
Consistent with these findings in goats are the data
reported here, in which IHC of formalin-fixed,
paraffin-embedded human brain samples was substantially less
sensitive than the CDI.

The CDI was developed to quantify PrPSc in tissue samples
from mammals producing prions. Concerned that limited PK
digestion was hydrolyzing some or even most of the PrPSc, we
developed a CDI that does not require PK digestion. The CDI
revealed that as much as 90% of PrPSc is sPrPSc; thus, it
was being destroyed during limited proteolytic digestion
used to hydrolyze PrPC. sPrPSc comprises 80% of PrPSc in the
frontal lobe and in the white matter (Fig. 4).

The CDI detected HuPrPSc with a sensitivity comparable to
the bioassay for prion infectivity in Tg(MHu2M) mice (Fig.
1). The high sensitivity achieved by the CDI is due to
several factors (8, 10, 11, 15). First, both sPrPSc and
rPrPSc conformers are specifically precipitated by PTA
(Table 5) (8, 9). PTA has also been used to increase the
sensitivity of Western blots enabling the detection of
rPrPSc in human muscle and other peripheral tissues (16,
17). Second, a sandwich protocol was used with the
high-affinity MAR1 mAb (11) to capture HuPrPSc and
Eu-labeled 3F4 mAb to detect HuPrPSc (12). Third, the CDI
detects PrPSc by Ab-binding to native and denatured forms of
the protein and, therefore, does not depend on proteolytic
degradation of PrPC. We chose not to perform Western blots
on most of the samples used in this study because such
immunoblots require denaturation of the sample, which
eliminates measurement of the native signal corresponding to
PrPC (Table 5). Moreover, a comparison between the CDI and
Western blotting on brain samples from sCJD and variant CJD
patients showed that the CDI was 50- to 100-fold more
sensitive (15). Additionally, Western blots combined with
densitometry are linear over a 10- to 100-fold range of
concentrations, whereas the CDI is linear over a >104-fold
range. The CDI has been automated, which not only improves
accuracy and reproducibility (10) but also allows numerous
samples to be analyzed, as reported here. Western blots are
difficult to automate and are labor intensive.

Our studies show that only the CDI detected PrPSc in all
regions examined in 24 sCJD and 3 fCJD(E200K) brains (Figs.
2 and 6). Comparative analyses demonstrated that the CDI was
vastly superior to histology and IHC. When 18 regions of 8
sCJD and 2 fCJD(E200K) brains were compared, we discovered
that histology and IHC were unreliable diagnostic tools
except for samples from a few brain regions. In contrast,
the CDI was a superb diagnostic procedure because it
detected PrPSc in all 18 regions in 8 of 8 sCJD and 2 of 2
fCJD(E200K) cases (Tables 1 and 2).

Histologic changes in prion disease have been shown to
follow the accumulation of prions as measured by bioassay of
infectivity and by PrPSc accumulation (18–22). Because low
levels of PrPSc are not associated with neuropathologic
changes, some discrepancy between vacuolation and PrPSc was
expected. In contrast to histology, IHC measures PrP
immunostaining after autoclaving tissue sections exposed to
formic acid. Because IHC measures PrP, we expected the
sensitivity of this procedure might be similar to the CDI,
but that proved not to be the case. Whether exposure of
formic acid-treated tissue sections to elevated temperature
destroys not only PrPC but also sPrPSc and only denatures
rPrPSc remains to be determined. Such a scenario could
account for the lower sensitivity of IHC compared with CDI
or bioassay (Tables 1 and 2).

Studies of the white matter in CJD brains were particularly
informative with respect to the sensitivity of the CDI,
where PrPSc levels were low but readily detectable, 10- to
100-fold above the threshold value (Fig. 4). Because animal
studies have shown that PrPSc and infectivity are
transported anterogradely from one brain region to another
along neuroanatomical pathways (23–25), we expected to find
PrPSc in white matter as demonstrated by the CDI but not
IHC. Axonal transport of PrPSc is also suggested by
diffusion-weighted MRI scans of CJD cases, which show
high-intensity signals in analogous neocortical regions of
the right and left cerebral hemispheres (26). This symmetry
of neuroradiological abnormalities is consistent with spread
of PrPSc to the contralateral cortex by means of callosal
commissural pathways.

Most immunoassays that detect HuPrPSc do so only after
subjecting the sample to limited proteolysis to form PrP
27–30, followed by denaturation. Because the CDI measures
the immunoreactivity before and after denaturation to an
epitope that is exposed in native PrPC but buried in PrPSc,
limited proteolysis to eliminate PrPC is unnecessary. Assays
based on limited proteolysis underestimate the level of
PrPSc because they digest sPrPSc, which represents 80–90% of
PrPSc in CJD and scrapie brains (Fig. 4 and Table 5).

Gerstmann–Sträussler–Scheinker, an inherited human prion
disease, is caused by the P102L mutation in the PRNP gene.
In mice expressing the Gerstmann–Sträussler–Scheinker mutant
PrP transgene, the CDI detected high levels of sPrPSc(P101L)
as well as low levels of rPrPSc(P101L) long before
neurodegeneration and clinical symptoms occurred (9).
sPrPSc(P101L) as well as low concentrations of rPrPSc(P101L)
previously escaped detection (27). Whether a similar
situation applies in other genetic forms of prion disease,
sCJD, or variant CJD remains to be determined. Because most
of the PrPSc in the brains of sCJD patients is
protease-sensitive (Fig. 4), it is likely that the lower
sensitivity of IHC is due to its inability to detect sPrPSc.
Presently, we have no information about the kinetics of
either sPrPSc or rPrPSc accumulation in human brain. Limited
information on the kinetics of PrPSc accumulation in
livestock comes from studies of cattle, sheep, and goats
inoculated orally, but most of the bioassays were performed
in non-Tg mice (28–30) in which prion titers were
underestimated by as much as a factor of 104 (10).

The studies reported here are likely to change profoundly
the approach to the diagnosis of prion disease in both
humans and livestock (31–33). The superior performance of
the CDI in diagnosing prion disease compared to routine
neuropathologic examination and IHC demands that the CDI be
used in future diagnostic evaluations of prion disease.
Prion disease can no longer be ruled out by routine
histology or IHC. Moreover, the use of IHC to confirm cases
of bovine spongiform encephalopathy after detection of
bovine PrPSc by the CDI (10) seems an untenable approach in
the future. Clearly, the CDI for HuPrPSc is as sensitive or
more sensitive than bioassays in Tg(MHu2M) mice (Fig. 1).

Our results suggest that using the CDI to test large numbers
of samples for human prions might alter the epidemiology of
prion diseases. At present, there is limited data on the
frequency of subclinical variant CJD infections in the U.K.
population (34). Because appendixes and tonsils were
evaluated only by IHC, many cases might have escaped
detection (Tables 1 and 2). Equally important may be the use
of CDI-like tests to diagnose other neurodegenerative
disorders, such as Alzheimer's disease, Parkinson's disease,
and the frontotemporal dementias. Whether IHC underestimates
the incidence of one or more of these common degenerative
diseases is unknown. Moreover, CDI-like tests may help
determine the frequency with which these disorders and the
prion diseases occurs concomitantly in a single patient (35,


We thank the staff of the Hunters Point Animal Facility for
their expert mouse studies and ZLB Behring for making the
MAR1 mAb available. This work was supported by National
Institutes of Health Contract NS02328 and National
Institutes of Health Grants AG02132, AG010770, and AG021601.
M.D.G. is supported by the John Douglas French Foundation
for Alzheimer's research, the McBean Foundation; National
Institute on Aging Grants AG021989, AG019724, and AG023501;
the Alzheimer's Disease Research Center of California; and
National Institutes of Health Grant M01 RR00079 to the
General Clinical Research Center. B.L.M. is supported by
National Institute on Aging Grants AG019724 and AG023501.


Author contributions: J.G.S. and S.B.P. designed research;
M.D.G., C.D., S.D., M.S., H.S., and S.J.D. performed
research; A.S., M.V., and H.B. contributed new
reagents/analytic tools; J.G.S., K.G., B.L.M., S.J.D., and
S.B.P. analyzed data; and J.G.S., S.J.D., and S.B.P. wrote
the paper.

Abbreviations: CDI, conformation-dependent immunoassay; CJD,
Creutzfeldt–Jakob disease; sCJD, sporadic CJD; fCJD,
familial CJD; iCJD, iatrogenic CJD; IHC,
immunohistochemistry; PrP, prion protein; PrPC, normal
cellular PrP; PrPSc, disease-causing PrP; HuPrP, human PrP;
MHu2M, chimeric mouse–human transgene; sPrPSc,
protease-sensitive PrPSc; rPrPSc, protease-resistant PrPSc;
PTA, phosphotungstate; PK, proteinase K; H&E, hematoxylin
and eosin; Tg, transgenic; (D - N), difference in Ab-binding
between native and denatured samples.

J.G.S., S.J.D., A.S., K.G., and S.B.P. have financial
interest in InPro Biotechnology, Inc.

To whom correspondence should be addressed at: Institute for
Neurodegenerative Diseases, University of California, Box
0518, San Francisco, CA 94143-0518. E-mail:

© 2005 by The National Academy of Sciences of the USA


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