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Journal of Clinical Microbiology, June 1998, p. 1750-1755, Vol. 36, No. 6
Animal Disease Research Unit, Agricultural
Research Service, U.S. Department of Agriculture,
Pullman, Washington 99164-70301;
Department of Veterinary Microbiology and Pathology, Washington
State University, Pullman, Washington
99164-70402;
Washington Animal Disease
Diagnostic Laboratory, Washington State University, Pullman,
Washington 991643;
National Animal
Disease Center, Agricultural Research Service, U.S. Department of
Agriculture, Ames, Iowa 500104;
and
Colorado State Diagnostic Laboratory, College of
Veterinary Medicine, Colorado State University, Fort Collins,
Colorado 805235
Received 19 August 1997/Returned for modification 11 December
1997/Accepted 27 January 1998
The transmissible spongiform encephalopathies are a heterogeneous
group of fatal neurodegenerative disorders occurring in humans, mink,
cats, and ruminant herbivores. The occurrence of novel transmissible
spongiform encephalopathies in cattle in the United Kingdom and Europe
and in mule deer and elk in parts of the United States has emphasized
the need for reliable diagnostic tests with standardized reagents.
Postmortem diagnosis is performed by histologic examination of brain
sections from affected animals. The histopathological criteria for
transmissible spongiform encephalopathies include gliosis,
astrocytosis, neuronal degeneration, and spongiform change. These
lesions vary in intensity and anatomic location depending on the host
species and genetics, stage of disease, and infectious agent source.
Diagnosis by histopathology alone may be ambiguous in hosts with early
cases of disease and impossible if the tissue is autolyzed. Deposition
of the prion protein (an abnormal isoform of a native cellular
sialoglycoprotein) in the central nervous system is a reliable marker
for infection, and immunohistochemical detection of this marker is a
useful adjunct to histopathology. In the present paper we describe
monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein
in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the
transmissible spongiform encephalopathies in these ruminant species.
The transmissible spongiform
encephalopathies (TSEs) are a heterogeneous group of fatal
neurodegenerative disorders characterized by deposition of an abnormal
isoform (prion protein Sc [PrP-Sc]) of a normal cellular glycoprotein
(PrP-C) in neural tissue. PrP-Sc, either alone or in association with
another protein, may represent a novel transmissible agent, the prion
(28), which propagates by catalyzing the conversion of PrP-C
to PrP-Sc through a nucleation or polymerization event (9,
14). Data in support of this "protein-only" hypothesis are
based largely on rodent models of the ovine TSE, scrapie, in which
PrP-Sc is the major component of infectious tissue extracts
(3). PrP-C and PrP-Sc are derived from the same single-copy
host gene (25) but differ in their physicochemical qualities
including solubility in detergent and relative resistance to digestion
by proteinase K (PK) (22). PK hydrolysis removes only the 60 to 70 residues at the amino terminus of the protein, leaving two or
three fragments representing the unglycosylated peptide and one or more
differentially glycosylated forms migrating between 19 and 28 kDa.
Expression of PrP-C by host genes, by transgenes, or in engrafted
tissue is required for the development of clinical disease, PrP-Sc
propagation, and brain lesions (2, 4, 5, 7). Conversion of
PrP-C to PrP-Sc has been demonstrated in a cell-free system
(19) and by direct contact of recombinant PrP-C with PrP-Sc
in frozen brain slices (1). The mechanisms of neurotoxicity
in the TSEs have not yet been delineated. Morphologic and functional
changes have been reported in neurons, microglia, and astrocytes in
vivo and in vitro in response to infection or exposure to neurotoxic
peptide fragments of PrP (6, 11, 29, 30).
TSEs occur naturally in humans, mink, cats, and ruminant herbivores.
Sheep scrapie is endemic in many parts of the world, and control
efforts have been hampered by the long incubation time and a lack of
tools for early diagnosis. Bovine spongiform encephalopathy (BSE), a
novel TSE of cattle and exotic ruminants (34), poses a more
serious threat because of its proposed causative relationship with a
new variant of human Creutzfeldt-Jakob disease (8). Chronic
wasting disease (CWD) is a relatively rare disorder reported in mule
deer, white tail deer, and elk originating from a small area of the
western United States (39). Diagnosis of ruminant TSEs is
based on the appearance of neuronal vacuolation, spongiform changes,
gliosis, and astrocytosis (15, 35, 37, 39) in neural tissue
collected postmortem. The histological lesion profiles vary in
intensity and anatomic location among species and individuals (13,
15); diagnosis by histopathology alone may be equivocal for hosts
with early cases of disease or autolyzed tissue (24).
Detection of PrP-Sc by immunoassay of fixed tissue is a useful
confirmatory assay (10, 12, 23, 24, 33). With one exception
(20), monoclonal antibodies (MAbs) and polyclonal antibodies
recognize PrP-C as well as PrP-Sc and immunodetection protocols must
include a process for the selective elimination of the reactivity of
PrP-C. PrP-C, which is sensitive to formalin fixation and routine
tissue processing procedures (21), is usually not detectable
in formalin-fixed tissue; epitopes on the PrP-Sc in these samples are
unmasked by heat, acid, or enzyme pretreatment (12, 16, 18).
The efficacies of the fixation and pretreatment protocols in which
PrP-C reactivity is eliminated and PrP-Sc staining is enhanced are
monitored by staining tissues from TSE-affected and healthy animals in
parallel. Under these conditions, immunohistochemical analysis with
validated reagents will provide useful diagnostic tests. Rabbit
antisera reactive with ruminant PrP-Sc cannot be standardized for
widespread use due to limitations in its quantity and specificity. In
this paper, we report the use of MAb F89/160.1.5 in the
immunohistochemical analysis of formalin-fixed central nervous system
samples from cattle, sheep, mule deer, and elk with naturally occurring
TSEs.
Antigen preparation and MAb production.
A synthetic
peptide, representing residues 146 to 159 of the bovine prion protein
(17) (NH2-SRPLIHFGSDYEDR-COOH), was
coupled to maleimide-activated keyhole limpet hemocyanin (Pierce
Chemical Company). Five 6-week-old BALB/c mice were each inoculated
subcutaneously at two sites with a total of 10 µg of conjugated
peptide emulsified in 200 µl of Freund's complete adjuvant. Two
booster inoculations of 10 µg of conjugated peptide in 200 µl of
Freund's incomplete adjuvant were administered at 14-day intervals.
Three days before cell fusion, the mice were immunized intravenously
with 10 µg of conjugated peptide in phosphate-buffered saline without
adjuvant. Cell fusion and cloning by limiting dilution were performed
by following standard protocols (41). Supernatants from
primary and cloned hybridomas were screened by a recombinant ovine
PrP-C enzyme-linked immunosorbent assay (ELISA). Clone 1.5 from cell line F89/160 was selected and was transferred to an artificial capillary cell culture system (CellMax; CellCo Inc.) for the in vitro
production of an MAb supernatant. The heavy-chain isotype was
identified by ELISA, and the MAb concentration was determined by
immunodiffusion.
Recombinant sheep PrP-C ELISA.
Supernatants from primary and
cloned hybridomas were screened by ELISA with recombinant sheep PrP-C
as the antigen.
(i) Production of recombinant sheep PrP-C in Escherichia
coli.
Genomic DNA was isolated from the peripheral blood
mononuclear cells of a Suffolk sheep. The PrP open reading frame was
amplified with flanking primers (38) modified to incorporate
EcoRI restriction sites (forward primer,
5'-ATCGAATTCAAGAAGCGACCAAAAC-3'; reverse primer,
5'-ATCGAATTCAGACACCACCACT-3'). The 786-bp PCR product was
digested with EcoRI, purified on agarose gels, and ligated into the vector pMal-cRI. Transformation of E. coli DH5 was
performed by conventional techniques. Transformants were screened by
PCR of colony minipreps with the cloning primers. One positive clone (pMal-1) was selected for large-scale fusion protein expression. The
fusion product ShPrP-maltose-binding protein (MBP) was isolated from
bacterial lysates by affinity chromatography on amylose resin columns
and was eluted with 10 mM maltose. Fractions were screened by Western
immunoblotting with a rabbit antiserum to PrP peptide NH2GQGGGTHNQWNKPSK (R2843) (26).
(ii) Recombinant ShPrP ELISA.
Each well of Immulon 2 plates
(Dynatech, Chantilly, Va.) was coated with 6.25 µg of the recombinant
ShPrP-MBP fusion protein in 50 µl of 0.05 M carbonate buffer (pH
9.6), and the plates were incubated overnight at 4°C. The plates were
blocked with a 1:15 dilution of commercially available milk-based
blocker (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 1 h. Fifty microliters of antiserum or hybridoma supernatant was
incubated in each well for 30 min at room temperature. The plates were
developed with goat anti-mouse immunoglobulin G (IgG)-horseradish
peroxidase (HRPO) and 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate
(6)] (ABTS; Kirkegaard & Perry Laboratories). The optical
density was read at 405 nm. Negative controls included supernatants
from isotype-matched MAbs of irrelevant specificity or tissue culture
medium adjusted to contain 15% fetal calf serum. Positive control
wells were incubated with rabbit anti-PrP peptide antiserum (R2843) and
were developed with goat anti-rabbit IgG-HRPO and ABTS. Positive wells
had optical densities at 405 nm higher than 2 standard deviations above
the mean for four negative control wells.
Source of brain tissue from ruminant herbivores with naturally
occurring TSEs and from control herbivores.
Brain tissues from 34 sheep with histopathological lesions of scrapie were tested for
reactivity with MAb F89/160.1.5 by immunohistochemical analysis. PrP-Sc
had been detected immunohistochemically with a rabbit anti-mouse PrP
polyclonal antiserum in 20 of these samples and by Western
immunoblotting in 6 of the 20 samples (23). Tissues from 3 sheep with no histological lesions of scrapie and no PrP-Sc detectable
by Western blot analysis were used as negative controls, as were
tissues from an additional 12 sheep with no clinical signs of scrapie
and no histological lesions. These tissues were provided by
pathologists in veterinary medical colleges and state diagnostic laboratories or by personnel from the Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Monoclonal Antibody F89/160.1.5 Defines
a Conserved Epitope on the Ruminant Prion Protein
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Western immunoblot analysis. PrP-Sc was isolated from the brains of sheep by differential centrifugation from a high-salt Sarkosyl buffer (31). Precipitated proteins were digested with 10 µg of PK per ml for 1 h at 37°C and were analyzed in aliquots equivalent to 125 mg of starting material on 15% polyacrylamide minigels (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes (Schleicher & Schuell). The filters were developed with 3 µg of MAb F89/160.1.5 per ml or a control antibody of the same isotype, goat anti-mouse IgG-HRPO, and a chemiluminescent substrate (Amersham). Filters were exposed to film (Amersham HyperFilm) for 8 to 20 min with no increase in background chemiluminescence.
Immunohistochemistry. Brains were fixed in 10% buffered formalin by immersion and were embedded in paraffin. One section from each block was stained with hematoxylin and eosin for routine histopathology. Additional tissue sections were mounted on positively charged glass slides (Probe-On Plus; Fisher Scientific) for immunohistochemical analysis. Sections for immunohistochemical analysis were deparaffinized and hydrated and then autoclaved in distilled water at 121°C for 30 min (16) and allowed to cool. The slides were immunostained, using capillary flow technology in an automated immunostainer (Code-On Slide Stainer; Fisher Scientific) as described previously (17, 18), with a biotinylated second antibody, streptavidin-alkaline phosphate complex (Biomeda Corp), and an alkaline phosphatase substrate-chromagen (Vector Red; Vector Laboratories). Additional sections of selected ovine samples were immunostained as described above except that bound primary antibody was detected with biotinylated horse anti-mouse IgG second antibody, avidin-biotin-HRPO complex (ABC-peroxidase; Vector Laboratories), and a peroxidase substrate-chromagen (AEC; Dako Corp). All slides were counterstained with Mayer's hematoxylin. Negative control procedures consisted of (i) substitution of MAb F89/160.1.5 with a similar concentration of an irrelevant control MAb of the same isotype and (ii) incubation of MAb F89/160.1.5 with brain tissue from scrapie-free sheep, cattle, or mule deer with no evidence of TSE as indicated by histopathology of samples from all three species and Western immunoblot analysis of ovine tissues.
Epitope mapping and PrP gene sequences.
An overlapping set
of octamer peptides spanning SRPLIHFGSDYEDR was synthesized on a
membrane support with commercial reagents and by following the
instructions of the manufacturer (SPOTs Test; Genosys Biotechnologies,
The Woodlands, Tex.). The ability of MAb F89/160.1.5 to bind to
individual octamer peptides was determined visually following
incubation with 
-galactosidase-conjugated secondary antibody and
substrate.
Mule deer and elk PrP gene sequences.
Three alleles of the
mule deer PrP sequence were identified. Alleles 138S2 and 138N1 encode
Ser and Asn at codon 138, respectively. Allele 138S1 differs from
allele 138S2 by a silent mutation. Two alleles of the elk PrP gene were
found and encode an M
L substitution at codon 132.
Nucleotide sequence accession numbers. The GenBank nucleotide sequence accession numbers of alleles 138S2, 138N1, and 138S1 and the two alleles of the elk PrP gene are AF009180, U97331, AF009181, AF016227, and AF016228, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Production of MAbs. Five mice were immunized with a keyhole limpet hemocyanin-conjugated synthetic peptide previously demonstrated to generate polyclonal antisera reactive with bovine and ovine PrP-C proteins in Western immunoblots (17). Antisera and hybridoma supernatants were screened by ELISA with a recombinant sheep PrP fusion protein as the antigen. Cell line 160 produced antibodies reactive in the ELISA and was selected for two rounds of cloning by limiting dilution and propagated in an in vitro artificial capillary cell culture production system. Pooled supernatants from cell line F89/160.1.5 (heavy-chain isotype immunoglobulin G1) had a concentration of 3.64 mg/ml. The MAb from this pool was further characterized by epitope mapping, Western immunoblot analysis, and immunohistochemical analysis.
Epitope mapping and sequence determination. The epitope recognized by MAb F89/160.1.5 was mapped with a panel of overlapping peptides (Table 1) immobilized on a derivatized cellulose membrane. Sequential deletion of amino-terminal residues S, R, P, and L (peptides 2, 3, 4, and 5, respectively) did not eliminate antibody binding. Peptide 6, lacking the I residue, and peptides 7 and 8, lacking IH and IHF, respectively, failed to bind to MAb F89/160.1.5. Therefore, only the sequence IHFG is common to all peptides bound by the MAb. This sequence is conserved in the deduced amino acid sequences reported to date for cattle, sheep, mule deer, and elk PrP in the samples from the present study for which frozen tissue was available (tissue from 12 sheep, 10 mule deer, and 2 elk) and for a larger sample of CWD-affected mule deer (n = 26). Samples with amino acid polymorphisms outside the antibody binding site (ovine codon 112, A to V; mule deer codon 138, N to S) showed no difference in the intensity or distribution of immunostaining with MAb F89/160.1.5.
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Western immunoblotting reactivity with PrP-Sc from TSE-affected sheep. MAb F89/160.1.5 was generated by inoculation into mice with a synthetic peptide and was selected by screening the MAb against a recombinant form of PrP-C. To determine whether MAb F89/160.1.5 binds to the disease-specific, protease-resistant fragments (PrP-Sc), brain extracts from sheep with natural scrapie and from healthy sheep were treated with PK, which hydrolyzes PrP-C and which leaves the hallmark multiple peptide bands of PK-resistant PrP-Sc (22). PK-treated extracts from scrapie-affected sheep typically showed two or three peptide bands with apparent molecular weights of between 19,000 and 28,000 (Fig. 1, lane 1). No bands were detected in extracts from healthy sheep brain (Fig. 1, lane 2) or when an isotype-matched control MAb was used to probe the Western immunoblots (data not shown).
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Immunohistochemical analysis of tissues from healthy and TSE-affected ruminants. Positive immunostaining by MAb F89/160.1.5 immunohistochemical analysis was detected for the brains of all TSE-affected animals examined (36 sheep, 19 cattle, 10 mule deer, and 4 elk); no immunostaining was detected for the brains of any healthy control animals (15 sheep, 15 mule deer, 12 elk, and 20 cattle). Examples of positive and negative immunostaining are shown in Fig. 2a to d. The pattern of immunoreactivity for TSE-affected animals was basically similar for all animals. PrP-Sc immunoreactivity was present in the brain stem and midbrain from the level of the hypothalamus rostrally to the obex caudally. Immunostaining was concentrated within specific neurologic nuclei. At low magnification, most immunostaining consisted of random dense granules, globules, and plaques within the gray matter neuropil admixed with spongiform lesions (Fig. 2a). The great majority of PrP-Sc immunoreactivity aggregated adjacent to or surrounding glial cell nuclei (Fig. 2b) and sometimes accumulated in a branching pattern around glial cells identified histologically as microglia (small, oval to angular hyperchromatic nuclei without a recognizable cytoplasm) (Fig. 2b) (32). There also was rim-like perivascular and subependymal immunostaining reminiscent of astroglial foot processes (Fig. 2c). The PrP-Sc immunoreactivities of neurons consisted of punctate immunostaining within neuronal perikarya (Fig. 2c) or distinct rimming around the periphery of neuronal perikarya (Fig. 2b) or around the peripheral membranes of intraneuronal vacuoles (Fig. 2a). Both neurons with and without intraneuronal vacuoles had PrP-Sc immunoreactivity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PrP-Sc is a marker protein for TSEs, and detection of PrP-Sc by immunohistochemical analysis is a useful adjunct to histopathology for the diagnosis of these diseases in ruminant animals. MAbs reactive with conserved epitopes on ovine, bovine, and cervid PrP-Sc proteins will be useful reagents for standardized diagnostic testing and comparative pathology studies. We immunized mice with a peptide representing an immunogenic region of the bovine PrP protein and screened the resulting MAbs for their reactivities to recombinant sheep PrP-C proteins to select cross-reacting antibodies. One of these MAbs, MAb F89/160.1.5, was shown to react with PrP-Sc by Western immunoblotting of PK-digested preparations from the brains of sheep with natural scrapie. The MAb was also shown to be reactive with PrP-Sc in formalin-fixed tissues from sheep, cattle, mule deer, and elk under tissue fixation and pretreatment conditions which eliminated the reactivity of PrP-C. The immunohistochemical staining pattern of MAb F89/160.1.5 was similar to the patterns described for the brains of scrapie-affected sheep and obtained with polyclonal rabbit antisera to ovine or mouse PrP (23, 24). The present data also demonstrate the utility of MAb F89/160.1.5 in the diagnosis of TSE from brain tissues of cattle and wild ruminants. We are characterizing the accumulation of PrP-Sc in extraneural tissues using MAb F89/160.1.5 and other MAbs to ovine PrP to determine the utility of antigen-based assays for antemortem and preclinical diagnosis of scrapie, CWD, and BSE. MAb reagents to conserved epitopes on PrP-Sc or cocktails of MAbs to multiple variable epitopes provide specific, reliable, and flexible tools for the accurate diagnosis of TSE in mammals.
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ACKNOWLEDGMENTS |
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This work was supported by the Agricultural Research Service, U.S. Department of Agriculture (grant CWU 5348-32000-011-00D).
We acknowledge the technical assistance of L. Mickelsen, W. Harwood, L. Kappmeyer, P. Dilbeck, T. McReynolds, D. Bradley, D. Orcutt, and J. Bulgin. E. Williams, M. Miller, and M. Wild provided blood and tissue samples from healthy and CWD-affected deer and elk. We thank A. Jenny, National Veterinary Services Laboratory, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, for identifying and providing tissues from scrapie-affected sheep.
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FOOTNOTES |
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* Corresponding author. Mailing address: Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 337 Bustad Hall, WSU, Pullman, WA 99164-7030. Phone: (509) 335-6020. Fax: (509) 335-8328. E-mail: korourke{at}vetmed.wsu.edu.
Present address: 560 Quail Ridge, Pullman, WA 99164.
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References
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Journal of Clinical Microbiology, June 1998, p. 1750-1755, Vol. 36, No. 6
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