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Use of capillary
electrophoresis and fluorescent labeled peptides to
detect the abnormal prion protein in the blood of
animals that are infected with a transmissible
spongiform encephalopathy.
Schmerr MJ,
Jenny AL,
Bulgin MS,
Miller JM,
Hamir AN,
Cutlip RC,
Goodwin KR.
National Animal Disease Center, MWA, ARS, US Department
of Agriculture, Ames, IA 50010, USA.
mschmerr@nadc.ars.usda.gov
Transmissible spongiform encephalopathies in humans and
in animals are fatal neuro-degenerative diseases with
long incubation times. The putative cause of these
diseases is a normal host protein, the prion protein,
that becomes altered. This abnormal prion protein is
found mostly in the brains of infected individuals in
later stages of the disease, but also can be found in
lymphoid and other tissues in lower amounts. In order to
eradicate this disease in animals, it is important to
develop a system that can concentrate the abnormal prion
protein and an assay that is very sensitive. The
sensitivity that can be achieved with capillary
electrophoresis makes it possible to detect the abnormal
protein in blood. A peptide from the carboxyl terminal
region, amino acid positions 218-232, was labeled with
fluorescein during the synthesis of the peptide at the
amino terminus. Antibodies that have been produced to
this peptide were affinity purified and used in a
capillary electrophoresis immunoassay. The amount of
fluorescein labeled peptide in the capillary was 50
amol. Blood was obtained from normal sheep and elk, from
sheep infected with scrapie and elk infected with
chronic wasting disease. Buffy coats and plasma were
prepared by a conventional method. After treatment with
proteinase K, which destroys the normal protein but not
the altered one, the blood fractions were extracted and
tested in the capillary electrophoresis immunoassay for
the abnormal prion protein. The abnormal prion protein
was detected in fractions from blood from infected
animals but not from normal animals. This assay makes a
pre-clinical assay possible for these diseases and could
be adapted to test for the abnormal prion protein in
process materials that are used for manufacture of
pharmaceuticals and products for human consumption.
PMID: 10486728 [PubMed - indexed for MEDLINE]
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