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.
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.
Schmerr
M J
MJ
National Animal Disease Center, MWA, ARS, US Department of Agriculture, Ames, IA 50010, USA. mschmerr@nadc.ars.usda.gov
Jenny
A L
AL
Bulgin
M S
MS
Miller
J M
JM
Hamir
A N
AN
Cutlip
R C
RC
Goodwin
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D017096:Prion Diseases / Q000097:blood*; D011328:Prions / Q000032:analysis / Q000097:blood*; D012756:Sheep; D013050:Spectrometry, Fluorescence
D011328:Prions