Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected witha transmissible spongiform encephalopathy

Transmissible spongiform encephalopathies in humans and in animals are fata l neuro-degenerative diseases with long incubation times. The putative caus e of these diseases is a normal host protein, the prion protein, that becom es altered. This abnormal prion protein is found mostly in the brains of in fected 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 di sease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensiti vity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl termin al region, amino acid positions 218-232, was labeled with fluorescein durin g the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillar y electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, f rom sheep infected with scrapie and elk infected with chronic wasting disea se. Puffy coats and plasma were prepared by a conventional method. After tr eatment with proteinase K, which destroys the normal protein but not the al tered one, the blood fractions were extracted and tested in the capillary e lectrophoresis immunoassay for the abnormal prion protein. The abnormal pri on protein was detected in fractions from blood from infected animals but n ot 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 p roducts for human consumption. (C) 1999 Elsevier Science BN. All rights res erved.