title: Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis.
abstract: An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.
authors.affiliation: Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
authors.forename: Roy
authors.initials: R
authors.lastname: Jackman
authors.forename: David J
authors.initials: DJ
authors.lastname: Everest
authors.forename: Mary Jo
authors.initials: MJ
authors.lastname: Schmerr
authors.forename: Mohammed
authors.initials: M
authors.lastname: Khawaja
authors.forename: Pat
authors.initials: P
authors.lastname: Keep
authors.forename: John
authors.initials: J
authors.lastname: Docherty
mesh_terms: D000818:Animals; D001665:Binding Sites; D019075:Electrophoresis, Capillary / Q000379:methods*; D005069:Evaluation Studies as Topic; D005838:Genotype; D007118:Immunoassay / Q000379:methods; D010455:Peptides / Q000737:chemistry; D017096:Prion Diseases; D011328:Prions / Q000097:blood; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012608:Scrapie / Q000097:blood* / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D012756:Sheep; D034561:Sheep, Domestic
chemical_list: D010455:Peptides; D011328:Prions