Evaluation of a Preclinical Blood Test for Scrapie
in Sheep Using Immunocapillary Electrophoresis
Author(s): Roy Jackman1, | David J. Everest2, | Mary Jo Schmerr3, | Mohammed
Khawaja4, | Pat Keep5, | John Docherty6
Journal of AOAC INTERNATIONAL
Print ISSN: 1060-3271
Volume: 89 | Issue: 3
Cover date: May/June 2006
Page(s): 720-727
Abstract text
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.
Author(s) affiliations
1Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom.
2Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom.
3Ames Laboratory, U.S. Department of Energy, Iowa State University, Ames, IA
50011.
mschmerr@ameslab.gov
4Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom.
5Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom.
6Immunochemistry Group, Veterinary Laboratories Agency, New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom.
http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.89.3.720