Molecular profiling of ovine prion diseases by using thermolysin-resistant PrPSc and endogenous C2 PrP fragments.

Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrPres signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrPres species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrPres in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrPres and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrPres. This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

Decreased cell surface prion protein in mouse models of prion disease.

Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by prions, composed of ordered aggregates of misfolded cellular prion protein. Neural antigen density of prion protein, Thy-1 and glial fibrillary acidic protein was analyzed using flow cytometry of dissociated mouse brain cells after inoculation with mouse-adapted transmissible spongiform encephalopathy agents. Transmissible spongiform encephalopathy gliosis was demonstrated by increased intracellular immunoreactivity for glial fibrillary acidic protein compared with controls. Immunoreactivity for cell surface prion protein was reduced 2.8-3.8-fold compared with control brain cells, whereas surface Thy-1 protein was reduced 1.5-4-fold. Double-staining protocols revealed loss of brain cells highly immunoreactive for prion protein and Thy-1, with a preferential reduction of prion protein, suggesting that prion protein expression, trafficking or consumption may be affected early in disease.

No abnormal prion protein detected in the milk of cattle infected with the bovine spongiform encephalopathy agent.

Milk specimens were collected from lactating cows that had previously been challenged with bovine spongiform encephalopathy (BSE)-infected brain at 4-6 months of age. One group of 10 animals received a single oral dose of 100 g, a second group received 1 g and the third was made up of unexposed controls. The cows were inseminated artificially, and calved at approximately 2 years of age and annually thereafter. Milking was done within the first week following calving and at 10-weekly intervals during the lactation period. Specimens were centrifuged to obtain a fraction enriched for somatic cells and these fractions were analysed for disease-associated, abnormal prion protein (PrP(BSE)) by using a modified commercial BSE ELISA and a different confirmatory assay. No abnormal prion protein has so far been identified in the cell fraction of milk from cattle incubating BSE by using these methods at their limits of detection.

Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis.

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.

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion.

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.

The use of non-prion biomarkers for the diagnosis of Transmissible Spongiform Encephalopathies in the live animal.

Scrapie and bovine spongiform encephalopathy (BSE) are major global concerns and the emergence of variant Creutzfeldt-Jakob disease (vCJD) has caused turmoil for blood transfusion services and hospitals worldwide. Recent reports of iatrogenic CJD (iCJD) cases following blood transfusions from Transmissible Spongiform Encephalopathies (TSE)-infected donors have fuelled this concern. Major diagnostic tests for BSE and scrapie are conducted post-mortem from animals in late stages of the disease. Although the lymphoreticular system is involved in the earlier pathogenesis of some forms of sheep scrapie and vCJD, which presents great opportunity for diagnostic development, other TSE diseases (some strains of scrapie, sporadic CJD (sCJD) and bovine BSE) do not present such a diagnostic opportunity. Thus, there is an urgent need for pre-mortem tests that differentiate between healthy and diseased individuals at early stages of illness, in accessible samples such as blood and urine using less invasive procedures. This review reports on the current state of progress in the development and use of prion and non-prion biomarkers in the diagnosis of TSE diseases. Some of these efforts have concentrated on improving the sensitivity of PrPSc detection to allow in vivo diagnosis at low abundances of PrPSc whilst others have sought to identify non-prion protein biomarkers of TSE disease, many of which are still at early stages of development. In this review we comment upon the limitations of prion based tests and review current research on the development of tests for TSE that rely on non-prion disease markers in body fluids that may allow preclinical disease diagnosis.

Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe.

A novel CE-based noncompetitive immunoassay for prion protein (PrP) was established. Fluorescein isothiocyanate (FITC)-labeled protein A (FITC-PrA) was used as a fluorescent probe to tag monoclonal antibody through noncovalent binding of FITC-PrA to the Fc region of the antibody. The FITC-PrA-Ab was incubated with the analyte, prion protein, under optimized condition, forming the immunocomplex FITC-PrA-Ab-PrP. The complex was separated and analyzed by capillary zone electrophoresis. The addition of carboxymethyl-beta-cyclodextrin in the running buffer as dynamical coating reagent improved the reproducibility and the resolution. The complex was isolated in less than 1 min with theoretical plates of 3.8 x 10(4). Relative standard deviations of peak height and migration time for the complex were 3.46 and 1.48%, respectively. A linear relationship was established for the bovine recombinant prion protein (rPrP) concentration in the range from 0.2 to 2.0 mug/mL and the peak height. The correlation factor was r2 = 0.9969. The estimated detection limit for rPrP was approximately 6 ng/mL, which is 3 times the signal-to-noise ratio. The method was successfully applied for testing blood samples from scrapie-infected sheep.

Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence.

A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.