Mary Jo Schmerr

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.

Ovine progressive pneumonia (Maedi-Visna) in sheep

Ovine progressive pneumonia (OPP) is a multi-systemic disease of sheep caused by a nononcogenic exogenous retrovirus belonging to the Lentiviridae subfamily. Characteristics of the disease are chronic lymphocytic pneumonitis, encephalitis, arthritis, mastitis and vasculitis associated with progressive wasting, dyspnea, lameness, indurated udder and, rarely, paralysis. Any one or all of the characteristics may be manifest. Transmission of the virus is predominantly through the colostrum to newborn lambs, however, transmission can occur by contact and in utero. Treatment of the disease is only symptomatic and prevention of infection is only by avoiding the virus.

Capillary isoelectric focusing of the scrapie prion protein

Prion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases. Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing. The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.

Failure of experimental vaccines to protect against infection with ovine progressive pneumonia (maedi-visna) virus

Cell culture medium was harvested from cells infected with ovine progressive pneumonia (OPP) virus and used to prepare killed virus vaccines. Virus was inactivated by either heat, formalin, or ethyleneimine and used either without adjuvant, with Freund incomplete adjuvant, or with aluminum hydroxide adjuvant to vaccinate sheep. The sheep produced precipitating antibody against the virus but were not protected against infection when challenged with live OPP virus.

Improvements in a competition assay to detect scrapie prion protein by capillary electrophoresis

Scrapie in sheep and goats is the prototype of transmissible spongiform encephalopathies found in humans and animals. A feature of these diseases is the accumulation of rod-shaped fibrils in the brain that form from an aggregated protein. This protein is a protease-resistant form of a normal host cell protein. When the aggregated protein is denatured in SDS and beta-mercaptoethanol, a monomer form (prion protein) with a molecular mass of 27 kDa is observed. Free zone capillary electrophoresis and peptides labeled with fluorescein were used to detect the prion protein through competition for a labeled peptide in immune complex formation. The separation of the immune complexes from the unbound peptide using 200 mM Tricine (pH 8.0) was faster and was better resolved than that obtained with phosphate or borate buffer systems. The amount of immune complex formation was dependent on the amount of antibody in the assay. The amount of bound labeled peptide and unbound labeled peptide could be measured directly by calculating the area of each respective peak. As increasing amounts of unlabeled peptide were added to the assay, a concentration dependent reduction in the immune complex peak was observed. The assay could detect less than 10.0 fmol of unlabeled peptide. There was a quantitative difference in the competition of preparations from scrapie infected sheep brain and normal sheep brain.

Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep

Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pellet was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCap SDS14-200 Beckman capillary). In infected sheep brain samples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on Western blot (22.4 kDa), a Ferguson plot was made to determine if there were abberations in the molecular mass determination. After correction, the major peak was estimated to be 19.2 kDa. This has a better correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was approximately 50 micrograms. For Western blot, the amount of brain sample was approximately 20 mg. For this assay, this is approximately 100 times less than that needed for Western blot for sheep samples.

Diagnosis of Preclinical and Subclinical Scrapie in a Naturally Infected Sheep Flock Utilizing Currently Available Postmortem Diagnostic Techniques

Scrapie is a naturally occurring transmissible encephalopathy of sheep and goats. Currently available methods for diagnosis are the presence of characteristic histopathologic changes and detection of an abnormal form of prion protein (PrPres) in the brains of affected animals. This study documents preclinical and subclinical scrapie in a flock of 16 sheep utilizing histopathology, immunohistochemistry (IHC), western blot, and electron microscopy (for scrapie-associated fibrils) for confirmation of the disease. Prior to necropsy, none of the sheep showed signs of clinical scrapie. Based on the results of histopathology and positive PrPres tests, 3 ewes were found to have subclinical scrapie. An additional ewe, which did not have histopathologic changes in the brain but was positive by IHC and western blot, was considered a preclinical case of scrapie. None of the sheep had amyloid in the brain stem.

Capillary electrophoresis of the scrapie prion protein from sheep brain

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrPsc) that has a molecular mass of ca. 27,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrPsc. The overall concentration of PrPsc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrPsc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrPsc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrPsc. After treatment with proteinase K and non-ionic detergents, PrPsc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of sheep and cattle immunoglobulins with antibody activity by high-performance liquid chromatography

High-performance anion-exchange chromatography (HPIEC) and high-performance size-exclusion chromatography (HPSEC) were used to purify serum IgM and IgG from sheep and cattle. Pooled serum from normal cattle and sheep and serum from sheep, infected with two different viruses, were prepared for HPIEC by chromatography on CM-Affi-Gel Blue. After HPIEC, fractions containing IgG and IgM were pooled and concentrated and further purified by HPSEC. The purity of fractions from HPIEC and HPSEC were evaluated by immunoelectrophoresis, protein-A-Sepharose affinity chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Monoclonal antibody specific for bovine IgG2 was used to assay for IgG2 and IgG2 contamination of other fractions. The antibody activity to ovine adenovirus 5 and to ovine progressive pneumonia virus was assayed by neutralization and immunodiffusion. Antibody activity was retained against both viruses in the fractions containing IgG1 and IgM. This high-performance liquid chromatography procedure was a rapid preparative method to produce specific immunoglobulins and could be used to evaluate the purity of immuno-reagents.

Conditions for binding bovine IgG1 to protein A-sepharose

Conditions were established so that both subclasses of bovine IgG were bound to Protein A-Sepharose. Increasing the pH of the starting buffer to pH 8.0 from pH 7.0 and increasing the starting phosphate concentration of the buffer to 0.5 M from 0.2 M enhanced the separation. Using these modifications in the buffer system, IgG1 was eluted from pH 7.0 to 7.8 and IgG2 at pH 5.0. Two major peaks were associated with IgG1 activity indicating heterogeneity of binding to protein A-Sepharose. One peak was found for IgG2. The molecular weights of the fractions were determined to be that of IgG by sodium dodecyl sulfate polyacrylamide gel electrophoresis.