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.
Schmerr
M J
MJ
National Animal Disease Center, US Department of Agriculture, Ames, IA 50010, USA.
Goodwin
K R
KR
Cutlip
R C
RC
Jenny
A L
AL
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001667:Binding, Competitive; D001933:Brain Stem / Q000737:chemistry / Q000276:immunology / Q000503:physiopathology; D019075:Electrophoresis, Capillary / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018620:PrPSc Proteins / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D012608:Scrapie / Q000276:immunology; D012680:Sensitivity and Specificity; D012756:Sheep
D000936:Antigen-Antibody Complex; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D010455:Peptides; D018620:PrPSc Proteins; C015296:5-iodoacetamidofluorescein