Abstract
Transmissible spongiform encephalopathies of animals and humans are infectious diseases that cause progressive degenerative disorders of the central nervous system. These diseases are caused by an accumulation in the lysosymes of a modified normal cellular protein. This protein is modified by a post translational modification which truncates a host cellular protein at the Nโterminus causing a conformational change in the protein. After modification, this protein becomes resistant to proteases and aggregates into rodโshaped fibrils in the brains of infected animals. When these aggregates are subjected to SDSโPAGE in the presence of 2โฮฒโmercaptoethanol, a monomeric form (prion protein) is observed with a molecular mass of ca. 27 kdaltons. Capillary electrophoresis was used to detect immunocomplex formation of the prion protein with an antiserum produced to a peptide of the prion protein. The synthesized peptide was labeled with fluorescein iodoacetamide at the free sulfhydryl group of cysteine that was added to Nโterminus of the peptide. When the fluorescein labeled peptide was incubated with increasing concentrations of the rabbit antibody, a new peak that was proportional to the amount of antiserum in the reaction mixture with a concurrent reduction in the labeled peptide peaks was observed. Incubation overnight at 4ยฐC enhanced immunocomplex formation. Competition of unlabeled peptide and of brain samples prepared from sheep for the fluorescein labeled peptide was carried out using a concentration of rabbit antibody that produced ca. 50% of the maximum amount of immunocomplex formation. Unlabeled peptide and brain samples prepared from scrapie infected sheep brain but not from normal sheep reduced immunocomplex formation. This reduction was dependent on the concentration of the peptide and the amount of scrapie infected brain sample. By using competition for labeled peptide instead of using direct binding of the antiserum to the prion protein as in a previous study, we increased the sensitivity of detection of the scrapie prion protein. ยฉ 1995 John Wiley & Sons, Inc.