A COMPETITION ASSAY TO DETECT SCRAPIE PRION PROTEIN BY CAPILLARY ELECTROPHORESIS

Transmissible spongiform encephalopathies of animals and humans are in fectious 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 i s modified by a post translational modification which truncates a host cellular protein at the N-terminus causing a conformational change in the protein. After modification, this protein becomes resistant to pr oteases and aggregates into rod-shaped fibrils in the brains of infect ed animals. When these aggregates are subjected to SDS-PAGE in the pre sence of 2-beta-mercaptoethanol, a monomeric form (prion protein) is o bserved with a molecular mass of ca. 27 kdaltons. Capillary electropho resis was used to detect immunocomplex formation of the prion protein with an antiserum produced to a peptide of the prion protein. The synt hesized peptide was labeled with fluorescein iodoacetamide at the free sulfhydryl group of cysteine that was added to N-terminus of the pept ide. When the fluorescein labeled peptide was incubated with increasin g concentrations of the rabbit antibody, a new peak that was proportio nal to the amount of antiserum in the reaction mixture with a concurre nt reduction in the labeled peptide peaks was observed. Incubation ove rnight at 4 degrees C enhanced immunocomplex formation. Competition of unlabeled peptide and of brain samples prepared from sheep for the fl uorescein labeled peptide was carried out using a concentration of rab bit antibody that produced ca. 50% of the maximum amount of immunocomp lex formation. Unlabeled peptide and brain samples prepared from scrap ie infected sheep brain but not from normal sheep reduced immunocomple x formation. This reduction was dependent on the concentration of the peptide and the amount of scrapie infected brain sample. By using comp etition for labeled peptide instead of using direct binding of the ant iserum to the prion protein as in a previous study, we increased the s ensitivity of detection of the scrapie prion protein. (C) 1995 John Wi ley & Sons, Inc.