Detection of the Prion Protein in a Liquid Phase Capture Assay Using Magnetic Beads Coupled to Protein A

Abstract

Detection of the abnormal prion protein in blood and cerebral spinal fluid of transmissible spongiform encephalophathy (TSE) infected individuals has not been possible by Western blot, immunohistochemistry or the present ELISA tests. We used an analytical approach in conjugation with a fluorescence immunoassay to develop methods to measure the abnormal prion in blood and cerebral spinal fluid. Monoclonal antibodies (mabs) and the corresponding fluorescein-labeled peptides and magnetic beads coupled to Protein A (PA) were used. The mabs, 12F10 and 3B5 reacted with the prion protein or the fluorescein-labeled peptides very rapidly in a liquid phase assay using a 0.15M TAPS buffer pH 8.8 containing 0.1% bovine serum albumin (BSA) and 0.1% N-Octyl-glucoside. The magnetic beads coupled to PA were mixed with the mab and recombinant prion protein (rPrP) or a sample and incubated for 30 min at 25 C. After washing the beads, the appropriate fluorescein labeled peptide was added and incubated for 15 min at 25 C. An aliquot of the supernatant fraction was removed and analyzed by capillary electrophoresis using laser-induced detection. Blood samples from sheep exposed to sheep scrapie were collected in 10 ml tubes containing 17.55 mg of EDTA. Buffy coats were prepared from the blood samples in the usual manner and were washed twice with 0.04 M Tris, pH 8.35 containing 0.135 M NH4Cl. The prepared buffy coats were frozen and thawed twice, incubated with 50 µg/ml of DNase and then incubated with Proteinase K (50µg/ml). The samples were then incubated with an equal volume of hexafluoro-2-propanol (HFIP) at 56 C for 5 min. The HFIP layer was dried in a vacuum centrifuge. The sample was re-suspended and used in temperature and pH. The optimal pH and temperature for the reaction of the antibody to the fluorescein labeled peptide was 8.8 and 25 C respectively. When a standard curve was made with the rPrP, 10 pg of rPrP could be easily detected. The blood samples correlated with the scrapie status of the sheep as was determined by postmortem examination of the brain by western blot. Further development of this assay and validation will make possible a rapid test for early detection of the TSEs.