Final Report for CRADA Agreement , AL-C-2006-01 with Microsens Biotechnologies: Detection of the Abnormal Prion Protein in Blood by Improving the Extraction of this Protein

Abstract

Several conditions were examined to optimize the extraction protocol using Seprion beads for the abnormal prion protein. Different combinations of water, hexafluro-2-propanol and formic acid were used. The results of these extraction protocols showed that the magnetic beads coated with Seprion reagents were subject to degradation, themselves, when the extraction conditions that would solubilize the abnormal prion protein were used. These compounds caused interference in the immunoassay for the abnormal prion protein and rendered these protocols incompatible with the assay systems. In an attempt to overcome this problem, another approach was then used. The coated beads were used as an integral part of the assay platform. After washing away denaturing agents, the beads with the 'captured' abnormal prion were incubated directly in the immunoassay, followed by analysis by the capillary electrophoresis. When a capillary electrophoresis electro-kinetic separation was attempted, the beads disturbed the analysis making it impossible to interpret. A pressure separation method was then developed for capillary electrophoresis analysis. When 20 samples, 5 of which were positive were analyzed, the assay identified 4 of the 5 positives and had no false positives. When a larger number of samples were analyzed the results were not as good - there were more » false positives and false negatives. It was then observed that the amount of beads that were loaded was dependent upon how long the beads were allowed to settle before loading them into the capillary. This resulted in unacceptable variations in the results and explained that when large numbers of samples were evaluated the results were not consistent. Because the technical difficulties with using the Seprion beads could not be overcome at this time, another approach is underway that is outside of the scope of this CRADA. No further agreements have been developed. Because the results were not favorable, no manuscripts were written nor intellectual property developed. « less