Fluorescent Immunoassay Development for PrPSc Detection and Antemortem Diagnosis of TSEs

摘要

The overall goal of our study is to develop methods of high- sensitivity and high-specificity for the antemortem diagnosis of prion diseases by detecting PrPSc in biological fluids using fluorescent immunoassay. During the second year of this contract, we have focused on the development of an immunological method to isolate blood PrP from normal and scrapie-infected animals and the assembly of an optical fiber-based fluorescence detection system. By using magnetic beds-based immunocapture protocols, plasma PrP from normal and scrapie-infected animals was successfully isolated and can be detected directly by Western blot. In terms of the resistance of PrP to proteinase K (PK) digestion, there was no marked difference in plasma PrPs between normal and infected animals although more extensive conditions should be tested. For instrumentation, the design and assembly of an optical fiber- based fluorescence detection system for use with immunoassays for PrP is completed. The key features of the detection system are maximum light collection efficiency through the use of 4 steradian collection for the detectors, sampling of the entire sample volume through axial excitation with one or more lasers, and small sample volume, appropriate for assay work on TSEs. In addition to this, the system is designed for ease of use by employing disposable micro-capillaries as a sample holder.