Physical Characterization of a Highly Infectious Monodisperse Preparation of TSE Infectivity as a Substrate for Diagnostic Development
Annual rept. 1 Sep 2004-31 Aug 2005
BALTIMORE RESEARCH AND EDUCATION FOUNDATION INC MD
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Rational development of diagnostics and therapies for the transmissible spongiform encephalopathy TSE diseases requires a better understanding of the structure of the elemental unit of infectivity. Our laboratory is using a novel preparation of the TSE agent in a highly dispersed form to purify infectivity away from other molecular contaminants. Nuclease and protease enzyme treatments reduced the levels of some contaminants in the preparation without inactivating infectivity or causing aggregation. We have applied density gradient equilibrium ultracentrifugation to our preparation to separate particles based on the buoyant density of the infectivity. CsCl, a common density medium, proved unsuitable for our purposes because it caused aggregation of PrP res. Despite this loss of dispersion, two sharp peaks of PrP res and TSE infectivity were formed in the CsCl gradient. Subsequent studies with low ionic strength media have succeeded in concentrating PrP res and purifying it away from greater than 99 of non-PrP protein while maintaining its small average particle size. The purified dispersed material is being titered by bioassay and the process is being scaled up. The purified particles are being carried forward to additional purification steps including equilibrium ultracentrifugation and sedimentation velocity ultracentrifugation.
- Medicine and Medical Research