NR AWNO
AU Sethi,J.; Lohman,K.; Silveira,J.; Caughey,B.W.; Rudolph,A.; Orser,C.
TI Defining interactions between pronucleonTM conformational ligands and the optimal aggregation state of PrP(TSE) substrates for the misfolded protein diagnostic (MPD) assay
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-20
PT Konferenz-Poster
AB The Misfolded Protein Diagnostic (MPD) Assay utilizes conformationally sensitive PronucleonTM peptide ligands for both the capture of the PrPres substrate and for amplification and detection. One recent focus has been to define the optimal conformational and aggregate state of our ligand and the PrPres target since the most infective form of the target protein may be a smaller, more soluble aggregate rather than the mature fibrils formed in late stage disease in the brain. This is in line with our observations in both tissue and blood that sample preparation can greatly influence the outcome of the MPD Assay. We have also examined synthetic and recombinant PrPc and fibril PrP materials. To investigate this further, we have created known aggregate states of PrP targets and evaluated their response in the MPD assay. Our fluorescent measurements of ligand folding for synthetic and recombinant PrPres demonstrate less reactivity than biologically derived PrPres aggregates. However, we can use aggregates of smaller PrP peptide sequences (eg. PrP106-126) to seed the unique MPD amplification reaction. We have also used field flow field flow-fractionation (FIFFF) fractionated PrPres material from scrapie-infected hamster brain and normal hamster brain equivalent aliquots and evaluated these materials in the MPD Assay. Fractions corresponding to the most infectious prion protein particles (fractions 10-12), having the highest specific infectivity, gave distinctly positive MPD assay results. We also observed reactivity with additional fractionated PrPres material (fractions 3, 16, and 23 and 29). The periodicity of positively-reacting fractions from the PrPres brain, corresponding from <30 kD up to 1,000 kD, suggests a unique hierarchical structural assembly of PrPres oligomeric units acting as unique substrates for the MPD specific ligand, while the normal brain equivalents generated no detectable response in the MPD Assay. Further investigations of FIFFF fractions are focused on the expanded utility of the technique to generate positive control PrPres material for the MPD Assay as well as for potential identification of inhibiting component fractions in human plasma.
AD J. Sethi, K. Lohman, A. Rudolph, C. Orser: Adlyfe Inc., 9430 Key West Ave., Suite 210, Rockville, MD 20850, USA; J. Silveira, B. Caughey: 2Rocky Mountain Laboratories, NIAID, Hamilton, MT 59840, USA
SP englisch
PO Italien