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TUESDAY, FEBRUARY 13
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Detection and Removal
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| 8:30 Morning Coffee
9:00 Comments by Session Chairperson
Larisa Cervenakova, M.D., Ph.D., Senior Scientist, Transmissible Diseases Department, J.H. Holland Laboratory for the Biomedical Sciences, American Red Cross
9:10 Prion Strain Identification by the Scrapie Cell Assay using Cell Panels
Sukhvir Mahal, Ph.D., Scientist, Infectology, Scripps
Research Institute
The scrapie cell assay (SCA; Kloehn et al., PNAS 100:11666-71, 2003) is a rapid, cell-based method for the quantification of mouse prions. With newly developed cell lines the assay can be completed within 10 days and can quantify RML brain homogenate to a 10-7 dilution, with a standard error of ± 20-30%. Moreover, it is possible to discriminate between different prion strains using a panel of appropriate cell lines.
9:40 Detection of PrPsc in Sheep Plasma using SRA
Seong An, Ph.D., Research Fellow, Research and Development, PeopleBio Inc.
We have developed a system called Multimer Detection System (MDS) for the detection of the PrPSc in blood, as previously presented on hamster plasma samples. We improved our MDS system to the Simultaneous Reaction Assay (SRA) using strepavidin conjugated magnetic beads to concentrate the sample. The SRA worked by adding the plasma sample, biotinylated entrapping antibody and HRP conjugated detection antibody simultaneously. SRA reduced the assay time by eliminating several incubation steps. We were able to differentiate the signal from scrapie from normal sheep blood samples. Hence, SRA is a very simple and robust method for detecting PrPSc in plasma.
10:10 Update on the 15B3 CJD Blood Test
Alex Raeber, Ph.D., Director of Research, Prionics AG
The detection of PrPSc in blood of TSE infected animals and humans have been hampered by the low levels and lack of protease resistance of PrPSc in blood. We have developed proprietary technologies based on the ELISA and FACS platform for the detection of disease associated prion proteins in plasma and serum using the conformation specific antibody 15B3 which recognizes both protease resistant and protease sensitive forms of abnormal, disease associated PrP. Analytical sensitivity studies using vCJD and scrapie brain homogenates suggest that the limit of detection is in the range of 10 - 100 infectious units per millilitre which is consistent with the levels of prion infectivity found in blood of infected animals and humans. Results of the analysis of serum and plasma panels from clinical and preclinical sheep infected with scrapie as well as screening of normal human plasma samples with the prototype blood test will be presented and the application of this test for use in the blood transfusion service and in the plasma fractionation process will be discussed.
10:40 Coffee Break, Poster and Exhibit Viewing in the
Exhibit Hall
11:10 Detection and Removal of Residual Contamination on Surgical Instruments
Helen Baxter, Ph.D., Senior University Fellow, School of Chemistry, University of Edinburgh
Residual contamination on reprocessed surgical and dental instruments is a significant problem in contamination control in the health service. Of particular concern is the risk of iatrogenic transmission of CJD since it is well documented that TSE infectivity adhering to metal surfaces can survive conventional cleaning and sterilization procedures. As there is now clear evidence of cases of vCJD arising from blood transfusion, the potential risk from undiagnosed CJD carriers in the population make it imperative that effective new procedures for cleaning 'high risk' instruments be developed and put into practice. The results of an interdisciplinary study on new methods of contaminant detection and decontamination for surgical instruments will be described. Specifically: (a) the effectiveness of RF gas-plasma methods in removing TSE infectivity from stainless steel using an animal model; (b) application of RF gas-plasma decontamination for removal of residual contaminants from surgical instruments and comparison of these procedures to current methods; (c) development of low background covalent fluorescent probes for derivatization of proteins on surfaces; (d) development and application of rapid surface fluorescent scanning techniques for the detection and quantification of residual protein contamination on surgical instruments at the sub femptomole level. |
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Conformational Transition and Infectivity
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11:40 Characterization of PrP-Derived Peptides Capable of Discriminating between
and PrPSc in Plasma
Anthony L. Lau, Ph.D., Post-Doctoral Fellow, Novartis Vaccines and Diagnostics, Inc.
Development of reagents that specifically bind PrPSc has been a long sought after milestone in the study of prion biology. Prion sequences grafted into an antibody scaffold have previously been shown by Morocini et al. to interact with PrPSC. Upon discovering that peptides were capable of capturing PrPSC without being conformationally constrained within scaffold, we hypothesized that other segments of the PrP peptide sequence may also bind preferentially to PrPSC. After screening the biotinylated PrP-derived peptides, we found two that were capable of binding full-length PrPSC in plasma, which contains a vast excess of PrPC and other proteins. Using paramagnetic beads coated with active peptide to selectively capture PrPSc followed by a sandwich ELISA for detection, a prototype assay was developed and used to study the mechanism of interaction between the active peptides and PrPSc. Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the most active peptides and PrPSC. Binding occurs through a structural domain that is only present in the abnormal conformer, is species independent, and is not affected by proteinase K digestion. PrPC levels in plasma and the amount of PrPSC captured from brain homogenate of four different species were quantitated. The limit of detection was calculated to be ~10 attomoles of PrPSc (based on the mass of PrP27-30) from a ~106-fold dilution of human vCJD brain homogenate. We believe the sensitivity of this assay and the elimination of the need for protease digestion will provide prion researchers a powerful new tool to study new areas of prion biology. Co-Authors: Alice Yam, Melissa Michelitsch, Xuemei Wang, Carol M. Gao, Robert Goodson, Robert Shimizu, Gulliver Timoteo, John Hall, Angelica Medina-Selby, Doris Coit, Colin McCoin, Celine Hu, Bruce Phelps, Ping Wu, David Chien, and David Peretz
12:10 Abnormal Prion Protein Conformers in Normal Human Brain
Wen-Quan Zou, M.D., Ph.D., Assistant Professor, Pathology, Case Western Reserve University
Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPc) through an á-helix to
b-sheet transition. Here we demonstrate, using sedimentation, gel filtration and enrichment of abnormal PrP by gene 5 protein, that small amounts of detergent-insoluble PrP aggregates and PK-resistant PrP species are present in normal human brains. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.
12:40 Controlling Amyloid Growth in all Dimensions
David G. Lynn, Ph.D., Center for the Analysis of Supramolecular Self-assemblies, Center for Neurodegenerative Diseases, Departments of Chemistry and Biology, Emory University
The great progress made in defining the structure of protein and peptide amyloid assemblies, particularly the arrangement of peptides in b-sheets, is counterbalanced by the still poor understanding of the higher organization of b-sheets within the fibril and overall fibril/fibril associations. The assembly pathway and basis of amyloid toxicity may well depend on these higher-order structural features. For example, significant evidence points to association between sheets as the rate limiting step in fibril assembly, and a critical metal binding site has now been identified that involves residues from different individual sheets. Here we review experiments that are identifying some of the issues associated with sheet-sheet association by investigating simple model peptides derived from the central core of the Ab peptide implicated in Alzheimer’s Disease. These peptides transition between fibril/ribbon/nanotube morphologies in response to assembly conditions, laying the foundation for understanding the folding landscape for these higher order assemblies, revealing potential targets for therapeutic intervention, and opening strategies for the design of highly ordered peptide self-assembled microscale morphologies. Co-Authors: Jijun Dong, Kun Lu, Anil K. Mehta, Yan Liang, W. Seth Childers, and James A. Simmons
1:10 Close of TSE Conference
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