1:20 Comments by Session Chairperson
Dr. Paul Brown
1:30 Detection of PrPsc in Blood using Newly Developed Multimer Detection System
Dr. Seong An, Research Fellow, Research & Development, PeopleBio Inc.
Transmissible spongiform encephalopathies(TSEs), also known as prion (PrP) diseases, can spread not only through the food supply, but through organ transplants, contaminated medical instruments, the blood supply, or pharmaceuticals made from animal products. Recently, two patients died of variant Creutzfeldt-Jacob disease (CJD) from probable blood transmission from an asymptomatic infected individual. We focused on the detection of the infectious form of prion (PrPsc) in blood. Thus, we have developed a new concept, called Multimer Detection System (MDS), using one epitope-specific antibody system to differentiate the infectious PrPsc (multimer) from cellular PrP (monomer). MDS detected the PrPsc in various infectious samples - brain homogenate, plasma spiked with PrPsc and plasma from a diseased hamster.
1:50 Towards Development of a Prion Blood Test
Dr. David Peretz, Senior Scientist, Blood Testing R&D, Chiron Corporation
We have developed proprietary prion related peptide reagents that bind the pathogenic prion protein isoform, PrPSc. When coated on magnetic beads, the reagents effectively capture and concentrate human and sheep PrPSc from brain homogenates spiked in negative plasma samples. Combined with an ultra-sensitive ELISA this assay allows PrPSc to be detected at a level of a million-fold dilution of human CJD and sheep scrapie brain homogenate. Circulating scrapie prion has been detected in some plasma samples from infected sheep. The assay does not require Proteinase K digestion or centrifugation steps, and is therefore easily automated. In conclusion, feasibility of a prion assay format has been demonstrated with the potential to achieve the sensitivity and throughput required for screening human blood and animal blood samples.
2:10 Highly Sensitive Assay Detection for PrP in Hamster Plasma
Dr. Luisa Gregori, Molecular Neurovirology Lab, BREF
The risk of iatrogenic transmission of TSE infections could be effectively managed and controlled with a sensitive and reliable blood-based diagnostic assay. The assay should be capable of concentrating, discriminating and detecting trace amounts of PrPres from blood of pre-clinical individuals. We developed an assay platform that could form the basis for a TSE diagnostic assay for human plasma. This PrP-based assay was developed using normal hamster plasma and it combines the specificity of an antibody capture method and the sensitivity of the ORIGEN analyzer system. We improved the assay sensitivity by exploiting key features of the ORIGEN analyzer technology. Using recombinant hamster PrP as the standard, we measured the concentration of PrP in normal hamster plasma and in infected and normal hamster brains. We also assessed the assay sensitivity with brain-derived PrPres mixed in PrP-depleted hamster plasma. The results of these studies are reported and discussed in the context of assay requirements for a blood-based TSE diagnostics.
2:30 Detection of Prion Protein in Blood Samples
Dr. Maureen Dyer, Biological and Chemical Sensor Engineer, Hanson Technologies, Inc.
Presence of the prion protein is currently determined post-mortem. A blood test would allow for early detection, possibly even before clinical symptoms develop. Using technology licensed from the government, we are developing a rapid, automated, fluorescence-based sandwich assay screen for prion protein in blood. Infectious prion can be discriminated from the normal form through use of conformation specific antibodies.
2:50 Afternoon Refreshment Break, Poster Exhibit Viewing
3:40 The Misfolded Protein Diagnostic (MPD)
Blood Assay: Current Status
Dr. Cindy Orser, Adlyfe, Inc. Rockville
Adlyfe has demonstrated the detection of misfolded prions in the blood of animals and humans using a novel blood based diagnostic called the Misfolded Protein Diagnostic (MPD) assay. Conformationally-dependent, labeled peptides that mimic the folding reaction of PrPC converting to PrPSC provide the basis for a blood-based diagnostic assay for PrPSC. Superior sensitivity is achieved due to signal amplification as additional labeled peptides are recruited in solution to undergo a similar conformation change. This has enabled the detection of PrPSC in blood of animals and humans infected with prion disease. The demonstration of the MPD Assay as a screening tool for the human blood supply is currently underway.
4:00 An Animal Model Evaluation and Assay Protocol for Screening Blood for vCJD
Dr. Stuart Wilson, Microsens Biotechnologies
Our Seprion ligand technology is specific for the abnormal form of the prion protein and does not bind the normal prion. The ligand has been built into an EU and USDA approved BSE test. We have developed the ligand further into a blood screening test and would like to present our latest protocol and latest data on evaluation of the test on the scrapie animal model.
4:20 Immunoassay for the Detection of Resistant Prion Protein (PrPres) in Human Plasma using an Original Combination of Chemical Ligands
Dr. Hervé Perron, Research Director, Senior Director Neurological Diseases Unit, R&D Immunoassay and Proteomics Department, bioMérieux
Detection of PrPres in human blood has been made possible by using the properties of two original ligands, Streptomycine and Claix-Arenes, with final specific antigenic detection using an anti-PrP monoclonal . Beyond the proof of principle of PrPres detection in human blood, detection in asymptomatic carriers and a test for excluding blood donations from such apparently healthy donors are the objectives of our R&D effort. Further studies and technical improvements are nonetheless required before a test prototype may be proposed for validation as a routine test for blood banking and/or human CJD diagnosis.
4:40 Rapid Blood Screening Test for CJD
Dr. Alex J. Raeber, Director of Research, Prionics AG
Two recent cases of vCJD in the UK caused by blood transfusion underscore the importance of implementing diagnostic blood tests for vCJD prions. We have developed rapid blood screening tests in two different assay formats, ELISA and fluorescence activated cell sorting (FACS) based on a capture and enrichment of PrPTSE from serum or plasma using the prion-specific antibody 15B3 and a subsequent detection step with a secondary
anti-PrP-specific antibody. Results from screening panels of TSE positive and negative sera will be presented and discussed.
5:00 Detection of Prions in Blood
Prof. Joaquin Castilla, Assistant Professor of Neurology, Protein Misfolding Disorders Lab, University of Texas Medical Branch, University of Texas, Galveston
Prion diseases are caused by an unconventional infectious agent termed prion, composed mainly of the misfolded prion protein (PrP(Sc)). The development of highly sensitive assays for biochemical detection of PrP(Sc) in blood is a top priority for minimizing the spread of the disease. Here we show that the protein misfolding cyclic amplification (PMCA) technology can be automated and optimized for high-efficiency amplification of PrP(Sc). We show that 140 PMCA cycles leads to a 6,600-fold increase in sensitivity over standard detection methods. Two successive rounds of PMCA cycles resulted in a 10 million-fold increase in sensitivity and a capability to detect as little as 8,000 equivalent molecules of PrP(Sc). Notably, serial PMCA enables detection of PrP(Sc) in blood samples of scrapie-afflicted hamsters with 89% sensitivity and 100% specificity. These findings represent the first time that PrP(Sc) has been detected biochemically in blood, offering promise for developing a noninvasive method for early diagnosis of prion diseases.
5:20 Question and Answer Period with all Session Speakers
5:45-6:45 Networking Reception in Exhibit Hall
