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The Seprion Separation System Development of a feasible blood screening protocol for abnormal prion protein Stuart Wilson Microsens Biotechnologies
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Immunohistochemistry Spongiform changes in cerebral cortex with plaques (arrow) Histopathology “Halo” vacuolation surrounding plaques Variant CJD
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Amyloid fibrils (aggregates of thousands of molecules) Large, insoluble. Visible by staining. Found deposited in tissues eg. brain and spleen. Readily available and used extensively for spiking studies. Amyloid oligomers (>2 molecules) Small, soluble. Most likely form in blood. Invisible by staining. From oligomers to fibrils
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Post-mortem tests and the use of protease The large aggregates of rogue prion protein in the brain are relatively resistant to protease allowing the normal prion protein (PrPc) to be digested away prior to testing. But: Problems with standardisation – lab to lab; sample to sample; tissue to tissue leading to false positives Problems with automation No guarantee that all rogue prion is protease resistant –Even post-mortem - atypical scrapie (nor98) and BSE –Ante-mortem - is rogue prion in blood resistant to protease? –Most post-mortem tests cannot easily be applied to ante-mortem testing
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Introduction to the Seprion Separation System
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Technology background There is a wealth of scientific literature demonstrating that polyionic polymers can bind to rogue prion protein: histopathological stains, curing infected cell lines, delaying or preventing disease http://www.priondata.org/ We have developed the use of polyionic polymers (Seprion) to specifically capture rogue prion protein and other amyloid type diseases and thus avoid the need for proteinase K
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EM analysis of Seprion-captured amyloid
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Western analysis of Seprion-captured material from BSE-infected and uninfected brain no protease used
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Western analysis of Seprion-captured material – effect of Proteinase K UninfectedInfected NPKAB NPK: Seprion captured material without proteinase K treatment A: Proteinase K treatment of captured material B: Proteinase K treatment of material prior to capture
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The Seprion Separation System works for all TSEs Results from 0.25 mg of each sample Amyloid in other diseases can be detected in liver, kidney and muscle
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Control brainsAlzheimer’s Disease brains brains Signal Seprion is a universal ligand for Protein Aggregation Diseases of the amyloid type
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vCJDnormalAlz 1Alz 2Alz 3 signal Method 0.2 mg of vCJD, normal or Alzheimer’s Disease brains were tested. After capture using Seprion coated beads the captured protein was eluted and analysed by anti-prion ELISA. Results Discussion A strong signal was obtained from the vCJD brain but, as expected, the anti-prion ELISA did not cross react with material captured from the Alzheimer’s Disease brain Investigation of the lack of cross-reaction of the anti-prion ELISA with Seprion-captured Alzheimer’s Disease brain
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Method 0.2 mg of vCJD brain was tested alone or mixed with 0.2 mg of Alzheimer’s Disease brain. After capture using Seprion coated beads the captured protein was eluted and analysed by anti-prion ELISA. Results Discussion A strong signal was obtained by the Seprion prion assay from the vCJD brain alone and when mixed prior to capture with Alzheimer’s Disease brain i.e. no interference was observed. Investigation of any potential interference in the Seprion prion assay by a CJD/Alzheimer’s Disease mixed infection vCJD + Alz 1 vCJD + Alz 2 vCJD + Alz 3
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The Idexx post-mortem assay
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TSE post-mortem commercial summary The Idexx BSE and CWD commercial assay has 100% specificity and 100% sensitivity compared to existing EU approved tests on brain and lymph nodes USDA approval for BSE and CWD EU approval for BSE and scrapie 100% sensitivity and specificity compared to IHC on ante-mortem scrapie RAMALT testing
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The Seprion Separation System Seprion capture using coated magnetic beads (30 min with shaking) Washing of beads by magnetic capture (10 min) (fits into standard automated magnetic bead handlers) Elution and denaturation of captured prion (5 min at 95 o C) (acid, alkali, salt elution alternatives) ELISA detection (2h 30 min – 3h 30 min) (standard automation) Total time 3h 15 min – 4h 15 min for each sample run – numbers determined by automated platform chosen
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The Seprion Separation System can be used for the investigation of therapeutics Results from 10 5 cells
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The Seprion Separation System applied to blood screening for TSEs
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Keeping blood safe The size of the problem is still unknown Blood related transmission has occurred Reduce risk by exclusion –Donor exclusion. Incomplete. –Leucodepletion. Animal models demonstrate that 55% of infectivity remains (Rohwer, 2004) –Filtration. Pall Corp, PRDT. Animal organ spiking models. Endogenous infection may be species specific. Prion in plasma may be complexed with other proteins/lipid rafts. Quality assurance? Epidemiology? Abrogate risk through blood testing
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Protocol for 225 microliters plasma Seprion capture using coated magnetic beads (30 min with shaking) Washing of beads by magnetic capture (10 min) (fits into standard automated magnetic bead handlers) Elution and denaturation of captured prion (5 min at 95 o C) (acid, alkali, salt elution alternatives) ELISA detection (2h 30 min – 3h 30 min) (standard automation) Total time 3h 15 min – 4h 15 min for each sample run – numbers determined by automated platform chosen
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NIBSC blind panel of vCJD spleen spiked into plasma Cut-off 10 mg/ml 1 mg/ml Day Signal The results returned to NIBSC Breaking the code Conclusion No false positives Assay sensitivity was 1-10mg vCJD spleen per ml plasma This equates to 10 2 – 10 3 IU/ml plasma
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Seprion assay results for 236 human plasma donations Signal 0- 0.10.1- 0.140.14- 0.20 cut-off One initial positive that did not retest positive
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Detection of PrPSc in sheep – symptomatic and asymptomatic
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Time from Seprion assay positive to clinical signs for four scrapie infected sheep
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Results of the Seprion plasma assay on a blind panel of sheep samples provided by the VLA *Sample from a suspect from the same farm as three previously confirmed positives ie. from a farm with endemic scrapie
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Scrapie time course – study months 1 and 2 4 3 3 2 4 4 1 Study month 1 Study month 2 4 Months prior to death Signal
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Summary The Seprion Separation System has been extensively validated on TSE post-mortem brain samples and the plate based system has been approved by the USDA and EU. A more flexible bead-based approach has been developed for use in therapeutic screening, for other tissues and for other protein aggregation diseases such as Alzheimer’s Disease. This same protocol was used to investigate scrapie in sheep plasma. Abnormal prion could be detected in symptomatic and asymptomatic pre-clinical animals. Scrapie blind panel studies have been completed and a time course study is underway. We are poised to receive the human plasma panel
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