the seprion separation system development of a feasible blood screening protocol for abnormal prion...
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The Seprion Separation System
Development of a feasible blood screening protocol for abnormal prion protein
Stuart Wilson
Microsens Biotechnologies
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
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
Introduction to the Seprion Separation System
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 diseasehttp://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
EM analysis of Seprion-captured amyloid
Western analysis of Seprion-captured material – effect of Proteinase K
UninfectedInfected
NPKA B
NPK: Seprion captured material without proteinase K treatment
A: Proteinase K treatment of captured material
B: Proteinase K treatment of material prior to capture
The Seprion Separation System works for all TSEs
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sCJDhumanbrain
vCJDhumanbrain
Humanbrain
control
vCJDhumanspleen
Humanspleencontrol
BSEinfected
cowbrain
Cowbrain
control
Scrapieinfectedsheepbrain
Sheepbrain
control
Results from 0.25 mg of each sample
Amyloid in other diseases can be detected in liver, kidney and muscle
Control brains Alzheimer’s Disease brains brains0
0.5
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1.5
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1 2 3 4 5
1-40 amyloid ELISA
1-42 amyloid ELISA
Tau ELISA
Signal
Seprion is a universal ligand for Protein Aggregation Diseases of the amyloid type
Collect and homogenise sample
Transfer to dilution plate
Run assay
Add diluent, transfer to capture plate
Read
Run automated PKstep
Spin plate in 4oC centrifuge
Remove supernatant, dry
Add buffer, incubateat 100oC
Run assay
Add buffer, transfer to capture plate
Read
Collect and homogenise sample
Transfer to deep-well plate
Detection
Sample prep
Seprion Proteinase K competition
Collect and homogenise sample
Transfer to dilution plate
Run assay
Add diluent, transfer to capture plate
Read
Run automated PKstep
Spin plate in 4oC centrifuge
Remove supernatant, dry
Add buffer, incubateat 100oC
Run assay
Add buffer, transfer to capture plate
Read
Collect and homogenise sample
Transfer to deep-well plate
Detection
Sample prep
Collect and homogenise sample
Transfer to dilution plate
Run assay
Add diluent, transfer to capture plate
Read
Run automated PKstep
Spin plate in 4oC centrifuge
Remove supernatant, dry
Add buffer, incubateat 100oC
Run assay
Add buffer, transfer to capture plate
Read
Collect and homogenise sample
Transfer to deep-well plate
Detection
Sample prep
Seprion Proteinase K competition
The Idexx post-mortem assay
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
The Seprion Separation System applied to blood screening for TSEs
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
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 95oC) (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
NIBSC blind panel of vCJD spleen spiked into plasma
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1
0 1 2
Cut-off
0.1
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0 1 2
Cut-off
10 mg/ml
10 mg/ml
1 mg/ml1 mg/ml
1 mg/ml1 mg/ml
Day
Signal
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 102 – 103 IU/ml plasma
Seprion assay results for 236 human plasma donations
Signal
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50
100
150
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250
43
192
0- 0.1 0.1- 0.14 0.14- 0.20
cut-off
One initial positive that did not retest positive
Detection of PrPSc in sheep – symptomatic
and asymptomatic
Time from Seprion assay positive to clinical signs for four scrapie infected sheep
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450
1 2 3 4
Animals
Da
ys
Results of the Seprion plasma assay on a blind panel of sheep samples provided by
the VLA
SEPRION ASSAY Positive Negative Total
Positive 2 0 2 VLA designation by Western blot Negative 1* 26 27 Total 3 26
*Sample from a suspect from the same farm as three previously confirmed positives ie. from a farm with endemic scrapie
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