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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vCJDhumanbrain

Humanbrain

control

vCJDhumanspleen

Humanspleencontrol

BSEinfected

cowbrain

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Scrapieinfectedsheepbrain

Sheepbrain

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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

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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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Cut-off

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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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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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Animals

Da

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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