fda advisory committee meeting

FDA Advisory Committee MeetingTransmissible Spongiform EncephalopathiesOctober 14, 2004

Steve Figard, Ph.D.Senior Scientist

Abbott Diagnostics

And a Host of Others…

Performance of the Enfer TSE Test with

Front End Automation

Overview

Brief Overview of the Enfer TSE AssayAutomation of “Front End”Brief Data Review – External Evaluation

– Specificity– Sensitivity– Autolyzed Samples

Enfer Assay Overview

Sample Preparation

Cut tissue

Homogenize

Clarify in centrifuge plate

Immune Reactions

Primary anti-PrP rabbit polyclonal

Secondary goat anti-rabbit IgG-HRP

Sample Treatment

Transfer to test platePK digestion of PrPc

nonspecific adsorption of PrPSc

NaCl wash

GuHCl/NaOH incubation

Signal Generation

Add substrate

Read signal

Front End Automation

USDA-APPROVED ENFER TESTAutomation of “Front End”

Safer Sample Cutting

– new plastic tool

Faster Sample Homogenization

– new automated instrument

Easier Sample Handling

– new tube replaces bag

Streamlined Sample Processing

– minimized transfer

Same High Performance

SAFER SAMPLE CUTTING

Plastic PunchBladereplaced by

• No sharps• Standard cuts

• Easy disposal• Rapid

• Inexpensive

TubeBagreplaced by

EASIER SAMPLE HANDLING

Time To Process 8 Samples:

2 Stomachers – 240 seconds (4 minutes) 1 ETDS – 10 seconds

ETDS VIIIStomacherreplaced by

FASTER SAMPLE HOMOGENIZATION

STREAMLINED SAMPLE PROCESSINGSame Tube Moves Through Process

Final component ofnew automation

Streamlined Sample Processing withTecan Genesis RSP

Automated sample pipetting– ETDS tube to centrifuge plate– centrifuge plate to test plate

Automated addition of EB2 (PK) to test plateApproximately 5 min for one plate (44 samples)ETDS VIII racks fit directly into TecanOther “standard” features of automation

– disposable tips – no cross contamination

– liquid detection system for tips– bar code reader (POSID)

HIGH PERFORMANCESpecificity Testing - LGC

HIGH PERFORMANCESensitivity/Autolyzed Samples

200 positive samples from OTM population– collected and frozen at least a year and in good condition– confirmed by BioRad/Prionics WB

200 negative samples from fallen stock– in state of “severe autolysis” – worst case sample type– confirmed by BioRad/Prionics WB

Autolyzed samples can have high levels of protease inhibitors which can affect Proteinase K digestion and possibly result in a false positiveAll samples were supplied as homogenates, not tissueTesting done at Veterinary Laboratories Agency, Newcastle, UK

HIGH PERFORMANCESensitivity/Autolyzed Samples

Positive vs. Negative (Autolyzed) Sampleswith Enfer Front End Automation

(n = 200 for each population)

Bins - S/CO's

0.0 1.020

0.040

0.060

0.080

0.010

00.0

1200

.0

Freq

uenc

y

012345

20

40

60

80

100

120

140

160

180

Neg (Autolyzed) Positives Cu

toff

Negative (Autolyzed) Positive

Mean 0.026 327Median 0.013 287

SD 0.073 -Range -0.114 - 0.899 12.3 - 1217

SD from Mean to Cutoff 13.3 -

100 % Sensitivity100% Specificity

in this samplepopulation

Questions?

Steve.Figard@Abbott.com