Blood Products Advisory Committee Meeting

April 26, 2007

Ben Marchlewicz, Ph.D.PRISM R&D, Program Manager

Abbott’s approach to Chagas Testing PRISM Chagas

Description of the assayPerformance

Chagas ConfirmatoryDescription of the assayPerformance

Abbott’s Strategy in Blood Screening for Chagas Disease

Develop a recombinant antigen based assay for antibodies to T. cruzi on the PRISM platform

Well-characterized antigens Presenting multiple epitopes Non-biohazardous

Develop a recombinant antigen based Confirmatory assay for resolution of PRISM Repeat Reactive specimens.

PRISM Chagas Assay

PRISM Chagas Screening Assay Design Configuration

+ * * *

* *

Anti- T. cruzi Ab in Sample

rAg-Coated P

*

ACR-Mouse Anti-Human IgG

Conjugate

H2O2

Light

TcF coated P

FP Co-coated P

+ Transfer

Wash

Conjugate Wash

Dual rAg Coated P, Single mAb Conjugate, Two–Step Assay

Multi-epitope rAg Amino acids Mol. Wt. (Kda)

TcF 85 8.3

FP3 693 76

FP6 994 110

FP10 722 78

Multi-epitope Recombinant Antigens used in PRISM Chagas Assay

Specificity

Study Total Repeatedly Reactive

Confirmed Positive

Resolved Specificity

In-House Feasibility

Testing7,755 4 1 99.96%

Field Study 4,100 8 3 99.88%

Total 11,855 12 4 99.93%

Random Donors

Specificity

Study Total Specificity

Leishmania visceral(sourced from India)

30 100%

Leishmania cutaneous(sourced from India)

11 100%

Malaria 10 100%

Cross Reactivity with other Parasitic Infections

PRISM Chagas Assay SensitivityChagas Positive Specimens (confirmed by RIPA)

RIPA Positive

RIPA Negative

PRISM Positive 419 0

PRISM Negative 0 0

Sensitivity

100% Sensitivity

Chagas Confirmatory Assay

Design of Chagas Confirmatory Immunostrip

Anti-hIgG (Sample Addition CTL)

hIgG-H CTL

hIgG-L CTL (Cut-off)

4 rAgs for anti-T. cruzi detection

TcF FP3 FP6 FP10

ID#

Test Bands On-board CTL bands

IMMUNOSTRIP HUMAN SPECIMEN 2 hr Incubation

Wash

Wash

1 hr Incubation ANTI-HUMAN AP CONJUGATE

10 min IncubationBCIP-NBTCOLOR

Chagas Confirmatory Assay Schematic

On-BoardControls

ChagasAntigens

Immunostrip

ID#

Visual Reading of Test Bands

Intensity Score

Absent - Faint or < on-board hIgG-L CTL +/-

= on-board hIgG-L CTL 1+ Between on-board hIgG-L CTL and hIgG-H CTL 2+

= on-board hIgG-H CTL 3+ > on-board hIgG-H CTL 4+

Sensitivity

RIPA Positive

RIPA Negative

Immunoblot Chagas

Confirmatory Positive

410* 0

Immunoblot Chagas

Confirmatory Negative

0 0

100% Sensitivity

* Also reactive in PRISM Assay & 2 reference ELISA’s

Country of Origin # of SamplesArgentina 41

Bolivia 53

Brazil 176

Chile 5

Colombia 1

Ecuador 1

El Salvador 12

Honduras 4

Mexico 33

Nicaragua 17

Surinam 1

USA 4

Venezuela 21

Unknown 41

Chagas Specimens Panel n = 410 (All are confirmed RIPA positive)

Specificity

RIPA Positive

PRISMChagas Assay Non-reactive

Immunoblot Chagas

Confirmatory Positive

1* 0

Immunoblot Chagas

Confirmatory Negative

0 500

100% Resolved Specificity

Unscreened Random Blood Donors

* Also reactive in PRISM Assay & 2 reference ELISA’s

Specificity

RIPA Positive

PRISMChagas Assay Non-reactive

Immunoblot Chagas

Confirmatory Positive

2* 0

Immunoblot Chagas

Confirmatory Negative

0 316

100% Resolved Specificity

Unrelated Medical Conditions

* Also reactive in PRISM Assay & 2 reference ELISA’s

Specificity

Study Total Specificity

Leishmania visceral(sourced from India)

30 100%

Leishmania cutaneous(sourced from India)

11 100%

Malaria 10 100%

Cross Reactivity with other Parasitic Infections

Summary

A fully automated blood screening assay for antibodies to T. Cruzi, on the PRISM system, is under development; Along with an Immunoblot Confirmatory assay, that can be performed on site, to confirm Repeat Reactive specimens to aid in donor management.