Update on Assay Development

George J. Dawson, Ph.D.

Infectious Diseases: Core R & D

Abbott Laboratories

West Nile Virus

West Nile Virus Strategies

Determine marker profile The overlap between RNA, antigen, and IgM detection Markers associated with acute, symptomatic infection

Develop prototype IgM class antibody test for detection of antibodies to WNV

Recombinant proteins have been obtained from two laboratories (Material Transfer Agreement’s executed)

EIA’s are being evaluated for IgM detection Primer pairs have been selected for in-house RT-PCR

Evaluate utility of IgM test Screening blood donors Reinstatement of WNV RNA positive donors Diagnosis of acute infection

Derived from core, preM regions

5'

3'

Structural

Nonstructural

C prM E 1 2A 2B 3 4A 4B 5

423

924

2427

3483

4176

4569

6426

6873

7638

1035

355

501aa

228 899

EprMC

Degenerate primers

Degenerate primers selected for PCR studies

West Nile specific primers

Primer pairs were selected for in-house studies to determine overlap between viremia and IgM

West Nile Virus PCR Primers

5'

3'Structural Nonstructural

C prM E 1 2A 2B 3 4A 4B 5

423

924

2427

3483

4176

4569

6426

6873

7638

1035

355

501aa

.Protein 1: Envelope gene (truncated) expressed in eukaryotic cells

E

EprM

Sig

nal

Seq

uen

ce

Protein 2: PreM/M/Env expressed in eukaryotic cells as vesiclesand secreted

Sig

nal

Seq

uen

ce

West Nile Virus Strain HNY1999

WNV Ag coatedsolid phase

Serum Added

Indirect Assay – WNV IgM/IgG Test

Protein 1

Anti-IgG Conjugate Added

Anti-IgM Conjugate Added

IgM Format

IgG Format

Anti-gamma added to specimen

diluent in IgM format

Anti-IgM Coated Solid Phase

Serum Added

IgM Capture EIA

Protein 2

Antigen Added Conjugate

Added

IgM and IgG assays were developed with protein 1 and 2; WNV RNA testing was performed on repeatably reactive human specimens and on all monkey sera

West Nile Virus Serologic Studies

Sample Category Number of Specimens

Volunteer blood donors 241

Confirmed positive specimens:

IgM only

IgM/IgG positives

8

9

Paired human samples 12 pairs

CDC Proficiency Panel 20

Experimentally Infected Rhesus Monkeys study in progress

5 animals

West Nile Virus Serologic Studies

Rationale for Assay Format Selection(s)

The original data demonstrating the utility of Protein 1 for WNV diagnostics utilized an indirect assay format

• Data generated in-house for Protein 1 with an IgM Capture format was not adequate

The original data demonstrating the utility of Protein 2 for WNV diagnostics utilized an IgM Capture assay format

• In-house studies indicated that performance of Protein 2 in an indirect assay format was not adequate

Additional studies are planned

Normal Population Distribution N = 241

1

32

123

28

125 4 1 1 1 3 1 1 1

5

0

20

40

60

80

100

120

140

S/N Value

Fre

qu

ency

Provisional Cutoff at:7SD + population mean

7 Repeat Reactives All were RNA Negative

and Negative by IgM Capture EIA

Utilizing Protein 2

Indirect IgM Assay Utilizing Protein 1

Volunteer Blood Donors (N=241)

37

182

10 5 1 2 2 2

020406080

100120140160180200

0.5

1.0

1.5

2.0

2.5 3.

03.

54.

04.

55.

05.

56.

06.

57.

07.

58.

08.

59.

09.

510

.0

S/N Value

Fre

qu

ency

Provisional Cutoff at :7SD + population mean

No repeatably reactive specimens were noted

IgM Capture of WNV Antibodies in Serum Samples

0

10

20

30

40

50

60

70

80

71 72 73 74 76 77 78 79

Sample

IgM

S/N

Protein 1 Indirect

Protein 2 IgM Capture

Cutoff

.

Note: All 8 specimens reactive using protein 2; only 5 specimens were reactive with EIA utilizing protein 1

Confirmed IgM positive and IgG positive

IgM Detection of WNV Antibodies in Serum Samples

Confirmed IgM positive and IgG negative

0

10

20

30

40

50

60

70

11 12 13 14 15 16 17 18 19

Sample

IgM

S/N

Protein 1 Indirect

Protein 2 IgM Capture

Cutoff

Note: All 9 specimens reactive using protein 2; only 3 specimens were reactive with EIA utilizing protein 1.

Sample 11 was WNV RNA positive

IgM Detection of WNV Antibodies in Serum Samples

Indirect Assay using Protein 1

0

20

40

60

80

100

120

140

Paired Specimens

S/N

Bleed 1Bleed 2

1121

7

11

957 5

19 2 62 243

6

24 7 26

6

20

20

503

21

12 55Cutoff

•The first sample from 5 of 12 patients was reactive in the IgM Indirect EIA using protein 1

• 5 of 12 follow-up bleed samples were reactive

IgM Detection of WNV Antibodies in Paired Serum Samples

Numbers at the top of the columns indicate days post onset of

symptoms

Paired Specimens

•The first sample from all 12 patients was reactive in the IgM Capture EIA using protein 2

•11 of 12 follow-up bleed samples were reactive

IgM Capture EIA using Protein 2

0

10

20

30

40

50

60

70

Patien

t 1

Patien

t 2

Patien

t 3

Patien

t 4

Patien

t 5

Patien

t 6

Patien

t 7

Patien

t 8

Patien

t 9

Patien

t 10

Patien

t 11

Patien

t 12

S/N 11

217 11 9

57

5

19

2

243

6

24

7

26

6

20

20

50 3

21

12

55

62

Cutoff

IgM Detection of WNV Antibodies in Paired Serum Samples

Numbers at the top of the

columns indicate days post onset

of symptoms

Bleed 1

Bleed 2

Note: Abbott’s IgM Capture EIA results using Protein 2 on the CDC Proficiency Panel generated equivalent or greater S/N values than those provided by CDC.

IgG Reactivity + ++ -++- ++ ++++I+-+ + I -

IgM Capture EIA using Protein 2

CDC Proficiency Panel (20 member panel)

05

101520253035404550556065707580

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Panel Member

S/N

Va

lue

Abbott Capture IgM Format

CDC Capture IgM Format

N N N N NI I

"N" denotes known negative specimens

"I" denotes indeterminate IgG only unconfirmed

SUMMARY

Protein 2 detected more acute phase WNV samples than Protein 1 Data suggest that conformational epitopes present in Protein 2 are important for

early IgM detection The cell line expressing Protein 2 is being scaled-up in-house

Preliminary evaluation of IgM testing indicates: For blood screening – additional studies are required The IgM response was robust in most symptomatic individuals

• IgM in conjunction with IgG could be part of the donor reinstatement algorithm • For diagnostics, IgM is the preferred method of detection

Prototype assay formats are being investigated EIA’s on polystyrene beads Microparticle based assays on AxSYM and IMx

Studies Planned or In Progress

Optimization of assay Incubation times, sample volume Concentration of rare reagents, washing steps Processing steps for rare reagents

Confirmatory strategies RT-PCR on early phase specimens IgG test with Protein 2 IgM and IgG tests with alternate protein Plaque reduction neutralization tests ( in collaboration with CDC or state labs)

External Studies Serologic studies on quarantined units from 2002 epidemic Donor reinstatement studies as companion to WNV NAT efforts Testing at state and/or hospital laboratories during 2003 WNV season

Additional proteins are being expressed in-house to determine their potential utility