# Presentation: 575313

- 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

## 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

- 10353

- 55

**501aa**

- 228

- 899

**E**

**prM**

**C**

-  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**

## West Nile Virus Strain HNY1999

- 5'

- 3'

- Structural

- Nonstructural

**C**

**prM**

**E**

**1**

**2A**

**2B**

**3**

**4A**

**4B**

**5**

- 423

- 924

- 2427

- 3483

- 4176

- 4569

- 6426

- 6873

- 7638

- 10353

- 55

**501aa**

- .

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

**E**

**E**

**prM**

** ****Signal Sequence**

**Protein 2: ****	****PreM/M/Env expressed in ****	****	****eukaryotic cells as vesicles**
**	****and secreted**

** ****Signal Sequence**

**West Nile Virus Strain HNY1999**

## Indirect Assay – WNV IgM/IgG Test

**WNV Ag coated**
**solid 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**

## IgM Capture EIA

**Anti-IgM Coated Solid Phase**

**Serum Added**

**IgM Capture EIA**

**Protein 2**

**Antigen Added**

**Conjugate Added**

## West Nile Virus Serologic Studies

**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

## Indirect IgM Assay Utilizing Protein 1

** **

**7 Repeat Reactives **
**All were RNA Negative **
**and **
**Negative by IgM Capture EIA **
**Utilizing Protein 2**

**Indirect IgM Assay Utilizing Protein 1**

## IgM Capture of WNV Antibodies in Serum Samples 

**Volunteer Blood Donors  (N=241****)**

- 37

- 182

- 10

- 5

- 1

- 2

- 2

- 2

- 0

- 20

- 40

- 60

- 80

- 100

- 120

- 140

- 160

- 180

- 200

- 0.5

- 1.0

- 1.5

- 2.0

- 2.5

- 3.0

- 3.5

- 4.0

- 4.5

- 5.0

- 5.5

- 6.0

- 6.5

- 7.0

- 7.5

- 8.0

- 8.5

- 9.0

- 9.5

- 10.0

**S/N Value**

**Frequency**

***Provisional Cutoff at :***

***7SD + population mean***

- No repeatably reactive specimens were noted

**IgM Capture of WNV Antibodies in Serum Samples **

## IgM Detection of WNV Antibodies in Serum Samples

- .

**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**

## IgM Detection of WNV Antibodies in Serum Samples

**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**

## IgM Detection of WNV Antibodies in Paired Serum Samples

**Indirect Assay using Protein 1**

- 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***

## IgM Detection of WNV Antibodies in Paired Serum Samples

**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**

**Patient**** 1**

**Patient**** 2**

**Patient**** 3**

**Patient**** 4**

**Patient**** 5**

**Patient**** 6**

**Patient**** 7**

**Patient**** 8**

**Patient**** 9**

**Patient**** 10**

**Patient**** 11**

**Patient**** 12**

**S/N**

- 11

- 21

- 7

- 11

- 9

- 57

- 5

- 19

- 2

- 2

- 43

- 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***

## IgM Capture EIA using Protein 2

**IgG Reactivity**

**+**

**+**

**+**

**-**

**+**

**+**

**-**

**+**

**+**

**+**

**+**

**+**

**+**

- I

**+**

**-**

**+**

**+**

- I

**-**

**IgM Capture EIA using Protein 2**

- "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**