# Presentation: 928242

## West Nile Virus Serology

-  Is ELISA challenged by the  arbovirus IFA test and new technologies?
- Susan J. Wong

## Current Standards for Serological Detection

- IgM Antibody Capture ELISA (MAC-ELISA) with CDC reagents
- IgG indirect (sandwich) ELISA with CDC reagents
- Verification by cross-species plaque neutralization testing on paired specimens against  likely viruses

## Comparison of Antibody Levels in Serum and CSF

## Why IgM antibody capture?

- If indirect (sandwich) ELISA technique were used for IgM, the **IgG would out compete** for available antigen binding sites on the WN Envelope antigen, and the small amount (10-100X less) of IgM would not be detected!
- Reaction (binding) kinetics of sandwich ELISA favor IgG

##  MRL (Focus Technologies) Arbovirus IFA Test

- Four different North American arboviruses are present in Vero cells dotted and fixed on each well of the slide
- Eastern Equine Encephalitis
- Western Equine Encephalitis
- La Cross Encephalitis
- St. Louis Encephalitis
- Screening for Alpha, Flavi- and Bunya viruses

## IFA Requirements

- Trained observer interprets pattern of fluorescence
- Recognizes  autoantibodies and non-specific binding
- Recognizes correct proportion of stained infected cells

## Antibody Response to WNV

- P/N IgM

- P/N IgG

**12**
**10**
**8**
**6**
**4**
**2**

**12**
**10**
**8**
**6**
**4**
**2**

## Slide 8

## Detection Windows of Opportunity

**30**
**25**
**20**
**15**
**10**
**  **
**  ****5**
**  **
**  ****0**

**0          50         100        150        200        250        300        350        400**

- IFA IgG
- IFA IgM
- MAC - ELISA “Window”
- IgG - ELISA “Window”

**Serum MACELISA P/N**

- Cut Off

- 

**Serum IgG ELISA P/N**

## 1999 WNV Outbreak Sera Comparison: IFA to ELISAs and PRN

## Review of 1999 IFA Results

- Three sera positive by IFA IgM test were collected 10 to 15 days post onset of symptoms
- Sera reactive in the MAC-ELISA were collected from 1 to 30 days post onset
- Good sensitivity of the IgG IFA compared to IgG ELISA
- Possibly a 10 to 15 day time advantage of MAC-ELISA?  Broader “window....

## 2001 IFA  IgG Results

- All sera (51) reactive in the IgG ELISA were reactive in the IgG IFA test to SLE
- 6 sera reactive in the IFA test, non-reactive in the IgG ELISA
- 1 patient WNV
- 5 patients no follow-up

## 2001 Arbovirus Antibody Comparison:  IFA to ELISAs and PRN

- 45 encephalitis patients (82 specimens) with at least one positive result (ELISA, IFA)
- 30 patients with IgG ELISA +ve or Eq
- 9  (30%)WNV
- 4 (13.3%)Flavivirus
- 3 (10%) Dengue
- 1(3.3%) Nonreactive by PRN
- 13 (43.3%) no follow up, no PRN

## 2001 IFA IgM Results

- All patients with a positive  or equivocal MAC ELISA tested on IgM IFA
- All patients (8) with reactive  IgG IFAs other than SLE were tested on IgM IFA
**One patient only reactive IgM to SLE in the IFA test (titer 2048, P/N 26), d 17,d 68**
- Mean P/N 17.78 (7.21-26.2)  first sera
- Mean P/N 15.53 (8.35-19.5) follow up sera

## Suspension Array Technology Sequence of Events

- Antigen - Black
- Patient’s Antibody - Blue
- PE Labeled Anti Antibody - Red

- Fluorescent Bead -
- Yellow

## Flow Cell

- Quantitative
- Measure of
- Extrinsic
- Fluorescence

- Bead
- Identification

- Laser 1

- Laser 2

## Luminex Analyzer

## Data Analysis of Multiplex Assay

## Reaction in Multiwell Filter Plates

## Pump, Filter Plate, Sonicator and Vortex

## Partial Plate in Use for Small Assays

## Black Strip Wells Carry Beads into Analyzer

## Data Review by Technologist

## Acknowledgements

- CDC ELC WNV grant
- CDC Arbovirus Diseases Branch
- Wadsworth Center
- Arbovirus Research Lab
- Viral Encephalitis Lab
- Diagnostic Immunology Lab

- NYSDOH BCDC Division of Epidemiology
- NYCDOH
- Yale U and L2Dx