An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection

Published: January 31, 2024

Abstract

Source: Valiant, W. G., et al. A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum. J. Vis. Exp. (2018).

This video demonstrates an assay to measure antibody-dependent enhancement (ADE) of Dengue virus infection using Zika Virus Convalescent Serum. The assay uses Dengue reporter viral particles (RVPs) encoding a green fluorescent protein. Serum antibodies against the Zika Virus cross-react with RVPs to form a complex. The bound antibodies enhance RVP uptake by cells expressing the Fc receptor, which is detected using flow cytometry.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Day 1 NOTE: Perform all the steps described below in a sterile laminar flow biosafety cabinet used for tissue culture in a BSL-2 laboratory. Thaw the serum samples at room temperature and transfer 100 µL of each serum sample to a sterile tube. Heat inactivate for 30 min at 56°C either in a wa…

Divulgations

The authors have nothing to disclose.

Materials

DENV 1-4 RVP Integral Molecular RVP-501
K562 cells ATCC CCL-243
RPMI Corning 10-040-CV
FBS GE lifesceinces SH 30910.03 10% FBS in RPMI-10
Penicillin-Streptomycin MP biomedicals 1670049 100 IU Pen/mL, 100 ug Strep/mL in RPMI-10
HEPES Cellegro 25-060-Cl 0.025M in RPMI-10
MEM Non-essential Amino Acid Solution (100×) Sigma M7145 1x in RPMI-10
Sodium Pyruvate Cellegro 25-000-Cl 1mM in RPMI-10
L-glutamine Fisher Scientific MT25005CI
Sterile V-bottom plates Thomas Scientific 333-8001-01V
BD Falcon Polypropylene 5 ml FACS tubes VWR 60819-728
Non-sterile U-bottom plates Falcon Ref 353910 A high throughput alternative to FACS tubes
5mL, sterile, serological pipette Denville P7127
200uL sterile pipette tips Denville P3020 CPS
20uL sterile pipette tips Denville P1121
50-200uL multichannel pipette Denville P3975-8-B
5-50uL multichannel pipette Denville P3975-9-B
20% formadehyde Tousimis #1008B
Water Bath ThermoFisher TSCOL35
thermomixer Eppendorf 2231000387 An alternative to a waterbath for heat inactivation
CO2 Incubator ThermoFisher 13-998-213
Ethanol Sigma Aldrich E7023
Liquid Bleach Fisher Scientific NC9724348
Flowjo 9.8 TreeStar, Inc. Flow cytometry analysis software
BD FACSDiva 6.1.2 Becton Dikinson
BD LSR II flow cytometer Becton Dikinson
Liquid Bleach Fisher Scientific NC9724348

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Citer Cet Article
An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection. J. Vis. Exp. (Pending Publication), e21927, doi: (2024).

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