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…
Divulgaciones
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 |
Tags
Citar este artículo
An In Vitro Assay to Examine the Antibody-Dependent Enhancement of Viral Infection. J. Vis. Exp. (Pending Publication), e21927, doi: (2024).