SARS-CoV-2 ADE Assay

ADE Introduction

Antibody-dependent enhancement (ADE) is a phenomenon by which non-neutralizing or sub-neutralizing antibodies bind to the virus and are then internalized by cells surface Fc Receptor, increasing virus entry. During the neutralizing process between SARS-CoV-2 and its antibody, the antibody Fab region will bind with receptor binding domain (RBD) on virus surface spike protein, blocking the virus entry. However, in ADE settings, the antibody Fc region interacts with cell surface Fc receptor, which leads to the entry of virus-antibody complex and causes ADE (Figure. 1).

Figure 1: ADE activity generation. Adapted from: Wu, F., Yan, R., Liu, M., Liu, Z., Wang, Y., Luan, D., ... & Huang, J. (2020). Antibody-dependent enhancement (ADE) of SARS-CoV-2 infection in recovered COVID-19 patients: Studies based on cellular and structural biology analysis. medRxiv.

ADE plays an essential role in SARS-CoV-2 vaccine and antibody drug development. ADE has exhibited dependency on antibody concentration and its neutralization ability, which are important aspects involved in vaccine design. Moreover, high ADE activity tightly correlates with high risks of lung injury in COVID-19 settings, which can result in failed vaccine development. In immunized macaques, a modified vaccinia Ankara viral vector expressing the SARS-CoV S protein had elicited anti-S IgG response which enhanced pulmonary infiltration of inflammatory macrophages and resulted in more severe lung injury compared to unvaccinated animals. Another experiment also confirmed that peptide vaccines induced antibodies can enhance infection in vitro and resulted in severe lung pathology in vivo. Regarding its impact on antibody responses and high risks in lung injury, ADE assay is an irreplaceable part of SARS-CoV-2 vaccine investigation.

How our ADE assays are done

Effector Cell：

• cells carrying the Fc Receptor (Raji cells, K562 cells, primary B cells etc.)
• 293T/ACE2 cells (ACE2 overexpression 293T cells) as the positive system control

Detection Method

• SARS-CoV-2 or pseudotyped SARS-CoV-2 infects effector cells after incubation with the serial dilution of test antibody or serum.
• ADE is measured by comparing the percentage of infected cells at different serum/antibody concentrations with untreated/non-enhancing controls.
• In the live virus-based ADE assay (BLS3), infection is quantified by immunostaining and flow cytometry analysis. In the Reporter (eg: GFP or Luciferase) viral particles (RVPs) based ADE assay, infection is quantified by flow cytometry analysis or luciferase assay system.
• Untreated and uninfected controls are included in each plate to control for assay variability across plates. The assay can be internally controlled using known concentrations of purified enhancing and isotype antibodies.
• The assay is optimized in a 96-well plate format to reduce the volume of testing material required, which in turn allows for parallel neutralization tests and/or multiple replicates/repeats for increased robustness.

Sample Requirements

• Sample: antibody (concentration> 0.2 mg/mL, sterile, pH 7.1-7.4) or inactivated serum.
• Positive control antibody or serum with ADE activity

Deliverable

• Project Report

Timeline

• 4-5 weeks

Example Schematic of the ADE assay based on Pseudotyped Luciferase rSARS-CoV-2 Spike (CD Cat # COV-PS01)

We also provide follow on services – read our SARS-CoV-2 Pseudovirus Neutralization Assay pages for more information.

We provide a full serial of SARS-CoV-2 and SARS-CoV-2 Mutation pseudovirus with GFP or luciferase reporter gene as well as cell lines carrying the Fc Receptor or ACE2. See below Related Products for more information.

Related Products

• Lentiviral SARS-CoV-2 Pseudovirus

• Human ACE2 Stable Cell Line - HEK293T

• SARS-CoV-2 Neutralizing Antibody ELISA Kit