Service Description
In vitro infection and neutralization assay

Pseudovirus
Expresses the SARS-CoV-2 spike protein.

Detection Sytem

1. GFP (Fluorescence via Fluorescence microscopy)
2. Luminescence (measured as relative luminescence units) (Renilla and firefly both are available)

Sample types
Purified antibodies/plasma/serum/inhibitors/proteins/short peptides

Replicates
The assay will be done in duplicates unless otherwise desired/requested. In case of serial dilutions, 6 dilutions can be tested.

Cell lines
• 293T overexpressing hACE2 receptor
• 293T cell line over expressing hACE2 receptor and hTMPRSS2* on the receptor.

*hTMPRSS2 is a serine protease that tends to prime the SARS Cov2 spike protein thereby facilitating the interaction with the ACE2 receptor and subsequent entry of virus/pseudotyped virus into the host cells.

Controls
Our service includes the following controls:
• ‘No sample‘ positive control (ACE2 cells incubated with pseudoviruses alone)
• positive serum/inhibitor control (neutralizing the pseudotyped virus with an IC50 of 239.7 to 945.4 (serum) and 0.001453 to 16.16 µM (serine protease inhibitor)
• ‘negative serum control‘ serum sample internally tested to be non-neutralizing
• negative control (no pseudovirus with an ACE2-expressing cell line)
Duplicate technical replicates will be performed for all controls unless otherwise requested.
Additional negative controls with cells that do not express the ACE2 receptor, can be added to this service upon request.

Deliverables
We offer three deliverables

1. An Excel-based report outlining the samples and their measured luminescence values. These values have already had background signal subtracted from them. The background signal is obtained from duplicate technical replicates of similar cells that have not had pseudovirus incubated with them. The Excel file will also include the inhibition rate(s) of the sample(s) at different dilutions compared to the "No Sample" positive control.
2. Graphical representation of assay along with IC50 values.
3. An elaborated report: a Word document summarizing the service background, controls, procedure, and references.

Example Data

Fig1. Pseudotyped SARS Cov2 neutralization by serum from Covid-19 recovered individual.

Fig.2. Infection/Transduction of pseudotyped SARS-Cov2 into different cell lines.

Fig.3.Neutralization of pseudotyped SARS Cov2 (GFP reporter) by anti-Spike Llama serum (Tyson) as analysed by fluorescence microscopy.

Fig. 4. Inhibition of pseudotyped SARS Cov2 transduction into hACE2 and hTMPRSS2 expressing cell lines by serine protease inhibitor (Nafamostat).