for Neutralizing Antibody Detection
Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. This article will show you a Strategy of Pseudovirus package for Neutralizing Antibody Assay.
Strategy of Pseudovirus package for Neutralizing Antibody Assay
To set up Pseudovirus for Neutralizing Antibody Assay, three importent component should be considered: 1) Non-replication for safety, 2) Spike protein for infection and 3) Reporter genes for analysis. Pseudovirus package use the SARS-CoV-2 (2019-nCoV, COVID-19 virus) S protein as a surface capsid glycoprotein and containing GFP and/or Luciferase reporter genes as a pseudovirus model. Therefore, this pseudovirus can infect cell lines overexpressing the ACE2 gene. Researchers can determine the infection efficiency by observing GFP through a microscope and detecting the value of Luciferase.
Non-replication Lentiviral Packaging System for Safety
ACE Biolabs suggest to use the lentiviral packaging system to generate SARS-CoV-2 pseudovirus.
Lentivirus is regarded as a biosafety level 2 material and safe to use due to its modified features (deletion of a number of accessory virulence genes , minimal genome of the viral particles, non-replicating and self-inactivation features), making it incapable of producing virus once infected into the host cell.
SARS-CoV-2 Spike Protein as a Surface Capsid Glycoprotein for Infection
Spike Protein used for Pseudovirus Neutralizing Antibody Assay most frequently is the first published sequence NC_045512.2. However, recently reports show that mutation of S protein improves infectivity or/and decreases sensitivity to neutralizing antibodies (ref: https://doi.org/10.1038/s41392-020-00302-8).
Wang, L., Wang, L. & Zhuang, H. Profiling and characterization of SARS-CoV-2 mutants' infectivity and antigenicity. Sig Transduct Target Ther 5, 185 (2020). https://doi.org/10.1038/s41392-020-00302-8
The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity https://doi.org/10.1016/j.cell.2020.07.012
Using mutant S protein as a surface capsid glycoprotein of pseudovirus will be an alternative choice. Here, we show you some examples using mutant S protein to improves pseudovirus infectivity.
D614G mutation improved infectivity -
A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. Plante, J.A. et al. reported that D614G enhances replication on human lung epithelial cells and primary human airway tissues through an improved infectivity of virions. ref: https://doi.org/10.1038/s41586-020-2895-3
C-ter modification improved infectivity -
Hu J. et al. generated pS-Mut plasmid bearing two mutations that destroy the endoplasmic reticulum (ER) retrieval signal (“KxHxx” motif) in the CT domain of the S protein and pS-C19del plasmid lacking the C-terminal 19 amino acids. The highest viral titer was observed in S-C19del pseudotyped virus, suggesting that the this mutant S facilitates lentivirus packaging. ref: https://dx.doi.org/10.1016%2Fj.gendis.2020.07.006
Reporter genes for analysis
Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes green fluorescent protein (GFP), red fluorescent protein (RFP) and the enzyme luciferase. ACE Biolabs suggests Pseudovirus package GFP and Luciferase reporter genes together. Therefore, you can determine the infection efficiency and neutralizing antibody efficacy by roughly observing GFP through a microscope and by quantitatively detecting the value of Luciferase.
To Set up a Pseudovirus Neutralizing Antibody Assay Platform, please refer to Pseudoviral Neutralizing Antibody Detection Platform.
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