PhenoSense® SARS-CoV-2 neutralizing Antibody Assay starts by generating pseudovirions by transfecting HEK293 cells with two different viral vectors. The first is in HIV genomic vector containing all the structural genes except envelope, which is instead replaced with a luciferase reporter gene. The second vector is an envelope expression vector carrying the SARS-CoV-2 spike protein open reading frame.
These pseudovirions are collected and incubated with patient samples and HEK293 cells expressing the ACE2 receptor. Virus that is not neutralized by the patient sample will be capable of replicating and producing luciferase, and the cells with glow. Patient samples with neutralizing antibodies will inhibit the virus from replicating and thereby prevent the luciferase from being produced.
The PhenoSense SARS-CoV-2 Neutralizing Antibody Assay has been adapted from the proprietary PhenoSense® Assay platform that was originally developed to evaluate antiretroviral drug susceptibility (Petropoulos et al., AAC 2000) and later modified to evaluate HIV-1 viral entry inhibitors, HIV-1, neutralizing antibody (nAb) activity (Richman et al, PNAS 2003) and HIV-1 co-receptor tropism (Whitcomb et al., 2007). The neutralizing antibody has been adapted to evaluate neutralizing antibodies for a number of viruses, including HIV, Influenza virus, RSV, Ebola virus, and now SARS-CoV-2.
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Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci USA. 2003 Apr 1;100(7):4144-4149.
Whitcomb JM, et.al. Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism. Antimicrobial Agents and Chemotherapy Jan 2007, 51 (2) 566-575.