Bioreporter Assay: A Sensitive Technique using a Bioluminescent Reporter System to Detect SARS-CoV-2 Antibodies
DEŞİFRE METNİ
During severe acute respiratory syndrome coronavirus 2, or SARS-CoV-2, infection, the virus spike protein's receptor binding domain, RBD, binds to host cell angiotensin-converting enzyme 2, ACE2, facilitating viral cell entry. In response, the immune system produces antibodies that bind to the viral RBD and prevent cellular entry, reducing viral infectivity.
To detect RBD-targeting SARS-CoV-2 antibodies, take a recombinant bioreporter suspension comprising small-sized protein fragments, fused to SARS-CoV-2 spike RBD. Add standard RBD-targeting SARS-CoV-2 antibodies and antibody-binding magnetic beads.
During incubation, the Fc antibody region binds to the beads' Fc-binding domains. Additionally, the recombinant bioreporter binds to the bead-bound RBD-targeting SARS-CoV-2 antibodies.
Post-incubation, centrifuge the mixture. Remove the unbound bioreporter-containing supernatant. Resuspend the bound beads in buffer. Transfer to a multi-well plate.
Add a solution containing large-sized protein fragments, having high affinity for small-sized protein fragments. Large and small-sized protein fragments bind together, forming an active engineered luciferase enzyme.
Add furimazine, a luciferase substrate. Luciferase catalyzes the oxidation of furimazine, causing light emission. Using a luminometer, measure the luminescence, indicative of the presence of neutralizing antibodies.
High signal intensities suggest high concentrations of RBD-targeting SARS-CoV-2 antibodies.
