This article discusses the detection of SARS-CoV-2 antibodies targeting the receptor binding domain (RBD) of the spike protein. The method utilizes a recombinant bioreporter system to measure luminescence, indicating the presence of neutralizing antibodies.
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.
In order to perform the HiBiT-RBD antibody detection assay, prepare the HiBiT-RBD bioreporter, and combine 50 microliters of the HiBiT-RBD-containing supernatant with 1 microgram of the commercial SARS-CoV-2-RBD antibody in a 1.5-milliliter microtube.
Add 20 microliters of immunoglobulin-binding protein, or protein G, to the solution. Bring the total volume to 300 microliters by adding PBS. Incubate the tubes on a tube shaker or rotator for 30 minutes, centrifuge at 12,000 x g for 30 seconds, then, discard the supernatant and wash with PBS. Repeat the process three times to remove free HiBiT-RBD.
Resuspend the cells in 50 microliters of PBS, and transfer it to a 96-well plate. Add 50 microliters of 1x LgBiT, and wait for 5 minutes. Then, add 50 microliters of 1x NanoLuc substrate. Immediately read the luminescence signal with a luminometer.
繁體中文
