Bioreporter Assay: A Sensitive Technique using a Bioluminescent Reporter System to Detect 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.