Bioreporter Assay: A Sensitive Technique using a Bioluminescent Reporter System to Detect SARS-CoV-2 Antibodies

1. Detecting anti-RBD antibody with a fast and sensitive assay

1. HiBiT-RBD antibody detection assay
   1. Prepare the HiBiT-RBD bioreporter.
      NOTE: It is recommended to use the supernatant for the following assay as it is simpler to collect and contains the mature glycosylated version of the protein. Moreover, the lysate has several other proteins that could interfere with the HiBiT-RBD-antibody interaction.
   2. Combine 50 µL of the HiBiT-RBD-containing supernatant with 1 µg of the commercial SARS-CoV-2-RBD antibody in a 1.5 mL microtube. Add 20 µL of immunoglobulin-binding protein (protein G) to the solution. Bring up the total volume to 300 µL by adding PBS.
      NOTE: The total volume of the mixture can be decreased to 150 µL. Lower total volumes are not recommended as it could result in inadequate mixing of antibodies with the bioreporter.
   3. Incubate the tube(s) on a tube shaker or rotator for 30 min. Centrifuge at 12,000 x g for 30 s, discard the supernatant and wash with PBS. Repeat the process three times to remove free HiBiT-RBD. Resuspend in 50 µL of PBS and transfer to a 96-well plate.
   4. Add 50 µL of 1x LgBiT and wait for 5 min. Then, add 50 µL of 1x NanoLuc substrate. Immediately read the luminescent signal with a luminometer.