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

Overview

In this video, we show the HiBit-RBD bioreporter system-based assay for detecting SARS-CoV-2 neutralizing antibodies. This system targets the viral RBD domain by using a bioluminescent reporter system containing HiBiT fused to the RBD domain.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
Bio-Plex Handheld Magnetic Washer	Bio-Rad	171020100	Device
Dual-Glo luciferase Assay System	Promega	E2940	100 mL kit
LgBiT	Promega	N3030	Assay kit
Pierce Protein G Magnetic Beads	Thermo Fisher Scientific	88848	Assay
SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab	SinoBiological	40592-MM57	Assay
Synergy Mx Microplate Reader	BioTek		96-well plate reader luminometer