1. Detecting anti-RBD antibody with a fast and sensitive assay
<ol>
	<li>HiBiT-RBD antibody detection assay
	<ol>
		<li>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.</li>
		<li>Combine 50 &#181;L of the HiBiT-RBD-containing supernatant with 1 &#181;g of the commercial SARS-CoV-2-RBD antibody in a 1.5 mL microtube. Add 20 &#181;L of immunoglobulin-binding protein (protein G) to the solution. Bring up the total volume to 300 &#181;L by adding PBS. 
		NOTE: The total volume of the mixture can be decreased to 150 &#181;L. Lower total volumes are not recommended as it could result in inadequate mixing of antibodies with the bioreporter.</li>
		<li>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 &#181;L of PBS and transfer to a 96-well plate.</li>
		<li>Add 50 &#181;L of 1x LgBiT and wait for 5 min. Then, add 50 &#181;L of 1x NanoLuc substrate. Immediately read the luminescent signal with a luminometer.</li>
	</ol>
	</li>
</ol>

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

bioreporter assay: a sensitive technique using a bioluminescent reporter system to detect sars-cov-2 antibodies

Source: Rezaei, R. et al., <a href='https://app.jove.com/v/62488'>Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021)
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.

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

Source: Rezaei, R. et al., Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021)
In this video, we ...

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.&#160;
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.&#160;
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.

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765 Views. 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...

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

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Bio-Plex Handheld Magnetic Washer	Bio-Rad	171020100	Device
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LgBiT	Promega	N3030	Assay kit
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