Name: detection of sars-cov-2 receptor-binding domain antibody using a hibit-based bioreporter
Uploaded: 2021-08-12T13:30:00
Description: Watch this Scientific Journal Video about Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter at JoVE.com

We appreciate and thank the technical assistance of Xiaohong He, Ricardo Marius, Julia Petryk, Bradley Austin, and Christiano Tanese De Souza. We also thank Mina Ghahremani for Graphic Design. We would also like to thank all the individuals who participated and donated their blood samples for this study. DWC is supported in part by uOttawa Faculty and Department of Medicine.

NOTE: The protocol described below adheres to all ethics guidelines according to protocol code 20200371-01H.
1. Production and evaluation of the HiBiT-RBD bioreporter
<ol>
	<li>Producing a sufficient quantity of HiBiT-RBD bioreporter
	<ol>
		<li>Prepare for cell culture
		<ol>
			<li>Prepare complete Dulbecco&#39;s modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Then, warm the media in a 37 &#176;C water bath.</li>
			<li>Turn the biological saf...

<ol>
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Worldometer Available from: <a target="_blank" href="https://www.worldometers.info/coronavirus/">https://www.worldometers.info/coronavirus/</a> (2021)</li><li>Cacciapaglia, G., Cot, C., Sannino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+wave+COVID-19+pandemics+in+Europe:+a+temporal+playbook.">Second wave COVID-19 pandemics in Europe: a temporal playbook.</a> Scientific Reports. 10 (1), 15514 (2020).</li><li>COVID-19 vaccines. World Health Organization Available from: <a target="_blank" href="https://www.who.int/emergencies/diseases/novel-coronavirus-2019/covid-19-vaccines">https://www.who.int/emergencies/diseases/novel-coronavirus-2019/covid-19-vaccines</a> (2021)</li><li>Hueston, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibody+response+to+SARS-CoV-2+infection.">The antibody response to SARS-CoV-2 infection.</a> Open Forum Infectious Diseases. 7 (9), (2020).</li><li>Lan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+SARS-CoV-2+spike+receptor-binding+domain+bound+to+the+ACE2+receptor.">Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.</a> Nature. 581 (7807), 215-220 (2020).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+for+SARS-CoV-2+vaccine+design:+fusion+of+Spike+glycoprotein+transmembrane+domain+to+receptor-binding+domain+induces+trimerization.">Implications for SARS-CoV-2 vaccine design: fusion of Spike glycoprotein transmembrane domain to receptor-binding domain induces trimerization.</a> Membranes. 10 (9), 215 (2020).</li><li>Piccoli, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+neutralizing+and+immunodominant+sites+on+the+SARS-CoV-2+Spike+receptor-binding+domain+by+structure-guided+high-resolution+serology.">Mapping neutralizing and immunodominant sites on the SARS-CoV-2 Spike receptor-binding domain by structure-guided high-resolution serology.</a> Cell. 183 (4), 1024-1042 (2020).</li><li>Premkumar, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+receptor-binding+domain+of+the+viral+spike+protein+is+an+immunodominant+and+highly+specific+target+of+antibodies+in+SARS-CoV-2+patients.">The receptor-binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients.</a> Science Immunology. 5 (48), (2020).</li><li>Walls, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elicitation+of+potent+neutralizing+antibody+responses+by+designed+protein+nanoparticle+vaccines+for+SARS-CoV-2.">Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.</a> Cell. 183 (5), 1367-1382 (2020).</li><li>Azad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoluciferase+complementation-based+biosensor+reveals+the+importance+of+N-+linked+glycosylation+of+SARS-CoV-2+Spike+for+viral+entry.">Nanoluciferase complementation-based biosensor reveals the importance of N- linked glycosylation of SARS-CoV-2 Spike for viral entry.</a> Mol Ther. , 0074-0075 (2021).</li><li>Bastos, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+accuracy+of+serological+tests+for+covid-19:+systematic+review+and+meta-analysis.">Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis.</a> BMJ. 370, 2516 (2020).</li><li>Bioluminescent Reporters | Reporter Gene Applications | An Introduction to Reporter Genes. Promega Available from: <a target="_blank" href="https://www.promega.ca/resources/guides/cell-biology/bioluminescent-reporters/#references-6d127eb8-eeae-40b7-86e9-fe300545e8fa">https://www.promega.ca/resources/guides/cell-biology/bioluminescent-reporters/#references-6d127eb8-eeae-40b7-86e9-fe300545e8fa</a> (2021)</li><li>Fleiss, A., Sarkisyan, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+bioluminescent+systems.">A brief review of bioluminescent systems.</a> Current Genetics. 65 (4), 877-882 (2019).</li><li>Nouri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinome-wide+screen+using+a+NanoLuc+LATS+luminescent+biosensor+identifies+ALK+as+a+novel+regulator+of+the+Hippo+pathway+in+tumorigenesis+and+immune+evasion.">A kinome-wide screen using a NanoLuc LATS luminescent biosensor identifies ALK as a novel regulator of the Hippo pathway in tumorigenesis and immune evasion.</a> The FASEB Journal. 33 (11), 12487-12499 (2019).</li><li>Boute, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+Luciferase+-+a+multifunctional+tool+for+high+throughput+antibody+screening.">NanoLuc Luciferase - a multifunctional tool for high throughput antibody screening.</a> Frontiers in Pharmacology. 7, 27 (2016).</li><li>Nouri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+celastrol+as+a+novel+YAP-TEAD+inhibitor+for+cancer+therapy+by+high+throughput+screening+with+ultrasensitive+YAP/TAZ-TEAD+biosensors.">Identification of celastrol as a novel YAP-TEAD inhibitor for cancer therapy by high throughput screening with ultrasensitive YAP/TAZ-TEAD biosensors.</a> Cancers. 11 (10), 1596 (2019).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+S1+NanoBiT:+A+nanoluciferase+complementation-based+biosensor+to+rapidly+probe+SARS-CoV-2+receptor+recognition.">SARS-CoV-2 S1 NanoBiT: A nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.</a> Biosensors and Bioelectronics. 180, 113122 (2021).</li><li>Brown, E. E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+critical+determinants+of+ACE2-SARS+CoV-2+RBD+interaction.">Characterization of critical determinants of ACE2-SARS CoV-2 RBD interaction.</a> International Journal of Molecular Sciences. 22 (5), 2268 (2021).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gain-of-functional+screen+identifies+the+Hippo+pathway+as+a+central+mediator+of+receptor+tyrosine+kinases+during+tumorigenesis.">A gain-of-functional screen identifies the Hippo pathway as a central mediator of receptor tyrosine kinases during tumorigenesis.</a> Oncogene. 39 (2), 334-355 (2020).</li><li>Schwinn, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Mediated+tagging+of+endogenous+proteins+with+a+luminescent+peptide.">CRISPR-Mediated tagging of endogenous proteins with a luminescent peptide.</a> ACS Chemical Biology. 13 (2), 467-474 (2018).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+NanoBiT-based+serological+assay+detects+SARS-CoV-2+seroconversion.">A high-throughput NanoBiT-based serological assay detects SARS-CoV-2 seroconversion.</a> Nanomaterials. 11 (3), 807 (2021).</li></ol>

The increasing number of people infected with the SARS-CoV-2 and the ongoing effort for global vaccination necessitates sensitive and fast serologic tests that can be used in large-scale serosurveys. Recent research shows that split nanoluciferase-based bioreporters can be used to develop such assays. We recently developed the HiBiT-RBD bioreporter to design a test that can be used to detect SARS-CoV-2-specific antibodies in patient serum in a fast and reliable fashion (Figure 4C).
<p cl...

The recent emergence of a new coronavirus, SARS-CoV21, has caused more than 2,800,000 fatalities and 128 million infections as of March 30th, 20212. Due to the lack of a reliable and well-established treatment procedure for SARS-CoV-2 clinical therapies, many endeavors have been made to restrict further viral transmission and more importantly, to develop an effective and robust treatment or a vaccine3. To date, there are more than 50 COVID-19 vaccine candidates in trials reported by the World Health Organization4. Detection of antibodies against SARS-CoV-2 is of paramount importance to determine the long-term stability of humoral response upon administration of the vaccine as well as in recovered patients of COVID-195. Some studies have demonstrated that there is a possibility that recovered SARS-CoV-2 patients lose most of the RBD-binding antibodies after 1 year5,6,7,8,9. Further investigation is required to better understand lasting immunity, and more sensitive antibody detection platforms can help further such work. Reports of sustained immunity of mild SARS-CoV-2 infections, which suggest long-term antibody responses, is also an interesting and worthwhile area of study. A fast and accurate method of detection is essential for monitoring antibodies in individuals&#39; sera to provide more information about immunity in the population.
Like other coronaviruses, SARS-CoV-2 uses protruding spike glycoprotein to bind to angiotensin-converting enzyme-2 (ACE2) to initiate a cascade of events that lead to the fusion of the viral and cell membranes6,7. Several studies have recently proved the RBD of the Spike protein to have a crucial role in eliciting powerful and specific antibody response against SARS-CoV28,9,10,11. In particular, correlations observed by Premkumar et al. between the titer of RBD-binding antibody and SARS-CoV-2 neutralization potency of patients&#39; plasma are consistent with RBD being an immunogenic compartment of the virus structure9. With that in mind, many diagnostic tests available for SARS-CoV-2 antibody detection are time and cost-intensive, require a lengthy procedure of incubation and washing (enzyme-linked immunosorbent assay [ELISA]), or lack sensitivity and accuracy (lateral flow immunoassay [LFIA])12. Therefore, a quantitative and rapid complementary serological method of COVID-19-derived antibody detection with high sensitivity, fast response, and relatively low cost would serve the need for a reliable serologic test for SARS-CoV-2 epidemiologic surveillance.
Collectively, the limitations of current serological assays prompted the investigation of the bioluminescent reporting system as a potential diagnostic agent in future serosurveys. Bioluminescence is a naturally occurring enzyme/substrate reaction, with light emission. Nanoluc luciferase is the smallest (19 kDa), yet the brightest system compared to Renilla and firefly luciferase (36 kDa and 61 kDa, respectively)13,14. Further, Nanoluc has the highest signal to noise ratio and stability among the previously mentioned systems. The high signal intensity of Nanoluc supports the detection of even very low amounts of reporter fusions15. Nanoluc Binary Technology (NanoBiT) is a split version of the Nanoluc system, which is comprised of two segments: small BiT (11 amino acids; SmBiT) and large BiT (LgBiT) with relatively low-affinity interactions (KD = 190 &#181;M ) to form a luminescent complex16. NanoBiT is extensively used in various studies involving the identification of protein-protein interactions15,17,18,19 and cellular signaling pathways11,20,21.
Recently, another small peptide with a distinctly higher affinity to LgBiT (KD = 0.7 nM ) was introduced, namely the HiBiT Nano-Glo system, in place of SmBiT. The high affinity and strong signal of the Nano-Glo &#34;add-mix-read&#34; assay makes HiBiT a suitable, quantitative, luminescent peptide tag. In this approach, the HiBiT tag is appended to the target protein by developing a construct imposing minimal structural interference. HiBiT-protein fusion would actively bind to the LgBiT counterpart, producing a highly active luciferase enzyme to generate detectable bioluminescence in the presence of detection reagents (Figure 1). Similarly, we developed a HiBiT Nano-Glo-based system to readily measure the neutralizing antibody titer in the sera of SARS-CoV-2 recovered individuals and recently developed a HiBiT-tagged SARS-CoV-2 RBD. This paper describes the protocol for producing the HiBiT-RBD bioreporter using standard laboratory procedures and equipment, and shows how this bioreporter can be used in a fast and efficient assay to detect SARS-CoV-2 RBD-targeting antibodies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5x Passive Lysis Buffer</td>
			<td>Promega</td>
			<td>E194A</td>
			<td>30 mL</td>
		</tr>
		<tr>
			<td>Bio-Plex Handheld Magnetic Washer</td>
			<td>Bio-Rad</td>
			<td>171020100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Glo luciferase Assay System</td>
			<td>Promega</td>
			<td>E2940</td>
			<td>100 mL kit</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr>
			<td>HiBiT-RBD Plasmid</td>
			<td></td>
			<td></td>
			<td>gacggatcgggagatctcccgatcccctatggt
				gcactctcagtacaatctgctctgatgccgcata
				gttaagccagtatctgctccctgcttgtgtgttgg
				aggtcgctgagtagtgcgcgagcaaaattta
				agctacaacaaggcaaggcttgaccgacaa
				ttgcatgaagaatctgcttagggttaggcgttttg
				cgctgcttcgcgatgtacgggccagatatacgc
				gttgacattgattattgactagttattaatagt
				aatcaattacggggtcattagttcatagcccat
				atatggagttccgcgttacataacttacggtaa
				atggcccgcctggctgaccgcccaacgaccc
				ccgcccattgacgtcaataatgacgtatgttccc
				atagtaacgccaatagggactttccattgacgtc
				aatgggtggagtatttacggtaaactgcccact
				tggcagtacatcaagtgtatcatatgccaagta
				cgccccctattgacgtcaatgacggtaaatgg
				cccgcctggcattatgcccagtacatgaccttat
				gggactttcctacttggcagtacatctacgtat
				tagtcatcgctattaccatggtgatgcggtttt
				ggcagtacatcaatgggcgtggatagcggtttg
				actcacggggatttccaagtctccaccccattg
				acgtcaatgggagtttgttttggcaccaaaatc
				aacgggactttccaaaatgtcgtaacaactccg
				ccccattgacgcaaatgggcggtaggcgtgta
				cggtgggaggtctatataagcagagctctctgg
				ctaactagagaacccactgcttactggcttatcg
				aaattaatacgactcactatagggagacccaa
				gctggctagcgtttaaacttaagcttggtaccga
				gctcggatccgccaccATGGAGACAGA
				CACACTCCTGCTATGGGTACTGC
				TGCTCTGGGTTCCAGGTTCCAC
				TGGTGACtctggctctagcggctctggctct
				agcggcggcATGGTGAGCGGCTG
				GCGGCTGTTCAAGAAGATTAGC
				tctagcggcGACTACAAGGACC
				ACGACGGTGACTACAAGGACCA
				CGACATCGACTACAAGGACGAC
				GACGACAAGggcagcggctccggca
				gcagcggaggaggaggctctggaggagga
				ggctctagcggcggcaacatcacaaatctgtg
				cccattcggcgaggtgtttaacgccaccagat
				ttgccagcgtgtatgcctggaaccggaagaga
				atctctaattgcgtggccgactatagcgtgct
				gtacaatagcgcctccttctctacctttaagt
				gctatggcgtgtcccccacaaagctgaacgac
				ctgtgcttcaccaacgtgtacgccgactcttttgt
				gatcaggggcgatgaggtgcgccagatcgc
				acctggacagacaggcaagatcgccgactac
				aactataagctgccagacgatttcaccggct
				gcgtgatcgcctggaatagcaacaatctggatt
				ccaaagtgggcggcaactacaattatctgtac
				cggctgttcagaaagagcaacctgaagccctt
				tgagcgggatatcagcacagagatctaccag
				gcaggctccaccccttgcaacggagtggagg
				gcttcaattgttattttcccctgcagagctacggc
				ttccagcctacaaatggcgtgggctatcagcca
				tacagggtggtggtgctgtcctttgagctgctg
				cacgcacctgcaaccgtgtcctctggacacatc
				gagggccgccacatgctggagatgggccatc
				atcaccatcatcaccaccaccaccactgatag
				cggccgctcgagtctagagggcccgtttaaac
				ccgctgatcagcctcgactgtgccttctagtt
				gccagccatctgttgtttgcccctcccccgtg
				ccttccttgaccctggaaggtgccactcccac
				tgtcctttcctaataaaatgaggaaattgcat
				cgcattgtctgagtaggtgtcattctattctgggg
				ggtggggtggggcaggacagcaaggggga
				ggattgggaagacaatagcaggcatgctggg
				gatgcggtgggctctatggcttctgaggcggaa
				agaaccagctggggctctagggggtatcccca
				cgcgccctgtagcggcgcattaagcgcggcg
				ggtgtggtggttacgcgcagcgtgaccgctac
				acttgccagcgccctagcgcccgctcctttcg
				ctttcttcccttcctttctcgccacgttcgccggctt
				tccccgtcaagctctaaatcgggggctcccttta
				gggttccgatttagtgctttacggcacctcgacc
				ccaaaaaacttgattagggtgatggttcacgta
				gtgggccatcgccctgatagacggtttttcgcc
				ctttgacgttggagtccacgttctttaatagtg
				gactcttgttccaaactggaacaacactcaacc
				ctatctcggtctattcttttgatttataagggatttt
				gccgatttcggcctattggttaaaaaatgagctg
				atttaacaaaaatttaacgcgaattaattctgt
				ggaatgtgtgtcagttagggtgtggaaagtccc
				caggctccccagcaggcagaagtatgcaaag
				catgcatctcaattagtcagcaaccaggtgtgg
				aaagtccccaggctccccagcaggcagaagt
				atgcaaagcatgcatctcaattagtcagcaac
				catagtcccgcccctaactccgcccatcccgc
				ccctaactccgcccagttccgcccattctccgcc
				ccatggctgactaattttttttatttatgcagaggc
				cgaggccgcctctgcctctgagctattccagaa
				gtagtgaggaggcttttttggaggcctaggcttttg
				caaaaagctcccgggagcttgtatatccattttc
				ggatctgatcaagagacaggatgaggatcgttt
				cgcatgattgaacaagatggattgcacgcagg
				ttctccggccgcttgggtggagaggctattcggc
				tatgactgggcacaacagacaatcggctgctct
				gatgccgccgtgttccggctgtcagcgcagggg
				cgcccggttctttttgtcaagaccgacctgtccgg
				tgccctgaatgaactgcaggacgaggcagcg
				cggctatcgtggctggccacgacgggcgttcct
				tgcgcagctgtgctcgacgttgtcactgaagcg
				ggaagggactggctgctattgggcgaagtgcc
				ggggcaggatctcctgtcatctcaccttgctcctg
				ccgagaaagtatccatcatggctgatgcaatg
				cggcggctgcatacgcttgatccggctacctgc
				ccattcgaccaccaagcgaaacatcgcatcg
				agcgagcacgtactcggatggaagccggtct
				tgtcgatcaggatgatctggacgaagagcat
				caggggctcgcgccagccgaactgttcgcca
				ggctcaaggcgcgcatgcccgacggcgagg
				atctcgtcgtgacccatggcgatgcctgcttg
				ccgaatatcatggtggaaaatggccgctttt
				ctggattcatcgactgtggccggctgggtgt
				ggcggaccgctatcaggacatagcgttggct
				acccgtgatattgctgaagagcttggcggcg
				aatgggctgaccgcttcctcgtgctttacgg
				tatcgccgctcccgattcgcagcgcatcgcc
				ttctatcgccttcttgacgagttcttctgagcg
				ggactctggggttcgaaatgaccgaccaag
				cgacgcccaacctgccatcacgagatttcgat
				tccaccgccgccttctatgaaaggttgggctt
				cggaatcgttttccgggacgccggctggatga
				tcctccagcgcggggatctcatgctggagt
				tcttcgcccaccccaacttgtttattgcagctta
				taatggttacaaataaagcaatagcatcacaa
				atttcacaaataaagcatttttttcactgcatt
				ctagttgtggtttgtccaaactcatcaatgtat
				cttatcatgtctgtataccgtcgacctctagct
				agagcttggcgtaatcatggtcatagctgtttc
				ctgtgtgaaattgttatccgctcacaattccacac
				aacatacgagccggaagcataaagtgtaaag
				cctggggtgcctaatgagtgagctaactcacat
				taattgcgttgcgctcactgcccgctttccagtc
				gggaaacctgtcgtgccagctgcattaatgaa
				tcggccaacgcgcggggagaggcggtttgcg
				tattgggcgctcttccgcttcctcgctcactgactc
				gctgcgctcggtcgttcggctgcggcgagcggt
				atcagctcactcaaaggcggtaatacggttatc
				cacagaatcaggggataacgcaggaaagaa
				catgtgagcaaaaggccagcaaaaggccag
				gaaccgtaaaaaggccgcgttgctggcgtttt
				tccataggctccgcccccctgacgagcatcac
				aaaaatcgacgctcaagtcagaggtggcgaa
				acccgacaggactataaagataccaggcgtt
				tccccctggaagctccctcgtgcgctctcctgtt
				ccgaccctgccgcttaccggatacctgtccgcc
				tttctcccttcgggaagcgtggcgctttctcat
				agctcacgctgtaggtatctcagttcggtgtag
				gtcgttcgctccaagctgggctgtgtgcacgaa
				ccccccgttcagcccgaccgctgcgccttatcc
				ggtaactatcgtcttgagtccaacccggtaag
				acacgacttatcgccactggcagcagccactg
				gtaacaggattagcagagcgaggtatgtaggc
				ggtgctacagagttcttgaagtggtggcctaact
				acggctacactagaagaacagtatttggtatc
				tgcgctctgctgaagccagttaccttcggaaa
				aagagttggtagctcttgatccggcaaacaaa
				ccaccgctggtagcggtggtttttttgtttgca
				agcagcagattacgcgcagaaaaaaaggat
				ctcaagaagatcctttgatcttttctacggggt
				ctgacgctcagtggaacgaaaactcacgttaa
				gggattttggtcatgagattatcaaaaaggatct
				tcacctagatccttttaaattaaaaatgaagtt
				ttaaatcaatctaaagtatatatgagtaaactt
				ggtctgacagttaccaatgcttaatcagtgagg
				cacctatctcagcgatctgtctatttcgttcatcca
				tagttgcctgactccccgtcgtgtagataactac
				gatacgggagggcttaccatctggccccagtg
				ctgcaatgataccgcgagacccacgctcacc
				ggctccagatttatcagcaataaaccagccag
				ccggaagggccgagcgcagaagtggtcctg
				caactttatccgcctccatccagtctattaattgtt
				gccgggaagctagagtaagtagttcgccagtt
				aatagtttgcgcaacgttgttgccattgctacag
				gcatcgtggtgtcacgctcgtcgtttggtatgg
				cttcattcagctccggttcccaacgatcaaggc
				gagttacatgatcccccatgttgtgcaaaaaag
				cggttagctccttcggtcctccgatcgttgtca
				gaagtaagttggccgcagtgttatcactcatggt
				tatggcagcactgcataattctcttactgtcatg
				ccatccgtaagatgcttttctgtgactggtgagta
				ctcaaccaagtcattctgagaatagtgtatgcg
				gcgaccgagttgctcttgcccggcgtcaatacg
				ggataataccgcgccacatagcagaactttaa
				aagtgctcatcattggaaaacgttcttcggggc
				gaaaactctcaaggatcttaccgctgttgagat
				ccagttcgatgtaacccactcgtgcacccaact
				gatcttcagcatcttttactttcaccagcgtttc
				tgggtgagcaaaaacaggaaggcaaaatgc
				cgcaaaaaagggaataagggcgacacgga
				aatgttgaatactcatactcttcctttttcaat
				attattgaagcatttatcagggttattgtc
				tcatgagcggatacatatttgaatgtattt
				agaaaaataaacaaataggggttccgcgca
				catttccccgaaaagtgccacctgacgtc</td>
		</tr>
		<tr>
			<td>LgBiT</td>
			<td>Promega</td>
			<td>N3030</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protein G Magnetic Beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>88848</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet In Vitro DNA Transfection Reagent</td>
			<td>Signagen</td>
			<td>SL100688.5</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab</td>
			<td>SinoBiological</td>
			<td>40592-MM57</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy Mx Microplate Reader</td>
			<td>BioTek</td>
			<td></td>
			<td>96-well plate reader luminometer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>2520056</td>
			<td>0.25%</td>
		</tr>
	</tbody>
</table>

detection of sars-cov-2 receptor-binding domain antibody using a hibit-based bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The signals from both the HiBit-RBD-containing cell lysate and supernatant of the transfected cells were recorded (Figure 2) to evaluate the appropriate protein source. HiBiT-RBD and LgBit were separately used as controls, and the data showed low background compared to a strong signal when both parts were combined. Hence, HiBiT-RBD interaction with LgBiT is necessary to generate active enzyme for substrate digestion and bioluminescence activity (Figure 1).
<...

Watch this Scientific Journal Video about Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter at JoVE.com

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

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