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

我们感谢何晓红、里卡多·马吕斯、朱莉娅·佩特里克、布拉德利·奥斯汀和克里斯蒂安诺·塔内塞·德索萨的技术援助。我们也感谢Mina Ghahremani的平面设计。我们还要感谢所有参与并为这项研究捐献血液样本的个人。DWC部分由uOttawa Faculty和 Department of Medicine 提供支持。

注意：下面描述的协议符合协议代码20200371-01H的所有道德准则。
1. HiBiT-RBD生物报告仪的生产和评估
<ol><li>生产足够数量的HiBiT-RBD生物报告机<ol>
<li>准备细胞培养<ol>
<li>准备含有10%胎牛血清和1%青霉素/链霉素的完整Dulbecco改良鹰培养基（DMEM）。然后，在37°C水浴中加热培养基。</li>
			<li>打开生物安全柜（BSC），并使用70%v / v乙醇对柜表面进行灭菌。</li>
		</ol>
</li>
		<li>培养细?...

<ol>
	<li>Ullah, H., Ullah, A., Gul, A., Mousavi, T., Khan, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+coronavirus+2019+(COVID-19)+pandemic+outbreak:+A+comprehensive+review+of+the+current+literature.">Novel coronavirus 2019 (COVID-19) pandemic outbreak: A comprehensive review of the current literature.</a> Vacunas. , (2020).</li><li>Coronavirus update (Live). 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>

感染SARS-CoV-2的人数不断增加，全球疫苗接种工作不断进行，因此需要进行灵敏和快速的血清学检测，这些检测可用于大规模血清调查。最近的研究表明，基于分裂纳米荧光素酶的生物报告人可用于开发此类测定。我们最近开发了HiBiT-RBD生物报告程序，以设计一种可用于以快速可靠的方式检测患者血清中SARS-CoV-2特异性抗体的测试（图4C）。
该测定中有几?...

截至20212年3月30日，最近出现的新型冠状病毒SARS-CoV21已造成超过2，800，000人死亡和1.28亿感染。由于缺乏可靠且完善的SARS-CoV-2临床治疗程序，因此已经做出了许多努力来限制进一步的病毒传播，更重要的是，开发一种有效而强大的治疗方法或疫苗3。迄今为止，世界卫生组织报告的试验中已有50多种COVID-19候选疫苗4。检测针对SARS-CoV-2的抗体对于确定接种疫苗后体液反应的长期稳定性以及COVID-195康复患者至关重要。一些研究表明，恢复的SARS-CoV-2患者有可能在1年后失去大部分RBD结合抗体5，6，7，8，9。需要进一步研究以更好地了解持久的免疫力，更灵敏的抗体检测平台可以帮助进一步开展此类工作。轻度SARS-CoV-2感染持续免疫的报告表明长期抗体反应，也是一个有趣且有价值的研究领域。快速准确的检测方法对于监测个体血清中的抗体以提供更多关于人群中免疫力的信息至关重要。
与其他冠状病毒一样，SARS-CoV-2使用突出的刺突糖蛋白与血管紧张素转换酶-2（ACE2）结合，引发一系列事件，导致病毒和细胞膜融合6，7。最近有几项研究证明，Spike蛋白的RBD在引发针对SARS-CoV28，9，10，11的强大和特异性抗体反应方面起着至关重要的作用。特别是，Premkumar等人观察到的RBD结合抗体滴度与患者血浆的SARS-CoV-2中和效力之间的相关性与RBD是病毒结构的免疫原性区室9一致。考虑到这一点，许多可用于SARS-CoV-2抗体检测的诊断测试都是时间和成本密集型的，需要漫长的孵育和洗涤过程（酶联免疫吸附测定[ELISA]），或缺乏敏感性和准确性（侧流免疫测定[LFIA]）12。因此，一种具有高灵敏度、快速反应和相对低成本的COVID-19衍生抗体检测的定量和快速互补血清学方法，将满足对SARS-CoV-2流行病学监测的可靠血清学检测的需求。
总的来说，当前血清学检测的局限性促使人们研究生物发光报告系统作为未来血清测量中的潜在诊断剂。生物发光是一种天然存在的酶/底物反应，具有光发射。Nanoluc萤光素酶是最小的（19 kDa），但与 Renilla 和萤火虫荧光素酶（分别为36 kDa和61 kDa）相比是最亮的系统13，14。此外，Nanoluc在前面提到的系统中具有最高的信噪比和稳定性。Nanoluc的高信号强度支持检测非常低量的报告聚变15。Nanoluc二进制技术（NanoBiT）是Nanoluc系统的分裂版本，它由两个部分组成：小BiT（11个氨基酸;SmBiT）和具有相对低亲和力相互作用（KD = 190μM）的大型BiT（LgBiT）形成发光配合物16。NanoBiT广泛用于涉及鉴定蛋白质 - 蛋白质相互作用的各种研究15，17，18，19 和细胞信号通路11，20，21。
最近，引入了另一种对LgBiT具有明显更高亲和力的小肽（KD = 0.7 nM），即HiBiT Nano-Glo系统，以代替SmBiT。Nano-Glo"添加-混合-读取"测定的高亲和力和强信号使HiBiT成为一种合适的定量发光肽标签。在这种方法中，HiBiT标签通过开发一种施加最小结构干扰的构建体附加到靶蛋白上。HiBiT蛋白融合将主动与LgBiT对应物结合，产生高活性荧光素酶，在检测试剂存在下产生可检测的生物发光（图1）。同样，我们开发了一种基于HiBiT Nano-Glo的系统，可以很容易地测量SARS-CoV-2回收个体血清中的中和抗体滴度，最近还开发了一种HiBiT标记的SARS-CoV-2 RBD。本文描述了使用标准实验室程序和设备生产HiBiT-RBD生物报告仪的方案，并展示了该生物报告书如何用于快速有效的检测中以检测SARS-CoV-2 RBD靶向抗体。

<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

COVID-19大流行的出现增加了对更好的血清学检测方法的需求，以确定严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）的流行病学影响。越来越多的SARS-CoV-2感染增加了对更好的抗体检测检测的需求。目前的抗体检测方法在速度方面会损害灵敏度，或者灵敏度高但耗时。很大比例的SARS-CoV-2中和抗体靶向受体结合域（RBD），这是SARS-CoV-2的主要免疫原性区室之一。我们最近设计并开发了一种高度灵敏的生物发光标记RBD（NanoLuc HiBiT-RBD）来检测SARS-CoV-2抗体。以下文本描述了产生HiBiT-RBD复合物的过程以及使用此工具评估RBD靶向抗体存在的快速测定。由于HiBiT-RBD蛋白产品在很宽的温度范围内具有耐用性，并且实验过程更短，可以在1小时内完成，因此可以考虑将该方案视为检测患者血清样品中SARS-CoV-2抗体的更有效的替代方案。

记录来自含有HiBit-RBD的细胞裂解物和转染细胞上清液的信号（图2）以评估适当的蛋白质来源。HiBiT-RBD和LgBit分别用作对照，当两个部分组合时，数据显示与强信号相比，背景较低。因此，HiBiT-RBD与LgBiT的相互作用对于产生用于底物消化和生物发光活性的活性酶是必要的（图1）。
添加蛋白G将有助于抗体沉淀（图3</stro...

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

概述的方案描述了生产HiBiT受体结合结构域蛋白复合物的程序及其在快速灵敏地检测SARS-CoV-2抗体中的应用。

使用基于HiBiT的生物报告器检测SARS-CoV-2受体结合域抗体

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

我的实验室一直从事COVID-19研究，我们最近开发了一种新型检测方法，使我们能够快速准确地检测SARS-CoV-2抗体。该协议概述了用于检测患者血清样本中抗SARS-CoV-2抗体的高通量测定方法，这在当前的大流行中至关重要。目前的大多数血清学检测会牺牲速度来提高灵敏度，反之亦然。我们的数据显示，我们的检测方法既灵敏又具有快速的响应时间。为了生产足够数量的高HiBiT-RBD生物报告书，首先准备细胞培养。首先，制备含有10%胎牛血清和1%青霉素和链霉素的完整Dulbecco改良鹰培养基。然后在37摄氏度的水浴中加热介质。打开生物安全柜，使用70%乙醇对柜表面进行灭菌。要培养细胞系，请将其从零下80摄氏度或液氮中取出，然后在37摄氏度的水浴中解冻。将解冻的细胞与至少10毫升的完整培养基混合。将细胞悬浮液移入15厘米培养皿中，并旋转板以均匀分布细胞。将培养皿置于37摄氏度，5%二氧化碳和85至95%湿度的细胞培养箱中。在显微镜下观察细胞，直到汇合水平达到80%至90%在高汇合度下，取出培养基，用温PBS洗涤细胞，并加入3至5毫升0.25%胰蛋白酶-EDTA以将细胞从表面分离。大约五分钟后，在显微镜下观察细胞。如果所有细胞都是漂浮的，则加入至少四毫升培养基，并将细胞悬浮液转移到新的无菌管中。使用血细胞计数器计数细胞，并将100万个细胞加入六孔板的每个孔中进行转染。要进行HiBiT-RBD质粒的转染，请使用一微克HiBiT-RBD表达质粒和合适的转染试剂。在室温下孵育10至15分钟，然后将总体积滴入板的每个孔中。第二天，用完整的培养基替换含有转染混合物的培养基。转染后48小时观察转染对照。将上清液收集在1.5毫升微管中。向细胞中加入500微升1X钝化裂解缓冲液或PLB，然后在室温下孵育并摇动板15分钟以进行细胞裂解。接下来，要使用荧光素酶测定法评估来自生物报告者的发光信号，首先制备反应组分并使用上清液作为生物报告者的来源。使用前将大的BiT和底物稀释至1X。将50微升上清液从每个孔或管转移到96孔板中，并向每个孔中加入50微升1X大BiT。在室温下孵育五分钟。打开光度计软件，向每个孔添加50微升的1X基板，将板放入发光计中并运行软件。为了进行HiBiT-RBD抗体检测测定，如前所述制备HiBiT-RBD生物报告器，并将50微升含有HiBiT-RBD的上清液与1微克商用SARS-CoV-2 RBD抗体在1.5毫升微管中混合。向溶液中加入20微升免疫球蛋白结合蛋白或蛋白G。通过添加 PBS 将总体积增加到 300 微升。将管子在管式振动器或旋转器上孵育30分钟。在12， 000 G下离心30秒。然后丢弃上清液并用PBS洗涤。重复该过程三次以删除免费的HiBiT-RBD。将细胞重悬于50微升PBS中，并将其转移到96孔板中。加入50微升1X大BiT，等待5分钟。然后加入50微升1X NanoLuc底物。立即使用发光计读取发光信号。为高通量测定制备大量HiBiT-RBD生物报告仪，并在每个样品的96孔板的每个孔中将20微升磁性蛋白G与50微升HiBiT-RBD上清液混合。向每个孔中加入10微升的血清样品，并通过添加PBS使总体积达到150微升。在室温下在摇摇杯上孵育30分钟。然后将板放在磁性垫圈上以沉淀蛋白G抗体复合物。将溶液丢弃在每个孔的中间部分，并加入PBS进行洗涤。重复洗涤步骤至少三次，以除去多余的HiBiT-RBD。加入50微升1X PBS和50微升1X大BiT，并在室温下孵育至少5分钟。准备发光计软件。然后加入50微升1X底物。将印版放入机器中，运行软件并记录信号。比较来自血清样品、对照样品和来自空孔的背景信号。记录来自含有细胞裂解物的HiBiT-RBD和转染细胞上清液的信号，以评估适当的蛋白质来源。数据显示，与两个部分组合在一起时的强信号相比，背景较低。添加蛋白G有助于抗体沉淀。该测定用于将来自商业SARS-CoV-2中和抗体的信号与对照IgG进行比较。特异性抗体信号信号强劲，而对照抗体接近发光背景水平。重组减毒溶瘤性水疱性口炎病毒或VSV在表达外源性RBD的M蛋白位置51处发生突变，用于为小鼠接种疫苗。从接种疫苗的小鼠收集的血清与注射对照VSV的小鼠相比产生了强大的信号。该协议可用于检测患者血清样本中的SARS-CoV-2抗体。与蛋白质G孵育后的洗涤步骤对于减少背景至关重要。此外，尽快添加基板以避免信号丢失也很重要。通过一些额外的优化步骤，相同的测定可用于分析血液样本，并且可能是确定患者血液样本中抗SARS-CoV-2抗体的有效方法。

Research

JoVE Journal

Immunology and Infection

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

科研

免疫与感染

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

使用报告基因表达重组SARS-CoV-2对K18 hACE2转基因小鼠的病毒感染进行实时成像和定量

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

利用开源移液机器人的高效SARS-CoV-2定量逆转录酶PCR唾液诊断策略

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Multiplex Immunoassay for Borrelia-Specific IgG and IgM in Serum

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research (Video) | JoVE

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

Quantifying Vaccine-Induced Neutralizing Antibodies in SARS-CoV-2 Variants

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

Author Spotlight: A Pseudotype Virus System for Assessing Omicron Subvariants and Neutralizing Antibodies in SARS-CoV-2 Research

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Studying Host-Virus Interactions with Pseudotyped Viruses (Video) | JoVE

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Smartphone-Integrated Dengue NS1 Paper-Based Diagnostic Device

Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen

Author Spotlight: Development of a Smartphone-Enhanced Paper-Based Device for Rapid Dengue NS1 Detection

便携式纸基免疫测定法与智能手机应用相结合，用于登革热NS1抗原的比色和定量检测

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an ...

Quick and Early Detection of Rabies Antibodies to Evaluate the Immune Response in a Patient

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test (Video) | JoVE

使用狂犬病间接荧光抗体检测狂犬病 IgG 和 IgM 抗体

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...