Name: <meta charset="utf8"></head><body>Forfatter Spotlight: Fremme af antivirale strategier gennem nye immunfangst- og massespektrometriteknikker
Uploaded: 2024-01-12T14:30:00
Duration: 1 min 27 s
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Antiviral Strategies through Novel Immunocapture and Mass Spectrometry Techniques at JoVE.com

<meta charset="utf8"></head><body>Analyse af SARS-CoV-2 ved hjælp af dual-reporter og multiplex-teknologi

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genesis+and+evolution+of+bead-based+multiplexing.">The genesis and evolution of bead-based multiplexing.</a> Methods. 158, 2-11 (2019).</li><li>Bystr&#246;m, S., 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=Affinity+proteomic+profiling+of+plasma+for+proteins+associated+to+area-based+mammographic+breast+density.">Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density.</a> Breast Cancer Research. 20 (1), 14 (2018).</li><li>Rudberg, A. -. S., 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+exposure,+symptoms+and+seroprevalence+in+healthcare+workers+in+Sweden.">SARS-CoV-2 exposure, symptoms and seroprevalence in healthcare workers in Sweden.</a> Nature Communications. 11 (1), 5064 (2020).</li><li>Liu, 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=Multiplex+reverse+transcription+PCR+Luminex+assay+for+detection+and+quantitation+of+viral+agents+of+gastroenteritis.">Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis.</a> Journal of Clinical Virology. 50 (4), 308-313 (2011).</li><li>Gadsby, N. J., Hardie, A., Claas, E. C. J., Templeton, K. 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International Standardization Organization Available from: <a target="_blank" href="https://nhiso.com/wp-content/uploads/2018/05/ISO-10993-5-2009.pdf">https://nhiso.com/wp-content/uploads/2018/05/ISO-10993-5-2009.pdf</a> (2009)</li><li>Poetz, O., 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=Sequential+multiplex+analyte+capturing+for+phosphoprotein+profiling.">Sequential multiplex analyte capturing for phosphoprotein profiling.</a> Molecular &amp; Cellular Proteomics. 9 (11), 2474-2481 (2010).</li><li>Dunbar, S. A., Vander Zee, C. A., Oliver, K. G., Karem, K. L., Jacobson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative,+multiplexed+detection+of+bacterial+pathogens:+DNA+and+protein+applications+of+the+Luminex+LabMAP+system.">Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system.</a> Journal of Microbiological Methods. 53 (2), 245-252 (2003).</li><li>Taniuchi, M., 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=Multiplex+polymerase+chain+reaction+method+to+detect+Cyclospora,+Cystoisospora,+and+Microsporidia+in+stool+samples.">Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples.</a> Diagnostic Microbiology and Infectious Disease. 71 (4), 386-390 (2011).</li><li>Wu, M., 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=High-throughput+Luminex+xMAP+assay+for+simultaneous+detection+of+antibodies+against+rabbit+hemorrhagic+disease+virus,+Sendai+virus,+and+rabbit+rotavirus.">High-throughput Luminex xMAP assay for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus, and rabbit rotavirus.</a> Archives of Virology. 164 (6), 1639-1646 (2019).</li><li>Dias, D., 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=Optimization+and+validation+of+a+multiplexed+Luminex+assay+to+quantify+antibodies+to+neutralizing+epitopes+on+human+papillomaviruses+6,+11,+16,+and+18.">Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18.</a> Clinical and Vaccine Immunology. 12 (8), 959-969 (2005).</li><li>Opalka, D., 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=Simultaneous+quantitation+of+antibodies+to+neutralizing+epitopes+on+virus-like+particles+for+human+papillomavirus+types+6,+11,+16,+and+18+by+a+multiplexes+lumina+assay.">Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexes lumina assay.</a> Clinical and Diagnostic Laboratory Immunology. 10 (1), 108-115 (2003).</li><li>Nolte-'T Hoen, E., Cremer, T., Gallo, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+and+viruses:+Are+they+close+relatives.">Extracellular vesicles and viruses: Are they close relatives.</a> Proceedings of the National Academy of Sciences. 113 (33), 9155-9161 (2016).</li><li>Ohmichi, 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=Quantification+of+brain-derived+extracellular+vesicles+in+plasma+as+a+biomarker+to+diagnose+Parkinson's+and+related+diseases.">Quantification of brain-derived extracellular vesicles in plasma as a biomarker to diagnose Parkinson's and related diseases.</a> Parkinsonism &amp; Related Disorders. 61, 82-87 (2019).</li><li>Ter-Ovanesyan, D., 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=Framework+for+rapid+comparison+of+extracellular+vesicle+isolation+methods.">Framework for rapid comparison of extracellular vesicle isolation methods.</a> Elife. 10, e70725 (2021).</li><li>Gamage, S. S. 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=Microfluidic+affinity+selection+of+active+SARS-CoV-2+virus+particles.">Microfluidic affinity selection of active SARS-CoV-2 virus particles.</a> Science Advances. 8 (39), (2022).</li><li>Renner, T. M., Tang, V. A., Burger, D., Langlois, M. -. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACE2-Biotin</td>
			<td>Acro Biosystems (Newark, DE)</td>
			<td>AC2-H82E6-25 ug</td>
			<td>Conc: 340 &#181;g/mL, LOT#BV35376-203HFI-2128</td>
		</tr>
		<tr>
			<td>Anti-Goat IgG, PE-conjugated</td>
			<td>Jackson ImmunoResearch (West Grove, PA)&#160;</td>
			<td>705-116-147</td>
			<td>Host species: Donkey&#160;</td>
		</tr>
		<tr>
			<td>Anti-Human IgG R-PE&#160;</td>
			<td>Life Technologies/Thermo Fisher (Waltham, MA)</td>
			<td>H10104</td>
			<td>Conc: 0.15 mg/mL, LOT#2079224, Host species: Goat</td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG, PE-conjugated</td>
			<td>Jackson ImmunoResearch (West Grove, PA)&#160;</td>
			<td>115-116-146</td>
			<td>Host species: Goat&#160;</td>
		</tr>
		<tr>
			<td>Anti-Rabbit IgG, PE-conjugated&#160;</td>
			<td>Jackson ImmunoResearch (West Grove, PA)&#160;</td>
			<td>111-116-144</td>
			<td>Host species: Goat&#160;</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>20RUO</td>
			<td>100 mM, pH 10 Conc. 1 mg/mL</td>
		</tr>
		<tr>
			<td>Blocker Casein in PBS</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>37528</td>
			<td>LOT#VD301372</td>
		</tr>
		<tr>
			<td>Blocker reagent for ELISA (BRE)</td>
			<td>Roche (Basel, Switzerland)</td>
			<td>11112589001</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-DYKDDDDK Tag Antibody (Anti-FLAG) 0.2 mg/ml, rat IgG2a, &#955;</td>
			<td>BioLegend (Amsterdam, The Netherlands)</td>
			<td>637321</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Saveen &#38; Werner (Limhamn, Sweden)</td>
			<td>B2000-500</td>
			<td>LOT#04D5865</td>
		</tr>
		<tr>
			<td>EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)</td>
			<td>Proteochem (Hurricane, UT)</td>
			<td>C1100-custom (65 mg)</td>
			<td>LOT# MK3857</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco/Thermo Fisher (Waltham, MA)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-ACE2 polyclonal antibody</td>
			<td>R&#38;D Systems/Bio-Techne (Minneapolis, MN)</td>
			<td>AF933</td>
			<td>Host species: Goat&#160;</td>
		</tr>
		<tr>
			<td>Goat IgG</td>
			<td>Bethyl Labs (Montgomery, TX)</td>
			<td>P50-200</td>
			<td>LOT#P50-200-6</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-bind 1.5 mL microfuge tubes&#160;</td>
			<td>VWR (Radnor, PA)</td>
			<td>525-0133</td>
			<td></td>
		</tr>
		<tr>
			<td>MagPlex-C Microspheres</td>
			<td>Luminex Corporation (Austin, TX)</td>
			<td>MC10XXX-01</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM tissue cuture media</td>
			<td>Gibco/Thermo Fisher (Waltham, MA)</td>
			<td>21430-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 96-Well, Polystyrene, Half-area, Clear</td>
			<td>Greiner Bio-One (Kremsm&#252;nster, Austria)</td>
			<td>675101</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Gibco/Thermo Fisher (Waltham, MA)</td>
			<td>25080-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin&#160;</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>31000</td>
			<td>LOT#UK292857</td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago AB (Uppsala, Sweden)</td>
			<td>09-9400-100</td>
			<td>LOT#272320-01</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco/Thermo Fisher (Waltham, MA)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma-Aldrich (St. Louis, MO)&#160;</td>
			<td>360627</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>Sigma-Aldrich (St. Louis, MO)&#160;</td>
			<td>437190</td>
			<td></td>
		</tr>
		<tr>
			<td>ProClin 300</td>
			<td>Sigma-Aldrich (St. Louis, MO)&#160;</td>
			<td>48915-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG</td>
			<td>Bethyl Labs (Montgomery, TX)</td>
			<td>P120-301</td>
			<td>LOT#12</td>
		</tr>
		<tr>
			<td>scFv-FAb1</td>
			<td>In-house production</td>
			<td></td>
			<td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 0.12 mg/mL.</td>
		</tr>
		<tr>
			<td>scFv-FAb2</td>
			<td>In-house production</td>
			<td></td>
			<td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc batch1: 0.38 mg/mL. Conc batch2: 0.45 mg/mL</td>
		</tr>
		<tr>
			<td>scFv-FAb3</td>
			<td>In-house production</td>
			<td></td>
			<td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 0.34 mg/mL.</td>
		</tr>
		<tr>
			<td>scFv-FAb4</td>
			<td>In-house production</td>
			<td></td>
			<td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc: 2.85 mg/mL.</td>
		</tr>
		<tr>
			<td>scFv-FAb5</td>
			<td>In-house production</td>
			<td></td>
			<td>Human Antibody Therapeutics Unit, Scilifelab, Sweden. Monoclonal scFv. Conc:2.7mg/mL.</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 infectious particles, Swedish isolate</td>
			<td>In-house production&#160;</td>
			<td></td>
			<td>The Public Health Agency of Sweden</td>
		</tr>
		<tr>
			<td>SARS-CoV-2 Spike Antibody (Hu-anti-S1)</td>
			<td>Novus Biologicals (Centennial, CO)</td>
			<td>NBP2-90980&#160;</td>
			<td>Monoclonal antibody. Conc: 1 mg/mL. Host: Human. Clone: CR3022. Isotype: IgG1 Kappa. LOT#T201B06</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic, anhydrous</td>
			<td>Sigma-Aldrich (St. Louis, MO)&#160;</td>
			<td>S3139</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>24510</td>
			<td>LOT# XH321563</td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>Thermo-Fisher Scientific (Waltham, MA)</td>
			<td>BP337-50</td>
			<td>LOT#194435</td>
		</tr>
		<tr>
			<td>Ultraviolet lamp</td>
			<td>Vilber Lourmat GmbH (Eberhardzell, Germany)</td>
			<td>VL-215.G</td>
			<td>Wavelength = 254 nm; 2 &#215; 15-watt bulbs</td>
		</tr>
		<tr>
			<td>Vero E6 cells</td>
			<td>ATCC (Manassus, VA)</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
		<tr>
			<td>xMAP INTELLIFLEX DR-SE (dual-reporter flow instrument)</td>
			<td>Luminex Corporation (Austin, TX)</td>
			<td>INTELLIFLEX-DRSE-RUO</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling of surface protein epitopes on viral particles by multiplex dual-reporter strategy

The most common pathogenic viruses, such as influenza, HIV, human cytomegalovirus, and SARS-CoV strains, are enveloped viruses. Cell infection by enveloped viruses requires the fusion of viral and host cell membranes, resulting in the release of the viral genome into the cytoplasm. Viral RNA will then replicate before being packed into a new viral particle1,2. During these processes, not only viral proteins but also host membrane proteins may be incorporated into the envelope, becoming an integral part of the new viral particle. Host cell membrane proteins incorporated into the virus envelope may facilitate virus entry into a new host cell, exploiting the mechanisms of cell-cell interactions, homing, and immune system escape3,4.
Despite the importance of investigating virus-associated proteins, most of the currently available techniques for virus analysis5 do not support high-throughput and high-multiplex characterization of virus surface antigen. Neither are they capable of detecting individual viral particles or of discriminating between infectious intact virus particles, non-infectious RNA, viral proteins, and virus subpopulations expressing different antigens. Recently, flow cytometry has been modified and adapted into a novel method for the analysis of viral particles, namely, flow virometry. Flow virometry allows the investigation of single viral particles and their surface antigens. However, limitations including low throughput, low multiplex capability, complicated experimental setup and data analysis, and limited detectability of small-sized viral particles remain6,7.
Microsphere-based multiplexed quantification of proteins and nucleic acid is a well-established technology with numerous applications ranging from protein quantification in body fluids, protein-protein interaction studies, and diagnosis of viral infections8,9,10,11,12,13. A recently introduced flow analysis instrument features a dual-reporter channel, allowing the measurement of two fluorescent reporter molecules in the same reaction well. This new capability has shown to be particularly useful for the parallel profiling of different immunoglobulin isotypes14. Here, it is described how the dual reporter system can be used to detect intact viral particles, targeting multiple surface antigens in parallel.
As a proof of concept, this report details the development of a triple-detection system for SARS-CoV-2 virus particles. SARS-CoV-2 consists of four main proteins, one is the spike protein (S), which consists of two subunits. The first subunit, S1, makes the primary binding to the ACE2 expressed in human cell membranes. The second subunit, S2, facilitates entry into the target cell by a fusion peptide, creating a pore in the target cell membrane that the virion can enter through15. The three remaining building blocks of SARS-CoV-2 are the nucleocapsid (N), the membrane protein (M), and the envelope protein (E). The nucleocapsid is responsible for the packaging of the viral genome by forming ribonucleoprotein structures with RNA, while the membrane and envelope proteins play central roles in the virus assembly.
The assay described here targets three independent epitopes of the S1 subunit expressed on the envelope surface of SARS-CoV-2. Serial dilutions of both SARS-CoV-2 infected and uninfected cell supernatants are used. Viral particles are captured via ACE2-conjugated microspheres binding the S1 subunit on the virus. Surface virus S protein is then detected in parallel with a commercialy-available tagged immunoglobulin single-chain variable fragment (scFv) and a human monoclonal anti-S1 antibody (Hu-anti-S1) together with an in-house developed FLAG-tagged scFv. The Hu-anti-S1 is detected by the first channel (RP1) in the dual-reporter system with orange R-phycoerythrin (PE)-conjugated anti-human IgG-Fc secondary antibody, and the scFv is detected by the second channel (RP2) with a blue Brilliant Violet 421 (BV421)-conjugated secondary anti-FLAG antibody. The virus particle assay is represented in Figure 1.

<meta charset="utf8"></head><body>Forfatter Spotlight: Fremme af antivirale strategier gennem nye immunfangst- og massespektrometriteknikker

Profilering af overfladeproteinepitoper på virale partikler ved hjælp af multiplex dual-reporter-strategi

Proteomic analysis of envelope virus revealed that host proteins are incorporated in new formed virus, affecting replication, infectivity, and pathogenesis. Our research focuses on profiling surface proteins in viral particles to find key elements in virus-host interaction, leading to new targets for antivirals and vaccines. Immunoelectron microscopy is the only imaging technique allowing the direct visualization of proteins suppressant virus.However, mass spectrometry immuno capture with magnetic beads and emerging flow biometry are now more commonly used to enable broader proteome profiling and to screen a large number of viral particles. Our method uses color-coded Luminex beads, and dual laser read out for immuno capture. It enables multiplex profiling of host surface proteins on intact virions and screening of antibodies and antiviral drugs on multiple viruses and antigens simultaneously.Mass spectrometry and affinity proteomics are powerful tools to identify both viral and host proteins on the virion surface. The strategy we describe will lead to a better understanding of the role of host proteins on virus particles, enabling high-throughput profiling of infected samples in various experimental setups.

Read this Scientific Article Protocol about Author Spotlight: Advancing Antiviral Strategies through Novel Immunocapture and Mass Spectrometry Techniques at JoVE.com

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

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Research

JoVE Journal

Immunology and Infection

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Conjugation of ACE2 and Control Antibodies to Magnetic Microspheres to Capture Viral Particles from SARS-CoV-2 Infected Cells

To begin, prepare the working solution of NeutrAvidin in MES buffer in a 1.5-milliliter low protein binding microfuge tube. Then prepare the working solution of goat immunoglobulin G control antibody in MES buffer. Vortex the diluted antibody solution, and centrifuge the tube.Next, take the Microtiter plate containing activated beads and place it on a magnetic plate separator for 30 seconds to immobilize the beads. With the Microtiter plate still positioned on the magnetic separator, aspirate the supernatant from magnet immobilized beads. Then add 100 microliters of NeutrAvidin solution, antibody solution, and MES buffer to appropriate wells containing beads.Seal the Microtiter plate and incubate for two hours on an orbital shaker at 650 RPM in the dark at room temperature. Then wash beads with PBST using an automated washer. After overnight incubation, prepare the recombinant human biotinylated ACE2 and biotin working solution in 10 millimolar PBS.Immobilize the microspheres on the magnetic plate separator for 30 seconds, and aspirate the supernatant from magnet immobilized microspheres, as shown earlier. Remove the Microtiter plate from the magnetic separator, and add 50 microliters of 10 millimolar PBS to each well. Then add 100 microliters of the biotinylated ACE2 and biotin working solution to appropriate wells containing NeutrAvidin conjugated microspheres.Seal the Microtiter plate, and incubate for one hour on an orbital shaker, as shown earlier. After washing the microspheres, store the ACE2 and biotin conjugated microspheres.

Konjugering af ACE2 og kontrolantistoffer mod magnetiske mikrosfærer for at fange virale partikler fra SARS-CoV-2-inficerede celler

Detection of SARS-CoV-2 Viral Particles in SARS-CoV-2 Infected Vero E6 Cell Supernatant

Begin by mixing casein, polyvinyl alcohol, polyvinylpyrrolidone and BSA with pH seven to prepare assay buffer. Next, prepare 10%rabbit immunoglobulin G in assay buffer to make the sample dilution buffer. Prepare the volume of the working bead mixture necessary for testing.Then thaw the prepared SARS-CoV-2 and control supernatants at four degrees Celsius for one hour. Label and arrange eight 1.5-milliliter microfuge tubes each for SARS-CoV-2 and control supernatants. To create the highest dilution of each supernatant, combine 600 microliters of the supernatant with the sample dilution buffer in appropriately labeled tubes, followed by briefly vortexing the tube to mix.Sequentially transfer 400 microliters of the one is to one diluted supernatant to the next dilution tube. Briefly vortex each diluted supernatant before proceeding with the next dilution. Then take the pre-prepared working bead mixture after vortexing for 30 seconds and add five microliters to each assigned well of a flat-bottom, 384-well microtiter plate.Add 45 microliters of the prepared supernatant dilution to assigned wells containing microspheres in the 384-well plate. Seal the plate and incubate overnight on an orbital shaker at 650 RPM in the dark at room temperature. After removing the microtiter plate from the orbital shaker, centrifuge the plate at 931g for one minute.Then remove the plate sealer. To immobilize the beads, place the microtiter plate on a magnetic plate separator for 30 seconds. With the microtiter plate still positioned on the magnetic separator, aspirate the supernatant from magnet immobilized beads.After removing the microtiter plate from the magnetic separator, add 60 microliters of PBST to each bead-containing well. Next, take five 1.5-milliliter tubes to prepare different detection mixes. Add human monoclonal anti-S1 antibody and FLAG-tagged, single-chain variable fragments targeting the spike protein on the SARS-CoV-2 particle to each tube.Add 50 microliters per well of the appropriate single-chain, variable fragment-specific, spike-detection mix to the washed microspheres. Seal the microtiter plate and incubate as shown earlier. Then centrifuge the microtiter plate.Wash excess spike detection reagent from beads with 60 microliters of PBST three times. Next, prepare a fluorescent solution consisting of PE-conjugated, anti-human immunoglobulin G together with brilliant violet for 21 conjugated anti-FLAG antibody. Add 50 microliters per well of fluorescent solution mix to the washed microspheres, and incubate as shown earlier.Then spin down the microtiter plate. Wash the excess fluorescent solution mix from the microspheres with 60 microliters of PBST. Suspend the microspheres in 60 microliters of PBST from the last wash step.Then analyze the plate on a dual-reporter flow analysis system with the desired settings. In the dilution of SARS-CoV-2-infected cell supernatants in both reporter channels, a concentration-dependent signal was observed. Three out of the five single-chain variable fragments can detect the virus in dilution as low as one to 18.For the remaining two single-chain variable fragments, the virus is detectable in dilutions as low as one to six.