<meta charset="utf8"></head><body>Análise do SARS-CoV-2 usando a tecnologia Dual-Reporter e Multiplex

<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. -. 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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. 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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. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+viral+particle+counts+measured+by+flow+virometry+provide+insight+into+the+infectivity+and+genome+packaging+efficiency+of+moloney+murine+leukemia+virus.">Intact viral particle counts measured by flow virometry provide insight into the infectivity and genome packaging efficiency of moloney murine leukemia virus.</a> Journal of Virology. 94 (2), e01600-01619 (2020).</li><li>Niraja, 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=A+flow+virometry+process+proposed+for+detection+of+SARS-CoV-2+and+large-scale+screening+of+COVID-19+cases.">A flow virometry process proposed for detection of SARS-CoV-2 and large-scale screening of COVID-19 cases.</a> Future Virology. 15 (8), 525-532 (2020).</li><li>Lipp&#233;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+virometry:+A+powerful+tool+to+functionally+characterize+viruses.">Flow virometry: A powerful tool to functionally characterize viruses.</a> Journal of Virology. 92 (3), e01765 (2018).</li><li>Drobin, K., Nilsson, P., Schwenk, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+antibody+suspension+bead+arrays+for+plasma+protein+profiling.">Highly multiplexed antibody suspension bead arrays for plasma protein profiling.</a> Methods in Molecular Biology. 1023, 137-145 (2013).</li></ol>

<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>Autor em destaque: Avançando estratégias antivirais por meio de novas técnicas de imunocaptura e espectrometria de massa

Perfil de epítopos de proteínas de superfície em partículas virais por estratégia de repórter duplo multiplex

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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JoVE Journal

Immunology and Infection

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

407 visualizações.  KTH Royal Institute of Technology. A análise proteômica do vírus do envelope revelou que as proteínas do hospedeiro são incorporadas ao vírus recém-formado, afetando a replicação, a infectividade e a patogênese. Nossa pesquisa se concentra no perfil de proteínas de superfície em partículas virais para encontrar elementos-chave na interação vírus-hospedeiro, levando a novos alvos para antivirais e vacinas. A microscopia imunoeletrônica é a única técnica de imagem que permite...