We acknowledge at SciLifeLab, Sweden, the Affinity Proteomics-Stockholm Scilifelab Unit team for developing and applying the method described here, the Human Antibody Therapeutics Unit for providing scFvs and Fab reagents, and Jonas Klingstr&#246;m for the VeroE6 cells infected with SARS-CoV-2 isolates stemming from clinical samples. The authors thank Sherry Dunbar, PhD, MBA of Luminex Corporation (Austin, TX), for research support, and Matt Silverman MSci, PhD of Biomedical Publishing Solutions (Panama City, FL; mattsilver@yahoo.com) for scientific and writing assistance. This work was supported by funds from the Knut and Alice Wallenberg Foundation and Science for Life Laboratory (SciLifeLab) (VC2020-0015 to Claudia Fredolini and Francesca Chiodi and VC-2022-0028 to Claudia Fredolini).

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

<ol>
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The authors declare no conflict of interest.

Bead-based multiplex technology has been shown to be a valuable platform for high-throughput pathogen detection in a number of clinical applications. The high flexibility of the platform, based on flow-cytometry principles, allows targeting antibodies, proteins, and nucleic acids18,19,20,21,22, multiplexing hundreds of analytes simultaneously. However, to our knowledge, this technology has not previously been applied to detect intact viral particles. In this report, the technology was applied for the detection of intact viral particles by targeting three independent surface epitopes of SARS-CoV-2.
Enveloped RNA viruses show high structural similarity to extracellular vesicles (EVs), small phospholipid membranes carrying viral RNA and proteins along with host proteins23. Sandwich immunoassays have previously been applied to the detection of EVs, using an antibody pair targeting two distinct surface proteins24,25. The limitation of sandwich assays to simultaneously detect only two proteins is removed with multiplex approaches that allow the simultaneous detection of more than two proteins per reaction.
The three-laser dual-reporter detection system described here is the most advanced bead-based flow analysis instrument to date. With respect to single-reporter readout systems, the dual-reporter (RP1 and RP2 channels) allows the detection of three surface proteins/epitopes in parallel. Targeting multiple viral surface proteins and epitopes provides a more accurate representation of the viral protein load, which, beyond confirming that the virus is in fact, intact, also opens up the opportunity to further investigate viral surface antigens and the mechanisms of the viral and host protein interactions.
During the COVID-19 pandemic, the importance of promptly identifying individuals carrying active viral particles was important in efforts to contain virus spread. Genomic RNA is detected by quantitative RT-PCR regardless of its origin (intact virus particles or free). However, only an intact envelope with accessible S protein can mediate cell entry and subsequent virus replication. Previous studies with microfluidic chips in patient samples have shown how the detection of intact viral particles combined with point-of-care testing would enable frequent testing and enhanced surveillance of disease spread, including a more informed choice of individuals to be quarantined26. The application of a multiplexed microsphere-based assay would allow for the design of assays aimed at the screening of multiple viruses and their surface antigen variants, obtaining a more accurate picture of virus spread in the population.
Flow virometry is a recent development of flow cytometry aiming at the analysis of viral particles. Despite being capable of detecting discrete viral particles, the analysis of small viruses poses a current issue for flow virometry27,28. Similarly to the method described here, flow virometry involves the capture of intact virions by gold nanoparticles coupled to antibodies. Limitations for both methods include (i) the dependence on high-affinity capture and detection reagents for surface-expressed antigen targeted by the microspheres or nanoparticles, (ii) limited capability to discriminate between virus particles and extracellular vesicles, and (iii) lack of standards for proper particle quantification.
Cells secrete EVs into their surroundings, and when infected by a virus, they may also secrete virions that are similarly sized as the EVs and may eventually express the same antigens29. Because the EVs will have similar membrane compositions as the virus, it could be hard to distinguish them from each other using only affinity-based methods such as the dual laser single-reporter approach. However, strategies described here feature a higher multiplex capacity, enabling a broader and deeper investigation of particles&#39; protein composition. Flow-based methods allow tracking of discrete particles, providing opportunities for digital quantification. One strategy to address the quantification issue in our method would be to use well-characterized synthetic vesicles expressing antigens of interest as virus-like particles (VLPs) for preparing standard curves.
A common path of entry and exit of SARS-CoV-2 from the host cells is through the interaction of the virus and host cell membrane2,15. In this process, the probability of host membrane proteins being incorporated into the virus surface is high. By screening incorporated host proteins, one can track the pathway of the infection and potentially predict disease&#160;course for different risk patients, allowing earlier treatment decisions. It also allows for the characterization of the viruses across different sample batches in research laboratories. This can be explored further by testing if different characteristics are related to different levels of viral infectivity and for screening of antibodies and drug molecules that target viral surface proteins.
An important aspect concerning the method described is that it relies on the affinity of the capture and detection reagents against their target proteins on the virus. The choice of affinity reagents is, therefore, a determining factor in assay performance. Possibly, multiple affinity reagents should be screened and tested for capture and detection to select those with the highest affinity. Here, the performance of ten scFvs and twelve Fab fragments was preliminarily assessed using recombinant RBD and on viral particles from the supernatant of SARS-Cov-2 infected Calu-3 lung epithelial cells (VeroE6 cells were used to culture/assess cytotoxicity in all subsequent studies). Anti-FLAG PE was used to detect the FLAG-tagged scFvs (Supplementary Table 1 and Supplementary Table 2). The five best-performing scFvs were then selected to be applied in the dual-reporter assay, together with commercial Hu-anti-S1 (Table 1), on supernatants from infected VeroE6 African green monkey kidney epithelial cells.
Another critical factor for the protocol&#39;s success is the procedure selected for microsphere coupling. The coupling method should be efficient and, at the same time, keep conformational epitopes or amino acid residues involved in the protein binding intact and unmodified. Here, the EDC-NHS reaction was applied to couple neutravidin directly to microspheres, adapting a protocol previously described30 and a neutravidin + biotin system to bind recombinant ACE2 to the coupled microspheres. Alternative coupling methods and their efficiency can be tested and compared. Finally, it was observed that different fluorescently labeled detection reagents (e.g., anti-FLAG PE (phycoerythrin) and anti-FLAG Brilliant Violet 421) may result in different MFI levels that may affect assay sensitivity.
In conclusion, the method described allows the detection of intact viral particles in solution, applying a dual-reporter strategy. The analysis of three surface determinants in parallel provides a more specific tool to characterize viral particles and eventually discriminate them from other EVs (e.g., not containing viral antigens). This strategy is an alternative to flow virometry. Although the current approach does not discriminate particle sizes, magnetic bead strategies using color-coded microspheres offer a broader capability in surface antigen profiling and experimental design by high-multiplex and high-throughput analysis. The assay shows high precision and robustness and can be extended to the analysis of any type of extracellular vesicle and any other type of bioparticle exposing surface antigens in body fluids or other liquid matrices. This was a proof-of-concept study that demonstrated the utility of using scFvs as one detection reagent in a multiplex analysis of multiple protein epitopes on viral particles. Future studies are necessary to determine the specific characteristics of scFvs (e.g., binding affinities, cross-reactivity with other reagents and targets) if they are to be used for quantitative or clinical purposes.

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.

<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

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.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

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

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.

Conjugation test 
The conjugation test showed that goat-IgG and neutravidin-biotinylated ACE2 were successfully conjugated to the microspheres. The assay detection specificity was confirmed by probing ACE2-conjugated microspheres with PE-labeled secondary antibodies generated in different animal species (Figure 2). No cross-reactivity between the different detection antibodies was observed. When the bead mixtures were probed with goat anti-ACE2 + anti-goat IgG PE, a median fluorescence intensity (MFI; arbitrary units) value above the background was detected for both ACE2 and goat IgG-conjugated microspheres but not for the unconjugated microsphere (bare) or for the biotin-coated microspheres. Anti-mouse IgG PE and anti-rabbit IgG PE were used as negative controls to check for false-positive signals. A negligible fluorescence signal was generated upon incubation with the microspheres, indicating that the positive signals for the ACE2 and the goat IgG were specific.
Viral particle detectability in cell supernatants 
Magnetic beads coupled to recombinant human ACE2 were used to capture SARS-CoV-2 viral particles from infected and control (no virus) VeroE6 cell culture supernatants and were then simultaneously probed for two distinct viral spike regions using a monoclonal antibody and one of five distinct scFvs. A concentration-dependent signal in the dilutions of SARS-Cov-2 infected cell supernatants was observed in both reporter channels (RP1 and RP2) (Figure 3), indicating that both the commercial Hu-anti-S1 antibody and the different scFvs detected the viral particle bound to the ACE2-conjugated microsphere. With three out of five scFvs, the virus is detectable in dilutions down to 1:18 (scFv2, scFv3, scFv5); for the remaining two scFvs (scFv7 and scFv9), it is detectable down to 1:6 dilutions. This could be attributable to a different affinity for the target. As shown in Figure 3 and Table 1, scFv3 provides the highest MFI intensity, followed by scFv5, scFv2, scFv7, and scFv9, respectively.
Globally, scFvs detection results in lower MFI in comparison to the Hu-anti-S1. This could indicate lower affinity, but it could also be an artifact due to the labeling with different fluorescent dyes (PE and BV421). Another trend that can be seen for scFv7 and scFv9 is that the MFI values are slightly lower for the RP1 channel (anti-spike) as well compared to the other three configurations. This could indicate that the scFvs are either cross-reacting or interfering in another way with the ACE2-Hu-anti-S1 interaction, which could also explain the lower signal in the RP2 channel. No viral particles were detected in the supernatant of the non-infected Vero E6 cells in either the RP1 or the RP2 channel.
The neutravidin-biotin conjugated microsphere, the goat-IgG microsphere, and the unconjugated microspheres are used as negative control beads. The viral particles were captured with magnetic microspheres coupled to ACE2 and tested with commercial human anti-spike in the RP1 reporter channel and with different scFvs in the RP2 reporter channel (scFv is indicated in the top left of each panel). No virus particles were detected in any of the infected and non-infected samples.
Assay precision and robustness 
To evaluate assay precision, all the conditions were run in triplicate. A coefficient of variance (CV) for the ACE2 microsphere was calculated for each dilution point. All of the calculated CVs for the assay were below 15%, where the highest measured CV was 13%, and the lowest CV was 1% (Table 2). As can be seen in the density plot (Figure 4) of the RP1 channel, PE detection of the commercial Hu-anti-S1 shows higher precision, mainly concentrated around a CV of 3%. The RP2 channel, BV detection of scFVs, shows higher CVs. However, as can be seen in Table 2, the higher range of CVs is driven by the samples with low concentrations of viral particles, such as the blank. To test the robustness of the protocol, the assay was repeated twice by different operators, using bead mixtures generated on different days and a lower sample volume (72% lower). A very good Pearson correlation, ranging between 0.98 and 1, was observed for both the RP1 and the RP2 channels (p-value &#60; 0.01), confirming assay robustness and the possibility of applying the assay when less sample is available (Figure 5). This flow analysis technology follows the &#34;ambient analyte theory&#34;17, making the assay sensitive to concentration but not volume.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66230/66230fig01.jpg" /> 
Figure 1: The virus particle assay. (A) Cell supernatant from both infected and un-infected Vero E6 cells are added in a serial dilution to either a 96-well or 384-well plate, together with magnetic microspheres conjugated with neutravidin, and then coupled to either biotinylated human ACE2 or Biotin. Unconjugated microspheres coupled with goat-IgG and bare microspheres are used as negative controls together with the neutravidin-biotin conjugated microsphere. (B) Microsphere-virus particle complexes that have formed are detected with a detection cocktail consisting of Hu-anti-S1 and one of the different scFvs with FLAG-tag. A fluorescent mix with anti-human IgG PE targeting the Hu-anti-S1 and anti-FLAG Brilliant Violet 421 targeting the scFvs is then added. (C) The three-laser, dual-detection system emits a red, green, and violet laser to detect the microparticle complex. The red laser detects the microsphere dye label, while the green and violet lasers detect the anti-S1 and the scFvs, respectively. The generated data are then analyzed. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66230/66230fig02.jpg" /> 
Figure 2: Conjugation confirmation plot. The bead mixtures consisted of four different microsphere IDs, each conjugated with a different protein: neutravidin-biotin-ACE2 (ACE2), unconjugated microsphere (Bare Bead), neutravidin-biotin (Biotin), and goat-IgG (Goat IgG). In the conjugation test three different configurations of detection fluorophores were used. Namely, goat anti-ACE2 + anti-goat IgG PE, anti-mouse IgG PE, and anti-rabbit IgG PE. The Y-axis shows the average measured MFI (median fluorescence intensity; arbitrary units) signal from each microsphere with the three different conditions. The X-axis shows the different capture antibodies applied. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66230/66230fig03.jpg" /> 
Figure 3: Multiplexed detection of surface proteins. Y-axis: Mean MFI (median fluorescence intensity; arbitrary units &#177; standard deviation) for each sample, analyzed in triplicate wells per condition. X-axis: Serial dilution points of cell supernatant. Orange: Virus particles in supernatant from Vero E6 infected with SARS-CoV-2 WT detected with human anti-spike + anti-human PE (phycoerythrin). Blue: Supernatant from Vero E6 infected with SARS-CoV-2 WT detected with the different scFvs + anti-FLAG Brilliant Violet 421. Grey: Non-infected cell supernatant detected with human anti-spike + anti-human PE. Black: Non-infected cell supernatant detected with the five different scFvs + anti-FLAG Brilliant Violet 421. The viral particles were captured with magnetic microspheres coupled to ACE2 and tested with commercial human anti-spike antibodies in the RP1 reporter channel and with different scFvs in the RP2 reporter channel (scFv is indicated in the top left of each panel). No virus particles were detected in any of the non-infected samples. The epitope targeted by scFv3 had the highest affinity. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66230/66230fig04.jpg" /> 
Figure 4: Variation dispersion plot. The Y-axis is the frequency of events, and the X-axis shows the coefficient of variance (CV) in percentage for each replicate of the different samples. RP1 and RP2 are the first and second reporter channels that detect fluorescence associated with phycoerythrin and Brilliant Violet 421, respectively. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66230/66230fig05.jpg" /> 
Figure 5: Run correlation matrix. (A,B) Y-axis: Pearson correlation matrix in log10-scale between three separate runs, run by three different operators and with different bead mixtures. A lower sample volume was applied in the third run. The histograms show the distribution of the different variable clusters based on measured MFI. (A) Correlation for the RP1 reporter channel between the different runs. (B) Correlation for the RP2 reporter channel between the different runs. MFI=median fluorescence intensity in arbitrary units. ***p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/66230/66230fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Detection</td>
			<td>Reactivity</td>
		</tr>
		<tr>
			<td>scFv2</td>
			<td>++</td>
		</tr>
		<tr>
			<td>scFv3</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>scFv5</td>
			<td>++</td>
		</tr>
		<tr>
			<td>scFv7</td>
			<td>+</td>
		</tr>
		<tr>
			<td>scFv9</td>
			<td>+</td>
		</tr>
		<tr>
			<td>Human anti-Spike IgG</td>
			<td>++++</td>
		</tr>
	</tbody>
</table>
Table 1: Ranking of scFvs in detection based on the MFI intensity obtained in the standard curves. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>RP1 (PE)</td>
			<td>RP2 (BV421)</td>
		</tr>
		<tr>
			<td>Sample Dilution</td>
			<td>CV range [%]</td>
			<td>CV range [%]</td>
		</tr>
		<tr>
			<td>Blank</td>
			<td>3&#8211;11</td>
			<td>2&#8211;13</td>
		</tr>
		<tr>
			<td>1:1458</td>
			<td>1&#8211;7</td>
			<td>2&#8211;7</td>
		</tr>
		<tr>
			<td>1:456</td>
			<td>4&#8211;6</td>
			<td>3&#8211;8</td>
		</tr>
		<tr>
			<td>1:162</td>
			<td>3&#8211;6</td>
			<td>3&#8211;7</td>
		</tr>
		<tr>
			<td>1:54</td>
			<td>2&#8211;4</td>
			<td>2&#8211;4</td>
		</tr>
		<tr>
			<td>1:18</td>
			<td>2&#8211;4</td>
			<td>1&#8211;4</td>
		</tr>
		<tr>
			<td>1:6</td>
			<td>2&#8211;6</td>
			<td>1&#8211;6</td>
		</tr>
		<tr>
			<td>1:2</td>
			<td>1&#8211;5</td>
			<td>1&#8211;3</td>
		</tr>
	</tbody>
</table>
Table 2: CV% (mean/standard deviation &#215; 100) range of each dilution point of the SARS-CoV-2 infected supernatant for both the RP1 and the RP2 reporter channels.
Supplementary File 1: Immunoglobulin single-chain variable fragment (scFv) generation. <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Screening scFvs in pairs with Fabs against serial dilution of recombinant Spike (RBD). To evaluate the performance of different detection peptides, 12 combinations of spike protein, Fab, were used as capture in buffer spiked with recombinant RBD. Ten (10) scFvs targeting different epitopes of the spike protein were applied as detection. Depending on the performance of the capture-detection pair, they were either marked as failed (-) or successful (+). <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Screening scFvs in pairs with Fabs against serial dilution of SARS-Cov-2 infected Calu-3 cell supernatant. For evaluation of different detection peptides performance, 12 combinations of spike protein, Fab, were used as capture in SARS-Cov-2 infected Calu-3 cell supernatant. Ten (10) scFvs targeting different epitopes of the spike protein were applied as detection. Depending on the performance of the capture-detection pair, they were either marked as failed (-) or successful (+). <a href="https://www.jove.com/files/ftp_upload/66230/Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>

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

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.

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

profiling-surface-protein-epitopes-on-viral-particles-multiplex-dual

Research

JoVE Journal

Immunology and Infection

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.

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.

407 Views.  KTH Royal Institute of Technology.  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.

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

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