The author acknowledges Dr. Christian Derntl from the Department for Biotechnology and Microbiology at the TU Wien and the Department of Medicine III, Division of Nephrology and Dialysis at the Medical University of Vienna, especially Dr. Markus Wahrmann for technical and scientific support. Protein expression in mammalian cells was supported by the Department of Biotechnology at the University of Natural Resources and Life Sciences (BOKU) Vienna. The author wants to express her deep acknowledgement to Dr. Nico Dankbar from XanTec bioanalytics in Duesseldorf, Germany, for helpful scientific discussions on performing the SPR binding assays.

For the present study, a CVN-small ubiquitin-like modifier (SUMO) fusion protein has been used in enzyme-linked immunosorbent assays instead of CV-N and is suitable for cell-based assays. Recombinant full-length influenza A virus HA H3 protein is obtained commercially (see Table of Materials) or expressed in mammalian HEK293 cell lines and baculovirus-infected insect cells according to standard protocols12. Wuhan-1 spike protein is expressed in mammalian HEK293 cells. The synthesis of monomannosylated peptide (MM) and dimannosylated peptide (DM) allows the detection of homogeneous ligands to CVN2 and monomannosylated small molecule10.
1. Creating CV-N constructs
<ol>
	<li>For each of the CVN2 variants and CVN2L0 protein (PDB ID 3S3Y), obtain the gene construct with an N-terminal pelB leader sequence and His-tag in pET27b(+) vector from commercial sources (see Table of Materials, Supplementary File 1).</li>
	<li>Obtain CVN2L0 and its variants (V2, V3, V4, and V5; Figure 2A,C) in the background of a CVN2L0 template gene consisting of two distinct DNA sequences for each CV-N repeat.</li>
	<li>Dissolve the lyophilized plasmid DNA in sterile deionized distilled water (ddH2O) to a final concentration of 100 ng/&#181;L.</li>
</ol>
2. Preparation of LB-agar plates with plasmid DNA transformed cells
<ol>
	<li>Prepare culture medium LB-Lennox by dissolving 10 g/L of peptone, 5 g/L of yeast extract, and 5 g/L of NaCl in ddH2O (see Table of Materials), and adjust the pH to 7.4. Perform the transformation into competent E. coli BL21 (DE3) for each variant (V2-V5) by chemical method following a previously published report10.</li>
	<li>Split the solution (900 &#181;L and 100 &#181;L), transfer 100 &#181;L on LB-agar plates (50 &#181;g/mL kanamycin), and gently use a sterile cell spreader. Incubate the agar plates overnight at 37 &#176;C.</li>
</ol>
3. Cloning
<ol>
	<li>Subclone the gene for CV-N into the NdeI and BamHI sites of pET11a (see Table of Materials) for transformation (electroporation) into electrocompetent cells following reference27.</li>
</ol>
4. Site-directed mutagenesis
<ol>
	<li>To generate CVN2L029 and mutant CVN-E41A in the background of a CVN2L0 template gene containing two distinguished DNA sequences for each CV-N repeat27.</li>
	<li>Make mutations using a site-directed mutagenesis kit (see Table of Materials) and specific mutagenic primers 5&#39;-gagaaccgtcaacgtttgcgataacagagttcagg-3&#39; and 5&#39;-cctgaactctgttatcgcaaacgttgacggttctc-3&#39; for running the PCR31.
	<ol>
		<li>Start a series of sample reactions using multiple concentrations of double-stranded DNA (dsDNA) template ranging from 5-50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template). Keep the primer concentration constant. 
		NOTE: The PCR mix and thermal cycling protocol are generally used as described in the instruction manual for the site-directed mutagenesis kit32.</li>
	</ol>
	</li>
	<li>Add the Dpn I restriction enzyme (1 &#181;L, 10 U/&#181;L, see Table of Materials) below the mineral oil overlay. Thoroughly and gently mix reactions, spin down in a table-top microcentrifuge for 1 min, and incubate immediately at 37 &#176;C for 1 h for digesting the parental supercoiled dsDNA.</li>
</ol>
5. Transformation of bacterial cells
<ol>
	<li>Thaw the XL1-Blue supercompetent cells (see Table of Materials) gently on ice. To transform each control and sample reaction, aliquot the supercompetent cells (50 &#181;L) to a prechilled polypropylene round-bottom tube (14 mL).
	<ol>
		<li>Transfer 1 &#181;L of the Dpn I-treated single-stranded DNA (ssDNA) from each control and sample reaction (mutated ssDNA) to separate aliquots of the supercompetent cells, which synthesize the complementary strand. Swirl the transformation reactions carefully to mix and incubate the reactions on ice for 30 min. 
		NOTE: Before transferring the Dpn I-treated DNA to the transformation reaction, it is recommended to remove any remaining mineral oil carefully from the pipette tip. As an optional control, the transformation efficiency of the XL1-Blue supercompetent cells needs to be checked by mixing 0.1 ng/&#181;L of the pUC18 control plasmid (1 &#181;L) with a 50 &#181;L aliquot of the supercompetent cells.</li>
	</ol>
	</li>
	<li>Apply heat pulse to the transformation reactions at 42 &#176;C for 45 s, and then place the reactions on ice for 2 min. 
	NOTE: The applied heat pulse has already been optimized for the mentioned conditions in polypropylene round-bottom tubes (14 mL).</li>
	<li>Add 0.5 mL of NZY+ broth (containing per liter: 10 g of NZ amine (casein hydrolysate), 5 g of yeast extract, 5 g of NaCl, 12.5 mL of 1 M MgCl2, 12.5 mL of 1 M MgSO4, 10 mL of 2 M glucose, pH 7.5, and preheated to 42 &#176;C) and incubate the transformation reactions at 37 &#176;C with shaking at 225-250 rpm for 1 h. Plate the correct volume of each transformation reaction (5 &#181;L from control plasmid transformation; 250 &#181;L from sample transformation) on LB-ampicillin agar plates. 
	NOTE: For the transformation controls and mutagenesis, spread cells on LB-ampicillin agar plates having 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal, 80 &#181;g/mL) and isopropyl-1-thio-&#946;-D-galactopyranoside (IPTG, 20 mM) (see Table of Materials). Inoculate 50 mL of the cell cultures with a single colony of transformed E. coli cells to purify mutated plasmid DNA for analyses. Mutagenesis is confirmed by DNA sequencing at an external facility.</li>
</ol>
6. Expression and protein purification
<ol>
	<li>For a large-scale culture, inoculate a small amount of LB (containing ampicillin) with a single colony from the transformed plate.</li>
	<li>Using the overnight culture, inoculate the expression culture with additives, such as 10 mM of MgCl2, 10 mM of MgSO4, and 20 mM of glucose, diluting the seed culture to 1/100.
	<ol>
		<li>Grow cells with vigorous shaking at 37 &#176;C. Grow cells to an Abs 600 nm between 0.4-0.6 (mid-log phase) before cooling the cells to 20 &#176;C. Induce with 1 mM IPTG and grow overnight.</li>
		<li>Then, harvest the cells by centrifuging at 4,000 x g for 15 min at 4 &#176;C, and discard the supernatant with a pipette.</li>
	</ol>
	</li>
	<li>Resuspend the cell pellet in phosphate-buffered saline (PBS) buffer and re-centrifuge at 4,000 x g for 15 min at 4 &#176;C. Then, discard the supernatant with a pipette. Resuspend the remaining pellet in 10 mL of lysis buffer and incubate the suspension for 1 h at 37 &#176;C. 
	NOTE: Composition of lysis buffer: (50 mM of NaH2PO4, 300 mM of NaCl, 2% Triton-X100, 500 ng/mL of lysozyme, 1 mM of phenylmethylsulfonylfluoride (PMSF), 1 mM of dithiothreitol, 1 mM of MgCl2, pH 8, see Table of Materials).
	<ol>
		<li>Subject the mixture to two freeze-thaw cycles (-80 &#176;C). Separate soluble and insoluble fractions by centrifugation at 4,000 x g for 15 min at 4 &#176;C and analyze them using polyacrylamide gel electrophoresis (PAGE), in particular, sodium dodecyl sulfate (SDS)-PAGE33(Figure 2B,C). 
		NOTE: Cells can be lysed in several ways, such as freeze-thaw, sonication, homogenization, enzymatic lysis, or a combination of these methods. Purification from inclusion bodies is recommended for collecting high protein yields26.</li>
	</ol>
	</li>
	<li>Purify proteins using Ni-NTA column chromatography (see Table of Materials). Load the soluble fraction onto a regenerated Ni-NTA bead column (1 mL/min). Wash the system with TBS buffer (50 mM of Tris, 150 mM of sodium chloride, pH 7.5) before starting a gradient (0-100% 500 mM imidazole in TBS over 60 min) and collecting fractions (1 mL/min). Dialyze the purified proteins for biochemical characterization against 100 mM PBS (Figure 2C,D).</li>
	<li>Alternatively, use His-Select Ni2+ affinity gel (see Table of Materials) in 14 mL tubes to bind and re-suspend his-tagged recombinantly expressed CV-N in buffer solutions with 20 mM imidazole and 250 mM imidazole, respectively. Incubate in batch for at least 30 min. 
	NOTE: Apply these semi-purified proteins to a single-use prepacked column for buffer exchange and cleanup of biological samples, for example, carbohydrates and proteins, which can load 1-1.5 mL eluate from immobilized metal affinity chromatography.</li>
	<li>Transfer the protein solutions to centrifugation tubes with a 10 kDa cut-off filter (see Table of Materials) and concentrate them by centrifuging for 10 min at 4,500 x g and 4 &#176;C. For SPR measurements, exchange the analyte solutions to 10 mM HEPES, 150 mM sodium chloride, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% Tween20, pH 7.4 (HBS-EP(+), see Table of Materials).
	<ol>
		<li>Add this SPR running buffer to a dilution factor of 1:10 and centrifuge four times to the initial volume for 10 min at 4,500 x g and 4 &#176;C.</li>
	</ol>
	</li>
	<li>Determine the protein concentration at 280 nm using a NanoDrop UV-Vis spectrophotometer (see Table of Materials) based on the calculated extinction coefficient (20,440 M-1 cm-1) for the main protein CVN2L0 showing a size of 23,474 Da34. Use PBS (100 mM, pH 7.0) or SPR buffer as a blank, and measure the protein concentration at three dilution steps (1:1, 1:10, and 1:100).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63541/63541fig02.jpg" /> 
Figure 2: CV-N sequences and expression. (A) CVN2 without a linker between each CV-N repeat (101 amino acids each) and four disulfide bridges is expressed in pET11a vector in E. coli. (B) Expressions of two independent colonies for CV-N (monomer) and CVN2 (dimer). (C) Disulfide bond variants are purified and analyzed on SDS-PAGE. A low molecular weight marker (6 &#181;L) is used as a reference. WT = CVN2L0 bearing four disulfide bridges as marked in (A). V2 is a variant with a disulfide bond replacement by polar residues at positions 58 and 73. V3-V5 are variants with two remaining S-S bonds and either polar (C58E-C73R) or non-polar (C58W-C73M) substituting amino acids or a combination of these residue pair substitutions. (D) HPLC chromatograms of purified CVN2L0 are elutated at a flow rate of 1 mL/min with a linear gradient from 5%-65% buffer B in buffer A over 30 min. Buffer A is: 0.1% (v/v) trifluoroacetic acid in ddH2O, buffer B is: 0.08% (v/v) trifluoroacetic acid in acetonitrile. Protein is analyzed on a high-performance silica gel 300-5-C4 (150 x 4.6 mm) column at 214 nm and 280 nm. <a href="https://www.jove.com/files/ftp_upload/63541/63541fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. SPR spectroscopy
<ol>
	<li>Use the Dual Channel SPR system (see Table of Materials) with running buffer HBS-EP(+) and 10 mM glycine HCl pH 1.5-1.6 as the regeneration buffer. Turn on the instrument, degasser, auto-sampler, and pump and wash the entire system with ddH20 for 1 h. Place ready-to-use running buffer in a separate bottle.</li>
	<li>Drop emersion oil onto the detector and mount a glass sensor chip (see Table of Materials) coated with a thin gold film and on the upper side functionalized with carboxymethyldextran hydrogel directly onto the detector below the three-port flow cell. Fix the setting by pulling down the handling. 
	NOTE: C19RBDHC30M 200 nm streptavidin derivatized carboxymethyldextran hydrogel with a medium density of biotinylated severe acute respiratory syndrome coronavirus-2 RBD protein, is a ready-to-use sensorchip with the pre-immobilized ligand.</li>
</ol>
8. SPR binding assay for CV-N binding to HA, S protein, and RBD
<ol>
	<li>Immobilize the proteinaceous ligands to sensor chips following the steps below.
	<ol>
		<li>Open a run table by clicking on Form in the menu bar and Run Table Editor in the integrated SPRAutoLink software (see Table of Materials). Choose and click on BASIC_Immobilization from the list of available run tables and follow the steps of the experimental procedure on the computer screen. The respective Sample Editor used is shown in the right upper corner.</li>
		<li>Click on Sample Set Editor in the Form section to fill out the reagents list for two racks placed in the autosampler for further analyses. Click on Autosampler Direct Control as a &#34;Tool&#34; in the menu bar to bring the racks forward or back home. Choose 4 &#176;C as the operating temperature. 
		NOTE: The SPR software allows for &#34;SPR Instrument Direct Control&#34; and &#34;Pump Direct Control&#34; via selecting the corresponding tools, as well as autosampler-handling, and by clicking on Form; also Run Table Editor, Data-Plot, or Post-Processing can be chosen to perform data analysis. Files are directly saved in the default directory and exported as scrubber.files from the Post-Processing window.</li>
	</ol>
	</li>
	<li>Start the pump to infuse ddH20 by clicking on Tools and Pump Direct Control and record data by clicking on SPR Instrument Direct Control, and each time Start&#160;in the newly appearing windows. Put the coupling reagents (step 8.3) in 300 &#181;L vials, put them into the autosampler racks and start the run table by clicking on Run. 
	NOTE: Chip surfaces are either conditioned with 10 mM glycine buffer pH 9.0 or may have been flushed with 1 M sodium chloride, 0.1 M sodium borate buffer pH 9.0 to condition carboxyl derivatized chip surface for EDC/NHS activation mix30.</li>
	<li>For this simple protein-protein interaction, use the CMD500D chip (see Table of Materials) to generate a micro-refractive index units (&#181;RIU) = 2500 - 3000 flow cell with immobilized HA and &#181;RIU = 400 flow cell with spike protein. At an infuse flow rate of 15 &#181;L/min, inject an aqueous and equal mixture of 0.4 M N-ethyl-N&#39;-(dimethylaminopropyl) carbodiimide hydrochloride (EDC*HCl) and 0.1 M N-hydroxysuccinimide (NHS) by applying the following sequential steps.
	<ol>
		<li>Refill pump refill at 25,000 &#181;L/min, perform baseline adjustment for 30 s, inject 90 &#181;L of sample activation solution (EDC/NHS) over 6 min contact time, and then hold for another 5 min.</li>
		<li>Repeat this cycle after baseline running for 1 min at 10 &#181;L/min on only the left flow cell (blue) to inject and immobilize chemically synthesized peptides10, HA, and spike protein at 20 &#181;g/mL, and allow for the subsequent baseline adjustment with ddH2O for 1.5 min before quenching the activated chip surface with 1 M ethanolamine HCl pH 8.5.</li>
	</ol>
	</li>
	<li>Switch the tubes from the liquid sampler to the degasser from ddH20 into the bottle with HBS-EP(+) (Supplementary Figure 1).</li>
	<li>Analyze the SPR sensorgrams.
	<ol>
		<li>To perform kinetic studies, use various analyte concentrations (10-5-10-8 M), with a regeneration step after each injection and blank measurements after different analytes. Change the flow rate to 10 &#181;L/min and start injections for 4 min contact time, then 5 min baseline generation, and two regeneration steps of 2 min each with an interval of 30 s.</li>
		<li>Inject the buffer solution for blank measurements whose sensorgrams are subtracted from sample runs to normalize different protein concentrations.</li>
	</ol>
	</li>
	<li>Click on Form, scroll down, and change to &#34;Post-Processing&#34; by clicking this operation mode. Click on Add to select binding curves generated over time in the Data Plot Form for each flow cell, and export the overlay as a scrubber file (.ovr). Click on File to open the file saving options. Obtain response curves by aligning left and right curves and subtracting signals of the second reference channel from those of the ligand channel. 
	NOTE: Data is operated in &#34;Post-Processing&#34; by defining sensorgrams computationally. It is put into an overlay of sensorgrams from left and right flow cells, or represented as sensorgrams from the difference of both channels.</li>
	<li>Clean the entire fluidics with 50-100 mM glycine buffer pH 9.5, water, and 20% ethanol before and after binding measurements to remove traces of salt or any protein contamination, or more stringent, with 0.5% SDS and glycine. 
	NOTE: To prevent instrument damage, it is recommended to check the mechanical stability of the glass chip before re-inserting the chip cartridge into the instrument if chips have been stored under buffer or at 100% humidity.</li>
</ol>

The author has nothing to disclose.

CV-N&#39;s binding affinity is correlated with the number of functional binding sites [2H on domains B, and 2L on domain(s) A when engineered as domain-swapped dimer]. A variant with an altered binding affinity (CVN2L0-V2, a homodimeric stable fold of CV-N comprising a disulfide bridge knock-out) is expressed in E. coli, purified, and positively tested for binding to HA-protein (H3N2) using SPR10, and shows a conformational change upon binding HA with either H or L carbohydrate-binding si...

Tetherin-associated antiviral activity is induced by interferon-&#945;, and it comprises protein-based tethers, that leads to the retention of fully formed virions on infected cell surfaces1. The necessity for tetherin glycosylation in the inhibition of virus release remains uncertain, implying the importance of glycosylation patterns on recombinantly expressed glycans for in vitro studies1,2, which depends on the conformation of (in the case of influenza virus) surface-expressed influenza hemagglutinin HA3,4. It has been noted that modification of oligosaccharide tethered to N-linked glycosylation is enough for tetherin-mediated restriction of HIV type-1 release2, while dimerization plays an essential role in preventing virus release, thereby involving the transmembrane domain or glycosyl-phosphatidyl-inositol (GPI)-anchor for tethering the budding virions5. Unique features are described for human and murine tetherin to block multiple enveloped viruses, retroviruses, and filoviruses. BST-2/tetherin is an interferon-inducible antiviral protein of the innate immunity1,6, acting with broad-spectrum antiviral activity and is antagonized by envelope glycoproteins5 to either translocate tetherin or disrupt the structure of tetherin6. For example, surface-expressed envelope glycoprotein HA and neuraminidase on influenza A virus are well known for tetherin antagonism in a strain-specific manner7, facilitating the recognition of host receptor binding sites8. Glycan-targeting antibodies are studied in the stoichiometry of their interactions with the rapidly customizing glycan shields on HA, resulting in binding affinity to influenza A H3N2 and H1N1 subtypes4.
To elucidate the binding mechanisms between antiviral agents and virus envelope spikes, i.e., carbohydrate ligands, and complementary immunological and spectroscopic methods, mono-, di- and tri-mannose moieties are chemically synthesized. The mannosylated peptides are created via azido glycosylation of glycosyl {beta}-peracetates to 1,2-trans glycosyl azides transformation9, mimicking the typically found N-acetyl glucosamine and high-mannose oligosaccharides on the surface of life-threatening viruses. Triazole bioisosteres are utilized to mimic linkages that form the mannosylated residue of HA peptide10 and facilitate site-specific interactions with antiviral CV-N derivatives around the second N-linked glycosylation spot on the HA head domain (HA top with 4 N-linked glycans N54, N97, N181, N301)8,11,12. Interactions between glutamic acid (Glu) and arginine (Arg) and the resulting helix dipole manifestated good stability of both model peptides and proteins but are visualized using SPR. If compared with recognizing a single chemically synthesized glycosylation site on HA10 by directly inhibiting receptor binding on the glycan moieties, a higher affinity of a four-site mutated Fc structure to its receptor is shown to elicit effector functions in vivo, revealing the unrelated composition of N-linked glycans attached to Fc mutant to be mechanistically determined13.
CV-N displays antiviral activity against HIV14,15, influenza16, and Ebola virus, which is mediated by nanomolar binding to high-mannose oligosaccharide modifications on envelope spike proteins12,17,18,19. Influenza HA binding to one high-affinity carbohydrate-binding site (H) in CV-N or two Hs in covalently linked dimeric CVN2 is determined to have equilibrium dissociation constants (KD) = 5.7 nM (Figure 1A) and KD = 2.7 nM, respectively. Both CV-N and CVN2 harbor another one or two low-affinity carbohydrate-binding sites (L)s12,17,20,21. Ebola GP1,2 binds to 2H of CVN2 with affinities in the lower nanomolar range (KD = 26 nM). CV-N WT binding to Ebola GP1,2 and HA exhibits affinities from KD = 34 nM to KD = 5.7 nM (A/New York/55/04)12. Lectins, such as CV-N, which specifically target high-mannose glycans on the viral envelopes, further inhibit replication of hepatitis C virus, SARS-CoV, herpesvirus, Marburg virus, and measles virus22.
The small CV-N molecule has been studied thoroughly for more than 20 years as it functionalizes to bind a wide range of viruses to inhibit viral entry16,18. Structural analyses and binding affinity assays indicate cross-linking of two Ls in a domain-swapped CVN2 dimer by bivalent binding in the micromolar range to enhance avidity to viral envelope glycoproteins10,19. Selective binding of Man&#945;1-2Man&#945; on Man(8) D1D3 arms and Man(9) comprises two binding sites of differing affinities located on opposite protein protomers20, thereby reaching nanomolar binding affinities (Figure 1B). Thus, CVN2 is considered a pseudo-antibody concerning its application to bind epitopes on HIV gp120, similar to virus-neutralizing antibodies17. Herein, the author is interested in investigating the potential binding of CVN2 to the SARS-CoV-2 spike via its receptor-binding domain (RBD). Binding curves of immobilized human angiotensin-converting enzyme (ACE)-2 with the SARS-CoV-2 RBD result in KD = 4.7 nM for this biologically relevant binding interaction23.
By contrast, selected immunoglobulin classes recognize specific and consistent structural protein patterns, which impart a substrate for affinity maturation in the membrane-anchored HA regions24. CV-N shows highly potent activity in almost all influenza A and B viruses16, and it is a broadly neutralizing antiviral agent. Our knowledge is incomplete on the location of targeted epitopes on the stem of HA1 and HA2 that possibly involve epitopic structures for glycan-targeting by highly neutralizing antibodies and as compared with lectin binding25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63541/63541fig1v5.jpg" /> 
Figure 1: Schematic representation of the SPR binding assay for CV-N to virus envelope spikes. (A) SPR Assay for CV-N binding to ligand: HA full-length protein (90 kDa). Kinetic data set (5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 0 nM) showing real-time double-referenced binding to influenza HA A/New-York/55/04 (H3N2). (B) CVN2L0 variant V2 binding to immobilized ligand DM within a concentration range of 500 nM to 16 &#181;M. Sequence: L residues are highlighted in yellow. H residues are highlighted in gray. E58 and R73 are a replacement for cysteines in the wildtype protein and make V2 a stable protein fold with three instead of four disulfide bonds <a href="https://www.jove.com/files/ftp_upload/63541/63541fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Whereas the glycan shield on the membrane-distal HA top part induces high-affinity binding to CV-N12, CVN2 binding to HA adjacent to a disulfide bridge of the HA top part has further been observed at its low-affinity sites10,12. Various polar interactions and interaction sites are identified in carbohydrate-binding by CV-N. These interactions are verified by generating knock-out variants in the binding site to correlate binding affinities to in silico predicted glycosylation12. Thus, the project aims to compare previously tested chemically mannosylated HA peptides in binding affinity and specificity with short peptide sequences from SARS-related 2019-nCoV spikes and SARS-CoV-2, naturally occurring modified by a small number of different N-linked glycosylation sites and O-linked glycosylation. Using cryo-electron microscopy and binding assays, Pinto and coworkers report a monoclonal antibody, S309, that potentially recognizes an epitope on SARS-CoV-2 spike protein containing a conserved glycan within the Sarbecovirus subgenus, without competing with receptor attachment26. The protocol of this study describes how designing, expressing, and characterizing CV-N variants are important to study how CV-N and CVN2 bind to glycosylated proteins and synthetic mannosylated peptides using the SPR technology10,12.
Tandem-linked dimer CVN2L027 and binding-site variants (V2-V5) are recombinantly expressed and variants are with disulfide bond replacements (C58E and C73R) (Figure 2A). Also, a mutant with a single-point mutation E41A is prepared because this position has been seen as an intermolecular cross-contacting residue. This mutant is another interesting molecule for SPR binding measurements between the lectin and high-mannose oligosaccharides deciphering binding domains and allows comparison with the dimeric form. The domain-swapped crystal structure of CVN2 shows a flexible linker, that extends between 49 and 54 residues. The two domains can continue moving around the hinge as rigid bodies, developing either a monomer through intramolecular domain interactions (domain A -residues 1-39;90-101- with domain B -residues 40-89) or a dimer by intermolecular domain swapping [domain A (of the first monomer) with domain B (of the second), and domain B (of the first monomer) with domain A (of the second copy)]. There are no close interactions between the two protomers&#39; A and B domains, except for Glu4128. The gene for CV-N can be developed using a repetitive PCR method with 40-mer synthesized oligos29 and is then subcloned into the NdeI and BamHI sites of pET11a for transformation (electroporation) into electrocompetent cells as described by Keeffe, J.R.27. The protein, which is used for achieving the respective crystal structure (PDB ID 3S3Y), includes an N-terminal 6-histidine purification tag followed by a Factor Xa protease cleavage site. Site-directed mutagenesis is utilized to make point mutations, switch codons, and insert or delete single or multiple bases or codons for amino acid exchange. These transformations provide invaluable insight into protein function and structure. Recombinantly expressed and purified CV-N, CVN2, and CVN3 have been biophysically well studied20,21,27, are cheap to produce, and therefore used to characterize binding assays to glycans immobilized onto SPR sensor chips. Conventional enzyme-linked immunosorbent assay (ELISA) provides less reproducibility concerning the immobilization technique of glycan ligands and transforms real-time binding of various binding-site variants, which is shown for SPR, into endpoint assays.
Binding-affinity variant CVN2L0-V2 (an intact fold of homodimeric CV-N with a disulfide bridge substitution10) is expressed with a His-tag in Escherichia coli (E. coli), purified over Ni-NTA column applying affinity chromatography and tested for binding to HA (H3N2), monomannosylated HA-peptide and dimannosylated HA-peptide using SPR. Chemically mannosylated peptides, or HA and S protein, all are ligands and amine coupled to the hydrophilic chip surface via reactive esters or biotin-streptavidin protein engineering. The same procedure of sequential runs is applied to those ligands, injecting various dilutions of CV-N and variants of CV-N (and CVN2) to obtain kinetic information for the molecular interaction analyses as described below30. RBD-immobilized SPR sensor chip is used for binding studies on CV-N to S peptides, and affinities are compared to SARS-CoV-2 binding with the human ACE2.

<table><tbody><tr><td>&amp;Auml;kta primeplus</td><td>Cytiva</td><td></td><td></td></tr><tr><td>Amicon tubes</td><td>Merck</td><td>C7715</td><td></td></tr><tr><td>Ampillicin</td><td>Sigma-Aldrich</td><td>A5354</td><td></td></tr><tr><td>Beckmann Coulter Cooler Allegra X-30R centrifuge</td><td>Beckman Coulter</td><td>B06320</td><td></td></tr><tr><td>Cell spreader</td><td>Sigma-Aldrich</td><td>HS86655</td><td>silver stainless steel, bar L 33&amp;nbsp;mm</td></tr><tr><td>Custom DNA Oligos</td><td>Sigma-Aldrich</td><td>OLIGO</td><td></td></tr><tr><td>Custom Gensynthesis</td><td>GenScript</td><td>#1390661&amp;nbsp;</td><td>cloning vector: pET27b(+)&amp;nbsp;</td></tr><tr><td>Cytiva&amp;nbsp;HBS-EP+ Buffer 10, 4x50mL</td><td>Thermo Scientific</td><td>50-105-5354</td><td></td></tr><tr><td>Dionex UlitMate 3000</td><td>Thermo Scientific</td><td>IQLAAAGABHFAPBMBFB</td><td></td></tr><tr><td>Dpn I restriction enzyme (10 U/&amp;mu;L)&amp;nbsp;</td><td>Fisher Scientific</td><td>ER1701</td><td></td></tr><tr><td>DTT</td><td>Merck</td><td>DTT-RO</td><td></td></tr><tr><td>EDC</td><td>Merck</td><td>39391</td><td></td></tr><tr><td>EDTA</td><td>Merck</td><td>E9884</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;Minispin&amp;nbsp;and MiniSpin Plus personal microcentrifuge</td><td>Sigma-Aldrich</td><td>Z606235</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>51976</td><td></td></tr><tr><td>Ethanolamine HCl</td><td>Merck</td><td>E6133</td><td></td></tr><tr><td>Falcon 50mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile, 25/Pack</td><td>Corning</td><td>352057</td><td></td></tr><tr><td>Glucose</td><td>Merck</td><td>G8270</td><td></td></tr><tr><td>Glycine HCl</td><td>Merck</td><td>55097</td><td></td></tr><tr><td>HA H3 protein</td><td>Abcam</td><td>ab69751</td><td></td></tr><tr><td>HEPES</td><td>Merck</td><td>H3375</td><td></td></tr><tr><td>His-select Ni2+</td><td>Merck</td><td>H0537</td><td></td></tr><tr><td>Imidazole</td><td>Merck</td><td>I2399</td><td></td></tr><tr><td>IPTG</td><td>Merck</td><td>I6758</td><td></td></tr><tr><td>Kanamycin A</td><td>Sigma-Aldrich</td><td>K1377</td><td></td></tr><tr><td>Kromasil 300-5-C4</td><td>Nouryon</td><td></td><td></td></tr><tr><td>LB agar</td><td>Merck</td><td>52062</td><td></td></tr><tr><td>LB agar</td><td>Merck</td><td>19344</td><td></td></tr><tr><td>LB Lennox</td><td>Merck</td><td>L3022</td><td></td></tr><tr><td>Lysozyme</td><td>Merck</td><td>10837059001</td><td></td></tr><tr><td>Magnesium chloride</td><td>Merck</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Merck</td><td>M7506</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;P0&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>S0751</td><td></td></tr><tr><td>NanoDrop UV-Vis2000c spectrophotometer</td><td>Thermo Scientific</td><td>ND2000CLAPTOP</td><td></td></tr><tr><td>NaOH</td><td>Merck</td><td>S5881</td><td></td></tr><tr><td>NHS</td><td>Merck</td><td>130672</td><td></td></tr><tr><td>NZ amine (casein hydrolysate)</td><td>Merck</td><td>C0626</td><td></td></tr><tr><td>PBS</td><td>Merck</td><td>806552</td><td></td></tr><tr><td>PD MidiTrap G-10</td><td>Sigma-Aldrich</td><td>GE28-9180-11</td><td></td></tr><tr><td>Peptone</td><td>Merck</td><td>70171</td><td></td></tr><tr><td>pET11a</td><td>Merck Millipore (Novagen)</td><td>69436&amp;nbsp;</td><td></td></tr><tr><td>PMSF</td><td>Merck</td><td>PMSF-RO</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (1000)</td><td>Qiagen</td><td>27106X4</td><td></td></tr><tr><td>Reichert Software Package Autolink1-1-9</td><td>Reichert</td><td></td><td></td></tr><tr><td>Reichert SPR SR7500DC Dual Channel System</td><td>Reichert</td><td></td><td></td></tr><tr><td>Scrubber2-2012-09-04 for data analysis</td><td>Reichert</td><td></td><td></td></tr><tr><td>SDS</td><td>Merck</td><td>11667289001</td><td></td></tr><tr><td>Site-directed mutagenesis kit incl pUC18 control plasmid</td><td>Stratagene</td><td>#200518</td><td></td></tr><tr><td>Sodim chloride</td><td>Merck</td><td>S9888</td><td></td></tr><tr><td>Sodium acetate.Trihydrate</td><td>Merck</td><td>236500</td><td></td></tr><tr><td>SPR sensor chip C19RBDHC30M</td><td>XanTec bioanalytics</td><td>SCR C19RBDHC30M</td><td></td></tr><tr><td>SPR sensor chip CMD500D</td><td>XanTec bioanalytics</td><td>SCR CMD500D</td><td></td></tr><tr><td>Sterilin Standard 90mm Petri Dishes</td><td>Thermo Scientific</td><td>101R20</td><td></td></tr><tr><td>TBS</td><td>Merck</td><td>T5912</td><td>10x, solution</td></tr><tr><td>Triton-X100</td><td>Merck</td><td>T8787</td><td></td></tr><tr><td>Tryptone</td><td>Merck</td><td>93657</td><td></td></tr><tr><td>Tween20</td><td>Merck</td><td>P1379</td><td></td></tr><tr><td>Vortex-Genie 2 Mixer</td><td>Merck</td><td>Z258423</td><td></td></tr><tr><td>X-gal</td><td>Merck</td><td>XGAL-RO</td><td></td></tr><tr><td>XL1-Blue Supercompetent Cells</td><td>Stratagene</td><td>#200236</td><td></td></tr><tr><td>Yeast extract</td><td>Merck</td><td>Y1625</td><td></td></tr></tbody></table>

engineering antiviral agents via surface plasmon resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

A dimeric domain-swapped CVN2L0 molecule is tested for binding to the HA top region in three separate SPR experiments and binding affinity is presented in KD values. Domain B is assumed to comprise H-binding sites, which are impacted by replacing a disulfide bond into ionic residues, and domain A forms L10,18. Single injections of CVN2L0 and variants V2 (three disulfide bridges) and V5 (two disulfide bridges) are first tested for binding to the HA-coup...

Watch this Scientific Journal Video about Engineering Antiviral Agents via Surface Plasmon Resonance at JoVE.com

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

engineering-antiviral-agents-via-surface-plasmon-resonance

Research

JoVE Journal

Bioengineering

2.2K Views.  University of California, Los Angeles. The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Video: Engineering Antiviral Agents via Surface Plasmon Resonance

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Chemistry

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in&#160;stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including&#160;the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Immunology and Infection

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance (SPR) Technology

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining blood types, and detecting food adulteration, among other biomolecular detections. This technology allows for the rapid identification of potential binding between biomolecules, facilitating fast and user-friendly, non-invasive screening of various indicators without the need for labeling. Additionally, SPR technology facilitates real-time detection for high-throughput drug screening. In this program, the application field and basic principles of SPR technology are briefly introduced. The operation process is outlined in detail, starting with instrument calibration and basic system operation, followed by ligand capture and multi-cycle analysis of the analyte. The real-time curve and experimental results of binding quercetin and calycosin to KCNJ2 protein were elaborated upon. Overall, SPR technology provides a highly specific, simple, sensitive, and rapid method for drug screening, real-time detection of related pharmacokinetics, virus detection, and environmental and food safety identification.

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance SPR Technology

The present study aims to elucidate the principle and methodology of surface plasmon resonance (SPR) technology, which finds versatile applications across multiple domains. This article describes SPR technology, its operational simplicity, and its remarkable efficacy, with the goal of fostering broader awareness and adoption of this technology among readers.

Surface plasmon resonance (SPR) technology is a sensitive precise method for detecting viruses, pathogenic molecular proteins, and receptors, determining ...

Medicine

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Biology

Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip

Source: Maier, I. <a href="https://app.jove.com/v/63541">Engineering Antiviral Agents via Surface Plasmon Resonance.</a> J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or SPR, technique for studying the interaction between two molecules using a sensor chip. The change in resonance angle and the intensity of the reflected light are detected as a function of the association and dissociation of two molecules, and this provides information about the interactions between the two molecules.

Source: Maier, I. Engineering Antiviral Agents via Surface Plasmon Resonance. J. Vis. Exp. (2022)
This video describes the surface plasmon resonance, or ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

A Viral Attachment Assay for Screening of Antiviral Compounds

For the viral attachment assay, plate 1 x 104 cells per well in a 96-well plate, and incubate overnight for cell attachment. The next morning, first, chill the 96-well plate containing the cell monolayer at 4 degrees Celsius for 1 hour.
Prepare the test compounds and virus mixture on ice. For example, HCV-CHLA, HCV - 1% DMSO, or positive control HCV-heparin solutions with 102 FFU virus for each treatment well. Aspirate the medium from the cell culture wells gently. Then, immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at 4 degrees Celsius for three hours.
Virus particles will attach on the cell surface at 4 degrees Celsius but will not be internalized. Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice-cold PBS. Add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours. Finally, for quantitating the released luciferase reporter from the infected cells, collect the supernatant from each sample well and perform the Gaussia luciferase assay.

Engineering Antiviral Agents via Surface Plasmon Resonance

Summary

Explore More Videos

Chapters in this video

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Real-Time Detection of Dynamic Affinity between Biomolecules Using Surface Plasmon Resonance (SPR) Technology

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Surface Plasmon Resonance to Study Biomolecular Interactions Using a Sensor Chip

A Viral Attachment Assay for Screening of Antiviral Compounds