Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) Average force profiles of WT (red), Alpha (blue), Beta (orange), Gamma (sky blue), Epsilon (green), Kappa (pink), and Delta (gray) variants as a function of the distance between the centers of mass of RBD and ACE2. (B) Initial snapshot of WT. Residues subjected to each mutation are shown as solid sticks (N501, K417, E484, L452, and T478). RBD and ACE2 are, respectively, colored in light gray and yellow. All N-glycans, water, and ions are hidden for clarity. (C) Initial snapshot of WT with clockwise 90° rotation along the normal from (B). All N-glycans are depicted in different colors. Any other residues, water, and ions are not shown for clarity.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Two-dimensional contact maps at D = 53 Å. (A) Interacting residue pairs between RBD WT and ACE2. RBD residues subjected to mutation are shown in colored boxes at the bottom: (B) blue for Alpha, (C) orange for Beta, and (D) green for Epsilon. The contact frequency is numbered with colors from light blue to dark blue. Dark red and yellow colors on the map, respectively, represent increased and decreased interactions between RBD and ACE2 upon mutations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Mutagenesis

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: (A) The average number of contacts between RBD residue 501 and ACE2. (B, C) Representative snapshots at D = 53 Å of (B) Alpha variant and (C) WT. (D) Average number of contacts between RBD residue 417 and ACE2 and (E, F) their interacting residue pairs at D = 53 Å of (E) Beta and (F) Alpha variants. (G) Average number of contacts between RBD residue 478 and ACE2 and (H, I) key interaction pairs at D = 78 Å of (H) Delta and (I) Epsilon variants. The overall color scheme is the same as in Figure , and each mutated residue in each variant is shown using the same colors (i.e., red for WT, blue for Alpha, orange for Beta, green for Epsilon, and gray for Delta). Interacting residues are depicted as solid sticks, and residues losing their interactions are shown as transparent sticks. RBD and ACE2 are presented in light gray and yellow, respectively.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Variant Assay

Journal: Journal of Chemical Theory and Computation

Article Title: Differential Interactions between Human ACE2 and Spike RBD of SARS-CoV-2 Variants of Concern

doi: 10.1021/acs.jctc.1c00965

Figure Lengend Snippet: Binding affinities between RBD variants and ACE2 and its comparison with the simulation results. K d is obtained from microscale thermophoresis experiments. F WT / F is a ratio, where F WT and F are the respective maximum pulling force of WT and of each variant obtained from the SMD simulations.

Article Snippet: The recombinant human ACE2 protein (GenBank accession: AF291820.1, Sino Biological 10108-H08H; Wayne, PA) was labeled with RED-NHS (second Generation) dye using the Monolith Protein Labeling Kit (NanoTemper Technologies, MO-L011, München, Germany).

Techniques: Binding Assay, Microscale Thermophoresis, Variant Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Identification of neutralizing antibodies with a PtY display platform. We first used our preconstructed naïve phage displayed human scFv library to screen binders with biotinylated SARS-CoV-2 RBD protein in the solution phase. After enrichment of phage binders, the scFv DNA from enriched binders was cloned into the yeast display plasmid, resulting in display of scFv on the yeast cell surface. We then performed FACS to isolate potential blocking antibodies that could prevent binding of the SARS-CoV-2 RBD to hACE2. The 0.013% gate contained blocking antibodies with high affinity toward RBD. That is, higher Y axis signal represented higher affinity to labeled RBD, whereas lower X signal represented higher potency in blocking the binding of differently labeled hACE2 to RBD. The potential blocking antibodies were sent for sequencing and transient expression. The purified antibodies were evaluated for affinity, blocking activity, biophysical properties, and virus-neutralizing activity

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Clone Assay, Plasmid Preparation, Blocking Assay, Binding Assay, Labeling, Sequencing, Expressing, Purification, Activity Assay

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characteristics of potential blocking antibodies

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Expressing, Binding Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of potential blocking antibodies. (a) Blocking assay was performed by immobilizing 1 µg/ml hACE2 on a plate. Serially diluted antibodies and biotinylated SARS-CoV-2 RBD protein were added for competitive binding to hACE2. IC 50 values were calculated with Prism V8.0 software using a four-parameter logistic curve fitting approach. (b) Epitope binning was carried out by BLI. Biotinylated SARS-CoV-2 RBD was immobilized onto the SA sensor, and a high concentration of the primary antibody was used to saturate its own binding site. Subsequently, a second antibody was applied to compete for the binding site on the SARS-CoV-2 RBD protein. Data were analyzed with Octet Data Analysis HT 11.0 software. (c) Neutralization activities of Ab2001.08 and Ab2001.10 were assessed by live virus assay. Live SARS-CoV-2 and serially diluted (3-fold) antibodies were added to VERO E6 cells. The PRNT 50 values were determined by plotting the plaque number (neutralization percentage) against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Blocking Assay, Binding Assay, Software, Concentration Assay, Neutralization

Journal: mAbs

Article Title: A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351

doi: 10.1080/19420862.2021.1930636

Figure Lengend Snippet: Characterization of JMB2002. Binding affinity of JMB2002 for the SARS-CoV-2 RBD (a)/S1 (b) prototype and its variants was determined by BLI. JMB2002 was loaded onto the AHC sensor, and serially diluted antigens were bound to JMB2002 on the biosensor. K D values were determined with Octet Data Analysis HT 11.0 software using a 1:1 global fit model. Blocking activity was assessed using ELISA with hACE2-coated plates. A mixture of biotinylated SARS-CoV-2 RBD (c)/S1 (d) proteins and JMB2002 was added for competitive binding to hACE2. IC 50 values were calculated by Prism V8.0 software using a four-parameter logistic curve fitting approach. Values are displayed as the mean ± standard deviations from three independent experiments. (e) The pseudovirus neutralization activity of JMB2002 was evaluated using a pseudotyped SARS-CoV-2 system, which contained a luciferase reporter. Pseudotyped viruses were preincubated with serially diluted antibodies for 1 h. The mixture was added to hACE2-expressing cells and incubated at 37°C for 20–28 h. Infection of cells with pseudotyped SARS-CoV-2 was assessed by measuring cell-associated luciferase activity. IC 50 values were calculated by plotting the inhibition rate against the log antibody concentration in Prism V8.0 software

Article Snippet: Specifically, the library was incubated with SARS-CoV-2 RBD containing a mouse Fc tag (Sino Biological, 40592-V05H), and biotinylated hACE2 (Kactus, ACE-HM401) was then added.

Techniques: Binding Assay, Software, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Luciferase, Expressing, Incubation, Infection, Inhibition, Concentration Assay

Journal: PLoS ONE

Article Title: Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

doi: 10.1371/journal.pone.0257089

Figure Lengend Snippet: (A) HEK-cell produced RBD fragment fused to Rabbit IgG Fc fragment (RBD::rFc) binding to immobilized biotinylated recombinant human ACE2 was detected by anti-Rabbit IgG-HRP antibodies and TMB chromogenic reaction. Data shown represent values from one experiment. (B) ACE2 Receptor binding competition assay between a constant concentration of partially purified ER-Golgi Retained Algae-Produced RBD::mClover (~40 nM) and increasing amounts of RBD::rFc or Bovine Serum Albumin showing specific competition. RBD::mClover binding was detected using anti-GFP HRP antibodies. Data points represent mean and error bars represent Standard Error of the Mean of normalized A 490 signal values over three independent experimental repeats.

Article Snippet: Strepavidin coated microtiter plates (Cat#15124, Thermo Fisher) were washed 3X in Receptor Assay Blocking buffer (25 mM TrisHCl, 150mM NaCl, pH 7.4, 0.1% wt/vol Bovine Serum Albumin, 0.05% vol/vol Tween-20) and then were coated with 50 ng per well of biotylated human ACE2 produced in HEK cells lines (Cat#10108-H08H-B Sino Biological) dissolved in 100 μL of PBS for one hour at room temperature with gentle shaking on an orbital table.

Techniques: Produced, Binding Assay, Recombinant, Competitive Binding Assay, Concentration Assay, Purification

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: Whole cell lysate from uninoculated Vero E6, CV-1, A549, Mv1Lu, CRFK, MDCK-NBL-2 and MDCK-SIAT1 cell lines were immunoblotted for endogenous ACE2 expression. Recombinant hACE2 (Sino Biological) was used as a positive control for detection of hACE2. 20 μg of cell lysates or 0.2 ng of recombinant hACE2 protein were loaded. β-actin was also immunoblotted from samples as a loading control.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques: Expressing, Recombinant, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 susceptibility of cell lines and substrates commonly used in diagnosis and isolation of influenza and other viruses

doi: 10.1101/2021.01.04.425336

Figure Lengend Snippet: ACE2 protein sequences from human, rhesus macaque, African green monkey, cat, dog, American mink, mouse, and chicken were aligned using MUSCLE. Residues involved in interaction with SARS-CoV-2 spike protein (based on ref ( – )) are shown using hACE2 numbering, and residues varying from hACE2 are highlighted in yellow. A gap in alignment is indicated with a dash. Percent identity to hACE2 across the entire protein is shown.

Article Snippet: Cell lysates and recombinant ACE2 protein control (Sino Biological) were immunoblotted for ACE2 and β-actin using primary antibodies (1:500 polyclonal goat anti-human ACE2, R&D Systems, AF933; 1:1000 monoclonal mouse anti-β-Actin, Abcam, AB8226) followed by secondary antibodies (1:4000 donkey anti-goat, Abcam; 1:4000 goat anti-mouse, Biorad).

Techniques:

Journal: Nature Communications

Article Title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

doi: 10.1038/s41467-020-18319-6

Figure Lengend Snippet: a Schematic of a SARS-CoV-2 particle, an enveloped ssRNA virus expressing at its surface the spike glycoprotein (S) that mediates the binding to host cells. b Structural studies have previously obtained a complex between the receptor-binding domain (RBD, a subunit of the S glycoprotein) and the angiotensin-converting enzyme 2 (ACE2) receptor. c Schematic of probing SARS-CoV-2 binding using atomic force microscopy (AFM). The initial attachment of SARS-CoV-2 to cells involves specific binding between the viral S glycoprotein and the cellular receptor, ACE2. The interactions are monitored by AFM on model surfaces, where the ACE2 receptor is attached to a surface and the S1 subunit or the RBD onto the AFM tip, and on A549 living cells expressing or not fluorescently labeled ACE2.

Article Snippet: PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1 subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H).

Techniques: Expressing, Binding Assay, Microscopy, Labeling

Journal: Nature Communications

Article Title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

doi: 10.1038/s41467-020-18319-6

Figure Lengend Snippet: a Binding of S-glycoprotein subunit (S1 or RBD) is probed on an ACE2-coated surface. b Retraction part of four force–distance curves showing either nonadhesive or specific adhesive curves. c Box plot of specific binding probabilities (BP) measured by AFM between the functionalized tip (S1, RBD, or PEG) and the grafted surface (ACE2 or OH-/COOH-terminated alkanethiol (bare surface)). One data point belongs to the BP from one map acquired at 1 µm/s retraction speed. The square in the box indicates mean, the colored box indicates the 25th and 75th percentiles, and the whiskers indicate the highest and the lowest values of the results. The line in the box indicates median. N = 12 (S1, RBD), 18 (PEG), and 9 (S1, RBD vs. bare surface) maps examined over 4 (S1, RBD), 6 (PEG), and 3 (S1, RBD vs. bare surface) independent experiments. d Bell–Evans model describing a virus-receptor bond as a two-state model. The bound state is separated from the unbound state by a single energy barrier located at distance x u . k off and k on represent the dissociation and association rate, respectively. e , f Dynamic force spectroscopy (DFS) plot showing the distribution of the rupture forces as a function of their loading rate (LR) measured either between the S1 subunit and the ACE2 receptor ( N = 1052 data points) ( e ) or between the RBD and the ACE2 receptor ( N = 1490 data points) ( f ). The error bar indicates s.d. of the mean value for a single interaction (0–200 pN). The solid line represents the fit of the data with the Bell–Evans fit. Experiments were reproduced at least four times with independent tips and samples. g , h The BP is plotted as a function of the contact time for S1 subunit and RBD on ACE2 model surfaces, and data points were fitted using a least-squares fit of a monoexponential growth. One data point belongs to the BP from one map acquired at 1 µm/s retraction speed for the different contact times. Experiments were reproduced three times with independent tips and samples. P values were determined by two-sample t test in Origin. The error bar indicates s.d. of the mean value. Source data are provided as a Source Data file.

Article Snippet: PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1 subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H).

Techniques: Binding Assay, Spectroscopy

Journal: Nature Communications

Article Title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

doi: 10.1038/s41467-020-18319-6

Figure Lengend Snippet: a Binding of S-glycoprotein subunit 1 (S1) is probed on A549 and A549–ACE2 cells. b Confocal microscopy (z stack) of A549–ACE2–eGFP (green) cell transduced with plasma membrane BFP (blue). c Overlay of eGFP and DIC images of a mixed culture of A549 and A549–ACE2–eGFP cells. d , e Force–distance (FD)-based AFM topography image ( d ) and the corresponding adhesion map ( e ) in the specified area in ( c ). The frequency of adhesion events is indicated. f Box plot of the binding probability between S1 and A549 cells (gray) or A549–ACE2 cells (green) without and after injection of cyclic RGD (cRGD, checked boxes) or sialic acid (SA, dashed boxes), respectively. The square in the box indicates mean, the colored box indicates the 25th and 75th percentiles, and the whiskers indicate the highest and the lowest values of the results. The line in the box indicates median. g Force versus time curves showing either a nonadhesive curve (bottom) or specific adhesive curves acquired at different LRs (LR1–LR3). h DFS plot showing the distribution or the rupture forces measured either between the S1 subunit and the ACE2 on model surfaces (black dots, extracted from Fig. ), and between the S1 subunit and ACE2-overexpressing A549 cells acquired at three different LRs (blue and red dots) ( N = 403). Blue dots belong to a data set acquired in fast-force volume mode, with a retraction velocity of 20 µm s − 1 (LR1). Red dots belong to data sets acquired in peak force tapping mode with 0.125 kHz peak force frequency and 375-nm amplitude (LR2) or at 0.25 kHz and 750 nm (LR3), respectively. The error bar indicates s.d. of the mean value. Histograms of force distribution on A549–ACE2 cells for LR1–LR3 are shown on the side. For experiments without injection of cRGD or SA, data are representative of at least N = 11 cells from N = 6 independent experiments. The data for blocking experiments with cRGD or SA were acquired for at least N = 4 cells from N = 2 independent experiments. P values were determined by two-sample t test in Origin. Source data are provided as a Source Data file.

Article Snippet: PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1 subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H).

Techniques: Binding Assay, Confocal Microscopy, Transduction, Injection, Blocking Assay

Journal: Nature Communications

Article Title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

doi: 10.1038/s41467-020-18319-6

Figure Lengend Snippet: a Efficiency of blocking peptides is evaluated by measuring the binding probability of the interaction between the S1 subunit and ACE2 receptor on model surface before and after incubation of the functionalized AFM tip with the four different peptides at increasing concentration (1–100 µM). b Histograms, with the corresponding data points overlaid in dark gray, showing the binding probability without peptide (0 µM) and upon incubation with 1, 10, or 100 µM of ACE2-derived peptides ([22–44], [22–57], [22–44–g–351–357], and [351–357]). The binding probability measured with a polyethylene glycol (PEG) tip enables to evaluate the nonspecific binding level. The prediction of the structure of the ACE2-derived peptides is shown in the inset. The structure of the peptides is based on the structure of the peptide in the crystal structure (PDB ID: 6m0j). For the [22–44–g–351–357] peptide, its structure was generated using homology modeling . The error bar indicates s.d. of the mean value. c Graph showing the reduction of the binding probability. Control with ddH 2 O is provided in the inset showing that repetitive measurements do not result in a similar decrease of the binding probability. Data are representative of at least N = 3 independent experiments (tips and sample) per peptide concentration. P value was determined by two-sample t test in Origin. The error bar indicates s.d. of the mean value. Source data are provided as a Source Data file.

Article Snippet: PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1 subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H).

Techniques: Blocking Assay, Binding Assay, Incubation, Concentration Assay, Derivative Assay, Generated

Journal: Nature Communications

Article Title: Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor

doi: 10.1038/s41467-020-18319-6

Figure Lengend Snippet: a Box plot showing that the reduction of binding probability measured the S1-subunit-derivatized tip and a mixed culture of A549 and A549–ACE2 cells upon injection of the [22–57] ACE2-derived peptide. The square in the box indicates mean, the colored box indicates the 25th and 75th percentiles, and the whiskers indicate the highest and the lowest values of the results. b Overlay of eGFP and DIC images of a mixed culture of A549 and A549–ACE2–eGFP cells. FD-based AFM topography images ( c , e ) and the corresponding adhesion map ( d , f ) recorded in the specified area in ( b ) (scanned with a scan angle) before ( c , d ) and after ( e , f ) incubation of the tip with the [22–57] ACE2-derived peptide. The frequency of adhesion events is indicated. Data are representative of at least N = 4 cells from N = 2 independent experiments. P values were determined by two-sample t test in Origin. Source data are provided as a Source Data file.

Article Snippet: PFQNM-LC and MSCT-D cantilevers (Bruker) were used to probe the interaction between S1 subunit (Genscript, #U5377FC120) or RBD protein (Genscript, #U5377FC120) and ACE2 protein (Sino Biological, 90211-C02H).

Techniques: Binding Assay, Injection, Derivative Assay, Incubation

Journal: bioRxiv

Article Title: Targeting ACE2-RBD interaction as a platform for COVID19 therapeutics: Development and drug repurposing screen of an AlphaLISA proximity assay

doi: 10.1101/2020.06.16.154708

Figure Lengend Snippet: Scheme describing the assays employed in this paper. A) The recombinant protein constructs ACE2-His-Avi (ACE2-Avi) (Acro Biosystems) and SARS-CoV-2 Spike Protein Receptor Binding Domain-Fc (RBD-Fc) (Sino Biological) were used to model ACE2-RBD binding. B) AlphaLISA assay system used to monitor ACE2-RBD interacts. Streptavidin donor beads recognize the Avi tag on ACE2. Protein A acceptor beads recognize the Fc tag on RBD. When in proximity the donor beads can be excited with light at 680 nm. This generates singlet oxygen which diffuses to the acceptor beads, causing the acceptor beads to luminesce at 615 nm. C) The TruHits counter-screen uses strep donor beads which directly interact with biotin acceptor beads. Because no intermediary molecule is needed to bring the donor and acceptor beads in proximity, the TruHits assay can identify compounds which directly interfere with the AlphaLISA readout.

Article Snippet: ACE2-His (Sino Biological, Wayne, PA) and S1-His (Sino Biological, Wayne, PA) are not recognized by either the Streptavidin Donor bead nor the Protein A Acceptor bead.

Techniques: Recombinant, Construct, Binding Assay

Journal: bioRxiv

Article Title: Targeting ACE2-RBD interaction as a platform for COVID19 therapeutics: Development and drug repurposing screen of an AlphaLISA proximity assay

doi: 10.1101/2020.06.16.154708

Figure Lengend Snippet: The AlphaLISA assay monitors ACE2-RBD interactions. A) ACE2-Avi and RBD-Fc were titrated against each other from 300 – 0.1 nM in matrix format to determine the optimal protein concentrations for the AlphaLISA assay. ACE2-Avi and RBD-Fc were mixed at the concentrations indicated and allowed to equilibrate at 25°C for 30 minutes. Streptavidin Donor beads and Protein A Acceptor beads were then introduced to the solutions to a final concentration of 5 µg/mL of each bead. After 40 minutes of incubation at 25°C the signal intensity was read using a PheraStar plate reader. B) Assay suitability to 1536-well format was determined by combining 4 nM ACE2-Avi with 4 nM RBD-Fc in PBS + 0.05 mg/mL BSA. The mixture was incubated at 25 °C for 30 minutes. Streptavidin donor beads (10 µg/mL) were added to columns 1-8 of a 1536-well plate. Protein A acceptor beads (10 µg/mL) were added to columns 5-8. The entire mixture was incubated at 25°C for 40 minutes before reading the low-signal control (columns 1-4) and high-signal control (columns 5-8) using a PheraSTAR plate reader with an AlphaLISA module. Z’ and signal-to-background (S:B) were calculated from these sample measurements. C, D) To confirm the AlphaLISA signal related to ACE2-RBD interactions, we tested the ability of His-S1 (another form of the SARS-CoV-2 Spike protein) and untagged ACE2 to lower the AlphaLISA signal in a dose-dependent manner. C) His-S1 was mixed in dose-response with 4 nM ACE2-Avi and allowed to incubate at 25°C for 30 minutes before adding 4 nM RBD-Fc. D) Untagged ACE2 was pre-incubated in dose-response with 4 nM RBD-Fc and allowed to incubate at 25°C for 30 minutes before adding 4 nM ACE2-Avi. Both His-S1 and untagged ACE2 showed dose-dependent signal loss, indicating the AlphaLISA signal is mediated by RBD-Fc binding to ACE2-Avi.

Article Snippet: ACE2-His (Sino Biological, Wayne, PA) and S1-His (Sino Biological, Wayne, PA) are not recognized by either the Streptavidin Donor bead nor the Protein A Acceptor bead.

Techniques: Concentration Assay, Incubation, Binding Assay