Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Comparison between RBDCoV-ACE2 and a virus neutralization test (VNT). Serum samples (n=16) of pre-pandemic (n=4) and COVID-19 convalescent (n=12) individuals were measured using both assays and analyzed by linear regression. The equation of the dashed regression line is shown next to the graph. VNT results are depicted as half-maximal inhibiting serum dilutions (VNT 50 ), RBDCoV-ACE2 results are shown in percentage inhibition of ACE2 binding. Correlation analysis was performed after Spearman and the correlation coefficient r is shown.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Neutralization, Inhibition, Binding Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between SARS-CoV-2 NeutraLISA and VNT and comparison to RBDCoV-ACE2. (a) Correlation and linear regression between NeutraLISA and VNT results for pre-pandemic (n=4) and COVID-19 infected (n=12) samples. Correlation analyses were performed after Spearman and correlation coefficients r are shown. (b) Descriptive statistics of the (c) correlation between NeutraLISA and RBDCoV-ACE2. One sample from each individual (n=168) was measured using both assays correlation was calculated after Spearman. Samples were classified as being negative (non-neutralizing) if they had a value below 20% (red lines).

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Infection

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: ACE2 binding inhibition varies between RBD mutants. Violin plots showing ACE2 binding inhibition (%) of individual serum samples from 7 to 49 days post PCR (n=50, depicted as dots) against RBD mutants. Black horizontal lines represent medians. Fold-decrease of ACE2 binding inhibition in comparison to wild-type corresponds to the ratio between the medians of wild-type and the respective RBD mutant. VOC-RBDs are shown in blue. Mutations of each RBD mutant are shown in the box above the violin plot.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Mutagenesis

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation between anti-RBD IgG MFI signals and ACE2 binding inhibition (%) of serum samples from COVID-19 patients for wild-type and 11 RBD mutants. Regression analysis comparing ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type and all RBD mutants included in the study. Each circle represents one sample (n=168). For longitudinal donors with more than one sample available, the sample closest to 20 days post positive PCR diagnosis was selected. The percentage next to the bracket indicates the proportion of samples with ACE2 binding inhibition ≤ 20% (in orange). Spearman’s correlation coefficient (r) is specified for every correlation.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Longitudinal analysis of ACE2 binding inhibition and anti-RBD IgG levels in Covid-19 patients. Mean ACE2 binding inhibition (%) and IgG responses (MFI) for wild-type RBD against time post positive PCR test for samples (n=149) taken from 1 to 92 days post PCR are shown (a, b). Black dots indicate mean responses with standard deviation indicated by the error bars. The same analysis is then shown for longitudinal samples of selected donors (n=6) for wild-type (c, d) and RBD delta (e, f). For all RBD mutants, mean ACE2 binding inhibition (%) and mean IgG responses (MFI) 1 to 92 days post PCR included in the study is shown (g, h). Each variant is illustrated by a different color according to the figure key.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Variant Assay

Journal: medRxiv

Article Title: COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants

doi: 10.1101/2021.08.20.21262328

Figure Lengend Snippet: Correlation of anti-RBD IgG levels and ACE2 binding inhibition with SARS-CoV-2 disease severity. Bar charts showing mean ACE2 binding inhibitions (%) against wild-type and RBD delta are correlated with WHO grades for disease severity for samples 7-49 days post PCR (a, b) and ≥ 50 days post PCR (c, d). Mean anti-RBD WT IgG and anti-RBD delta IgG levels are shown for samples 7-49 days post PCR (e, f) and ≥ 50 days post PCR (g, h). Individual samples are displayed as colored dots, bars indicate the mean of the dataset with error bars representing standard deviation. Number of samples is given below the columns (n). If no samples for a group were available, the column is labeled with “n/a”. WHO grade 1 - ambulatory / no limitations of activities, 2 - ambulatory / limitation of activities, 3 - hospitalized, mild disease / no oxygen therapy, 4 - hospitalized, mild disease / mask or nasal prongs, 6 - hospitalized, severe disease / intubation + mechanical ventilation, 7 - hospitalized, severe disease / ventilation + additional organ support (pressors, RRT, ECMO), 8 – Death. The study did not contain samples of WHO grade 5.

Article Snippet: Assay buffer (1:4 Low Cross Buffer (Candor Bioscience GmbH) in CBS (1x PBS + 1% BSA) + 0.05 % Tween20) was supplemented with biotinylated human ACE2 (Sino Biological, # 10108-H08H-B) to a final concentration of 342.9 ng/mL to produce ACE2 buffer.

Techniques: Binding Assay, Inhibition, Standard Deviation, Labeling

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: Sensors (Basel, Switzerland)

Article Title: Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

doi: 10.3390/s21144814

Figure Lengend Snippet: An MMB-based inhibitor screening assay of the S1-ACE2 interaction. Magnetic beads that are conjugated to anti-His antibodies are introduced to S1 recombinant protein. Then, biotinylated ACE2 protein is added and detected using a fluorescent molecule, such as streptavidin R-phycoerythrin (SA-PE).

Article Snippet: To assess the inhibition of the S1-ACE2 interaction by the anti-S1 antibody, the conjugated magnetic beads with the attached S1 protein were incubated for one hour at 37 °C on a rotator with 50 μL of 2000 ng/mL of the biotinylated ACE2 protein and 50 μL of increasing concentrations of anti-S1 antibody (AM001414, Active motif, Carlsbad, CA, USA), ranging between a final concentration of 0.05 nM and 100 nM ( ).

Techniques: Screening Assay, Magnetic Beads, Recombinant

Journal: Sensors (Basel, Switzerland)

Article Title: Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

doi: 10.3390/s21144814

Figure Lengend Snippet: Illustration of magnetically modulated biosensors (MMB) with and without an inhibitor. ( a ) Two electromagnets, positioned on opposite sides of a sample cell, aggregate the magnetic beads and transport them in and out of an orthogonal laser beam. Without an inhibitor, the S1 protein interacts with the fluorescently labeled ACE2 receptor protein, and thus, when the magnetic beads pass through the laser beam, the fluorescence emission is high. Consequently, the average peak-to-peak signal is high. ( b ) When an inhibitor interferes with the interaction, the magnetic beads are not attached to the fluorescently labeled ACE2, and therefore, when the beads pass through the laser beam, the fluorescence emission is low. Consequently, the average peak-to-peak signal is small.

Article Snippet: To assess the inhibition of the S1-ACE2 interaction by the anti-S1 antibody, the conjugated magnetic beads with the attached S1 protein were incubated for one hour at 37 °C on a rotator with 50 μL of 2000 ng/mL of the biotinylated ACE2 protein and 50 μL of increasing concentrations of anti-S1 antibody (AM001414, Active motif, Carlsbad, CA, USA), ranging between a final concentration of 0.05 nM and 100 nM ( ).

Techniques: Magnetic Beads, Labeling, Fluorescence

Journal: Sensors (Basel, Switzerland)

Article Title: Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

doi: 10.3390/s21144814

Figure Lengend Snippet: Detection of the S1-ACE2 interaction. ( a ) A dose response curve of S1 interaction with ACE2, measured by MMB. All measurements were normalized to the average signal of the conjugated beads. Using all the replicates, the curve was fitted to one site-total binding model. The extracted K D is reported as K D (CI 95%: lower limit–upper limit). The chi-square Goodness of fit is reported as χ 2 (degrees of freedom, sample size). The solid red line was fitted using a non-linear regression analysis to the log-log dose response curve, and it represents the analytical sensitivity of the assay. Error bars (in blue) represent the standard error of the mean (SEM) of blank measurements ( n = 5 ) and all other concentrations ( 5 ≤ n ≤ 8 ) . ( b ) Negative controls to the dose response. Abbreviations: “Exp”, conjugated magnetic beads with the attached S1 protein are introduced to 250 ng/mL ACE2 protein and SA-PE. “No S1”, conjugated magnetic beads without the attached S1 protein are introduced to 250 ng/mL ACE2 and SA-PE. “No ACE2”, conjugated magnetic beads with the attached S1 protein are introduced to SA-PE. “No proteins”, conjugated magnetic beads are introduced to SA-PE. Three asterisks (***) indicate a statistical significance of p < 0.001 . All measurements were normalized to the signal of the conjugated magnetic beads. The normalized fluorescence signal values are presented as the mean ± SEM.

Article Snippet: To assess the inhibition of the S1-ACE2 interaction by the anti-S1 antibody, the conjugated magnetic beads with the attached S1 protein were incubated for one hour at 37 °C on a rotator with 50 μL of 2000 ng/mL of the biotinylated ACE2 protein and 50 μL of increasing concentrations of anti-S1 antibody (AM001414, Active motif, Carlsbad, CA, USA), ranging between a final concentration of 0.05 nM and 100 nM ( ).

Techniques: Binding Assay, Magnetic Beads, Fluorescence

Journal: Sensors (Basel, Switzerland)

Article Title: Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

doi: 10.3390/s21144814

Figure Lengend Snippet: Assessing an anti-S1 antibody (414-1) as an inhibitor of the S1-ACE2 interaction. Binding curve of the S1-ACE2 interaction with increasing concentrations of the anti-S1 antibody. Measurements were normalized to the average signal (designated as 100% Response) when S1 binds with ACE2, at 0 nM of the anti-S1 antibody. The orange line was fitted using a non-linear regression analysis to the log (inhibitor) versus the normalized response using GraphPad. The chi-square Goodness of fit is reported as χ 2 (degrees of freedom, sample size). IC 50 and pIC 50 , and their SEM were extracted from the fitted line and are reported as pIC 50 ± SEM and IC 50 (CI 95%: lower limit–upper limit). For each concentration 3 ≤ n ≤ 5 .

Article Snippet: To assess the inhibition of the S1-ACE2 interaction by the anti-S1 antibody, the conjugated magnetic beads with the attached S1 protein were incubated for one hour at 37 °C on a rotator with 50 μL of 2000 ng/mL of the biotinylated ACE2 protein and 50 μL of increasing concentrations of anti-S1 antibody (AM001414, Active motif, Carlsbad, CA, USA), ranging between a final concentration of 0.05 nM and 100 nM ( ).

Techniques: Binding Assay, Concentration Assay

Journal: Sensors (Basel, Switzerland)

Article Title: Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

doi: 10.3390/s21144814

Figure Lengend Snippet: Assessing a small molecule (SSAA09E2) as an inhibitor of the S1-ACE2 interaction. In each binding curve, the signals were normalized to the signal at 0 µM of inhibitor, which was set as 100% Response. ( a ) A binding curve of S1-ACE2 interaction with increasing concentrations of DMSO. The experiment was repeated three times ( n = 3 ) . ( b ) A binding curve of S1-ACE2 interaction with increasing concentrations of SSAA09E2 in a final concentration of 1% v/v DMSO. The experiment was repeated three times ( n = 3 ). (Inset) Abbreviations: “No inhibitor”, conjugated magnetic beads with the attached S1 protein were incubated with biotinylated ACE2 protein and SA-PE. “No ACE2”, conjugated magnetic beads with the attached S1 protein were incubated with the inhibitor and SA-PE, but without the ACE2 protein. Measurements were normalized to the signal (designated as 100% Response) when S1 binds with ACE2 without an inhibitor and without DMSO. Error bars represent the standard error of the mean value of four experiments ( n = 4 ) . Four asterisks (****) indicate a statistical significance of p < 0.0001 . ( c ) A binding curve of S1-ACE2 interaction with increasing concentrations of SSAA09E2 Maleate dissolved in DDW. The experiment was repeated three times ( n = 3 ). (Inset) Abbreviations: “No inhibitor”, conjugated magnetic beads with the attached S1 protein were incubated with biotinylated ACE2 protein and SA-PE. “No ACE2”, conjugated magnetic beads with the attached S1 protein were incubated with the inhibitor and SA-PE, but without the ACE2 protein. Measurements were normalized to the average signal (designated as 100% Response) when S1 binds with ACE2 without an inhibitor. Error bars represent the standard error of the mean value of five experiments ( n = 5 ) . Three asterisks (***) indicate a statistical significance of p < 0.001 .

Article Snippet: To assess the inhibition of the S1-ACE2 interaction by the anti-S1 antibody, the conjugated magnetic beads with the attached S1 protein were incubated for one hour at 37 °C on a rotator with 50 μL of 2000 ng/mL of the biotinylated ACE2 protein and 50 μL of increasing concentrations of anti-S1 antibody (AM001414, Active motif, Carlsbad, CA, USA), ranging between a final concentration of 0.05 nM and 100 nM ( ).

Techniques: Binding Assay, Concentration Assay, Magnetic Beads, Incubation