A human monoclonal antibody blocking SARS-CoV-2 infection methods
Aim. Evidence-backed execution summary for A human monoclonal antibody blocking SARS-CoV-2 infection methods from A human monoclonal antibody blocking SARS-CoV-2 infection.
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human
Subject model for the experiment.
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- confirm full cohort details in the source paper
Antiviral and biochemical properties of the human mAb 47D11
reagent used in the protocol.
- Use
- The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infection of VeroE6 cells with SARS-S and SARS2-S pseudotyped VSV with IC 50 values of 0.061 and 0.061 µg/ml (Fig. ), respectiv...
47D11 targets a conserved epitope in the SARS2-S-S1 B domain
reagent used in the protocol.
- Use
- The SARS2-S1 B RBD (residues 338-506) consists of a core domain and a receptor-binding subdomain (residues 438-498) looping out from the antiparallel betasheet core domain structure that directly engages the receptor. Compared to the S1 B core domain, the protein sequence identity of the S1 B receptor in...
47D11 targets a conserved epitope in the SARS2-S-S1 B domain
reagent used in the protocol.
- Use
- In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful...
Methods
reagent used in the protocol.
- Use
- Coronavirus spike ectodomains (S ecto ) of SARS-CoV-2 (residues 1-1213; strain Wuhan-Hu-1; GenBank: QHD43416.1 ) and HCoV-OC43 (residues 15-1263; strain Paris; UniProtKB: Q696P8 ) were expressed transiently in HEK-293T cells with a C-terminal trimerization motif and Strep-tag using the pCAGGS expression...
Generation of H2L2 mAbs
reagent used in the protocol.
- Use
- H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the man...
Production of human monoclonal antibody 47D11
reagent used in the protocol.
- Use
- For recombinant human mAb production, the cDNA's encoding the 47D11 H2L2 mAb variable regions of the heavy and light chains were cloned into expression plasmids containing the human IgG1 heavy chain and Ig kappa light chain constant regions, respectively (InvivoGen). Both plasmids contain the interleukin-2 sig...
Immunofluorescence microscopy
reagent used in the protocol.
- Use
- Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) usi...
Flow cytometry-based receptor-binding inhibition assay
reagent used in the protocol.
- Use
- Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2...
Antiviral and biochemical properties of the human mAb 47D11
The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infection of VeroE6 cells with SARS-S and SARS2-S pseudotyped VSV with IC 50 values of 0.061 and 0.061 µg/ml (Fig. ), respectiv...
- Use
- The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infection of VeroE6 cells with SARS-S and SARS2-S pseudotyped VSV with IC 50 values of 0.061 and 0.061 µg/ml (Fig. ), respectiv...
Antiviral and biochemical properties of the human mAb 47D11
a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with bind...
- Use
- a ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. The average ± SD from two independent experiments with technical duplicates is shown. b Interference of antibodies with bind...
Immunofluorescence microscopy
Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) usi...
- Use
- Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) usi...
Flow cytometry-based receptor-binding inhibition assay
Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2...
- Use
- Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2...
Virus neutralization assay
Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications. In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 µl was mixed with 50 µl (500 TCID...
- Use
- Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications. In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 µl was mixed with 50 µl (500 TCID...
ELISA analysis of antibody binding to CoV spike antigens
NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial...
- Use
- NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial...
Pseudotyped virus neutralization assay
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells...
Virus neutralization assay
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications. In brief, mAbs were twofold serially diluted in culture medium starting at 40 µg/ml and 50 µl was mixed with 50 µl (500 TCID...
ELISA analysis of antibody binding to CoV spike antigens
Software used for acquisition, scoring, statistics, or reporting.
- Use
- NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial...
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47D11 targets a conserved epitope in the SARS2-S-S1 B domain
In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody-either alone or in combination-offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus.
Generation of H2L2 mAbs
H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the manufacturer's instruction for first injection, whereas boosting was done using Ribi (Sigma) adjuvant. Injections were done subcutaneously into the left and right groin each (50 µl) and 100 µl intraperitoneally. Four days after the last injection, spleen and lymph nodes are harvested, and hybridomas made by standard method using SP 2/0 myeloma cell line (ATCC#CRL-1581) as a fusion partner. Hybridomas were screened in antigen-specific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). For thi...
Immunofluorescence microscopy
Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000 (Invitrogen, Catalog# 11668019). Two days post transfection, cells were fixed by incubation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and stained for nuclei with 4,6-diamidino-2-phenylindole (Sigma, Catalog# D9542). Cells were subsequently incubated with mAbs at a concentration of 10 µg/ml for 1 hour at room temperature, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45...
Flow cytometry-based receptor-binding inhibition assay
Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2 - C-terminally fused to the GFP using Lipofectamine 2000 (Invitrogen). Two days post transfection, cells were dissociated by cell dissociation solution (Sigma-aldrich, Merck KGaA; Catalog# C5914). In all, 2.5 µg/ml of human Fc tagged SARS-S1 B and SARS2-S1 B was pre-incubated with mAb at the indicated mAb:S1 B molar ratios for 1 hour on ice and subjected to flow cytometry. Single-cell suspensions in FACS buffer were centrifuged at 400 × g for 10 min. Cells were subsequently incubated with S1 B and mAb mixture for 1 hour on ice,...
Pseudotyped virus neutralization assay
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells were infected with the VSV-G pseudotyped VSVΔG bearing the firefly ( Photinus pyralis ) luciferase reporter gene. Twenty-four hours later, supernatants containing SARS-S/SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney VeroE6 (ATCC#CRL-1586) cells. In the virus neutralization assay, mAbs were fourfold serially diluted at two times the desired final concentration in DMEM supplemented with 1% fetal calf serum (Bodinco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Lonza, Catalog# 17-602E). Diluted mAbs were...
ELISA analysis of antibody binding to CoV spike antigens
NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethyl...
Measurement outputs
What raw and processed outputs should exist?
The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infe...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
A ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. T...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Coronavirus spike ectodomains (S ecto ) of SARS-CoV-2 (residues 1-1213; strain Wuhan-Hu-1; GenBank: QHD43416.1 ) and HCoV-OC43 (residues 15-1263; strain Paris; UniPr...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infe...; A ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. T...; Coronavirus spike ectodomains (S ecto ) of SARS-CoV-2 (residues 1-1213; strain Wuhan-Hu-1; GenBank: QHD43416.1 ) and HCoV-OC43 (residues 15-1263; strain Paris; UniPr...; H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expressi...; Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications. In brief, mAbs wer...; NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and...
from paperReporting output
Report representative outputs alongside summary comparisons for The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2 (Fig. ). The 47D11 antibody was found to potently inhibit infe..., A ELISA-binding curves of 47D11 to S ecto (upper panel) or S1 A and S1 B (RBD: receptor-binding domain) (lower panel) of SARS-S and SARS2-S coated at equimolar concentrations. T..., Coronavirus spike ectodomains (S ecto ) of SARS-CoV-2 (residues 1-1213; strain Wuhan-Hu-1; GenBank: QHD43416.1 ) and HCoV-OC43 (residues 15-1263; strain Paris; UniPr..., H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and....
inferred from protocolStructured statistical methods
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expressi...; Neutralization of authentic SARS-CoV and SARS-CoV-2 was performed using a plaque reduction neutralization test as described earlier, with some modifications. In brief, mAbs wer...; NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody-either alone or in combination-offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus.
H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the manufacturer's instruction for first injection, whereas boosting was done using Ribi (Sigma) adjuvant. Injections were done subcutaneously into the left and right groin each (50 µl) and 100 µl intraperitoneally. Four days after the last injection, spleen and lymph nodes are harvested, and hybridomas made by standard method using SP 2/0 myeloma cell line (ATCC#CRL-1581) as a fusion partner. Hybridomas were screened in antigen-specific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). For this purpose, hybridomas are cultured in serum- and protein-free medium for hybridoma culturing (PFHM-II (1 × ), Gibco) with addition of non-essential amino acids 100 × NEAA, Biowhittaker Lonza, Catalog# BE13-114E). H2L2 antibodies were purified from hybridoma culture supernatants using Protein-...
Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000 (Invitrogen, Catalog# 11668019). Two days post transfection, cells were fixed by incubation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and stained for nuclei with 4,6-diamidino-2-phenylindole (Sigma, Catalog# D9542). Cells were subsequently incubated with mAbs at a concentration of 10 µg/ml for 1 hour at room temperature, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45 min at room temperature. The fluorescence images were recorded using a Leica SpeII confocal microscope.
Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2 - C-terminally fused to the GFP using Lipofectamine 2000 (Invitrogen). Two days post transfection, cells were dissociated by cell dissociation solution (Sigma-aldrich, Merck KGaA; Catalog# C5914). In all, 2.5 µg/ml of human Fc tagged SARS-S1 B and SARS2-S1 B was pre-incubated with mAb at the indicated mAb:S1 B molar ratios for 1 hour on ice and subjected to flow cytometry. Single-cell suspensions in FACS buffer were centrifuged at 400 × g for 10 min. Cells were subsequently incubated with S1 B and mAb mixture for 1 hour on ice, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45 min at room temperature. Cells were subjected to flow cytometric analysis with a CytoFLEX Flow Cytometer (Beckman Coulter). The r...
Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells were infected with the VSV-G pseudotyped VSVΔG bearing the firefly ( Photinus pyralis ) luciferase reporter gene. Twenty-four hours later, supernatants containing SARS-S/SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney VeroE6 (ATCC#CRL-1586) cells. In the virus neutralization assay, mAbs were fourfold serially diluted at two times the desired final concentration in DMEM supplemented with 1% fetal calf serum (Bodinco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Lonza, Catalog# 17-602E). Diluted mAbs were incubated with an equal volume of pseudotyped VSV particles for 1 hour at room temperature, inoculated on confluent VeroE6 monolayers in 96-well plate, and further incubated at 37 °C for 24 hours. Luciferase activity was measured on a Berthold Centro LB 960 plate luminometer using D...
NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808, Biotek). Half-maximum effective concentration (EC 50 ) binding values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism (version 8).
Machine-readable layer
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"name": "A human monoclonal antibody blocking SARS-CoV-2 infection methods",
"description": "Evidence-backed execution summary for A human monoclonal antibody blocking SARS-CoV-2 infection methods from A human monoclonal antibody blocking SARS-CoV-2 infection.",
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"name": "47D11 targets a conserved epitope in the SARS2-S-S1 B domain",
"text": "In conclusion, this is the first report of a (human) monoclonal antibody that neutralizes SARS-CoV-2. 47D11 binds a conserved epitope on the spike RBD explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor-binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody-either alone or in combination-offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus."
},
{
"@type": "HowToStep",
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"name": "Generation of H2L2 mAbs",
"text": "H2L2 mice were sequentially immunized in 2 weeks intervals with purified S ecto of different CoVs in the following order: HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-OC43, SARS-CoV, and MERS-CoV. Antigens were injected at 20-25 µg/mouse using Stimune Adjuvant (Prionics) freshly prepared according to the manufacturer's instruction for first injection, whereas boosting was done using Ribi (Sigma) adjuvant. Injections were done subcutaneously into the left and right groin each (50 µl) and 100 µl intraperitoneally. Four days after the last injection, spleen and lymph nodes are harvested, and hybridomas made by standard method using SP 2/0 myeloma cell line (ATCC#CRL-1581) as a fusion partner. Hybridomas were screened in antigen-specific ELISA and those selected for further development, subcloned and produced on a small scale (100 ml of medium). For thi..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Immunofluorescence microscopy",
"text": "Antibody binding to cell surface spike proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV was measured by immunofluoresence microscopy. HEK-293T (ATCC#CRL-3216) cells seeded on glass slides were transfected with plasmids encoding SARS-S, SARS2-S, or MERS-S - C-terminally fused to the green fluorescence protein (GFP) using Lipofectamine 2000 (Invitrogen, Catalog# 11668019). Two days post transfection, cells were fixed by incubation with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and stained for nuclei with 4,6-diamidino-2-phenylindole (Sigma, Catalog# D9542). Cells were subsequently incubated with mAbs at a concentration of 10 µg/ml for 1 hour at room temperature, followed by incubation with 1:200 diluted Alexa Fluor 594 conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, Catalog# A-11014) for 45..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Flow cytometry-based receptor-binding inhibition assay",
"text": "Antibody interference of S1 B binding to human ACE2 receptor on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10 5 cells per ml in a T75 flask. After reaching 70~80% confluency, cells were transfected with an expression plasmid encoding human ACE2 - C-terminally fused to the GFP using Lipofectamine 2000 (Invitrogen). Two days post transfection, cells were dissociated by cell dissociation solution (Sigma-aldrich, Merck KGaA; Catalog# C5914). In all, 2.5 µg/ml of human Fc tagged SARS-S1 B and SARS2-S1 B was pre-incubated with mAb at the indicated mAb:S1 B molar ratios for 1 hour on ice and subjected to flow cytometry. Single-cell suspensions in FACS buffer were centrifuged at 400 × g for 10 min. Cells were subsequently incubated with S1 B and mAb mixture for 1 hour on ice,..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Pseudotyped virus neutralization assay",
"text": "Production of VSV pseudotyped with SARS-S and SARS2-S was performed as described previously with some adaptations. Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding SARS-S or SARS2-S carrying a 28- or 18-a.a. cytoplasmic tail truncation, respectively. One day post transfection, cells were infected with the VSV-G pseudotyped VSVΔG bearing the firefly ( Photinus pyralis ) luciferase reporter gene. Twenty-four hours later, supernatants containing SARS-S/SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney VeroE6 (ATCC#CRL-1586) cells. In the virus neutralization assay, mAbs were fourfold serially diluted at two times the desired final concentration in DMEM supplemented with 1% fetal calf serum (Bodinco), 100 U/ml Penicillin and 100 µg/ml Streptomycin (Lonza, Catalog# 17-602E). Diluted mAbs were..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "ELISA analysis of antibody binding to CoV spike antigens",
"text": "NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar antigen amounts at 4 °C overnight. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% bovine serum albumin (Bio-Connect) in PBS containing 0.1% Tween-20 at room temperature for 2 hours. Fourfolds serial dilutions of mAbs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 hour at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human secondary antibody (ITK Southern Biotech) diluted 1:2000 in blocking buffer for 1 hour at room temperature. An HRP-conjugated anti-StrepMAb (IBA, Catalog# 2-1509-001) antibody was used to corroborate equimolar coating of the strep-tagged spike antigens. HRP activity was measured at 450 nanometer using tetramethyl..."
}
],
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"name": "Antiviral and biochemical properties of the human mAb 47D11"
},
{
"@type": "HowToTool",
"name": "Antiviral and biochemical properties of the human mAb 47D11"
},
{
"@type": "HowToTool",
"name": "Immunofluorescence microscopy"
},
{
"@type": "HowToTool",
"name": "Flow cytometry-based receptor-binding inhibition assay"
},
{
"@type": "HowToTool",
"name": "Virus neutralization assay"
},
{
"@type": "HowToTool",
"name": "ELISA analysis of antibody binding to CoV spike antigens"
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"@type": "HowToSupply",
"name": "Antiviral and biochemical properties of the human mAb 47D11"
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"name": "47D11 targets a conserved epitope in the SARS2-S-S1 B domain"
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{
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"name": "47D11 targets a conserved epitope in the SARS2-S-S1 B domain"
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"name": "Methods"
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{
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"name": "Generation of H2L2 mAbs"
},
{
"@type": "HowToSupply",
"name": "Production of human monoclonal antibody 47D11"
},
{
"@type": "HowToSupply",
"name": "Immunofluorescence microscopy"
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"@type": "HowToSupply",
"name": "Flow cytometry-based receptor-binding inhibition assay"
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"headline": "A human monoclonal antibody blocking SARS-CoV-2 infection",
"datePublished": "2020",
"author": [
{
"@type": "Person",
"name": "Chunyan Wang"
},
{
"@type": "Person",
"name": "Wentao Li"
},
{
"@type": "Person",
"name": "Dubravka Drabek"
},
{
"@type": "Person",
"name": "Nisreen M. A. Okba"
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{
"@type": "Person",
"name": "Rien van Haperen"
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"name": "Albert D. M. E. Osterhaus"
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"name": "Frank J. M. van Kuppeveld"
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"@type": "Person",
"name": "Bart L. Haagmans"
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{
"@type": "Person",
"name": "Frank Grosveld"
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"@type": "Person",
"name": "Berend-Jan Bosch"
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"identifier": "10.1038/s41467-020-16256-y"
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