Antigens used for ADCP and ADNP assay are as following: SARS-CoV-2 Spike protein and receptor-binding domain (RBD) protein from WT strain, Omicron BA.1, BA.2, BA.3, BA.4/5, BQ.1 and XBB.1. SARS-CoV-2 Spike protein of ancestral strain (cat# Z03481) and all RBD protein (cat# Z03483, cat# Z03728-100, cat# Z03740, cat# CP0007, cat# Z03745 for WT strain, Omicron BA.1, BA.2, BA.3, BA.4/5, respectively) were provided by Genscript (Jiangsu, China). BQ.1 Spike (cat# CG273) and XBB.1 Spike (cat# CG275) proteins were provided by Vazyme (Nanjing, China) Omicron BA.1 Spike ectodomain (GenBank: OL672836.1, residues 1-1205) was expressed as previously described [21 (link)]. The prefusion Omicron Spike ectodomain of BA.2, BA.3, BA.4/5 with proline substitutions at residues 983 and 984, a “GSAS” instead of “RRAR” at the furin cleavage site (residues 679-682), with a C terminal T4 fibritin trimerization motif, an HRV-3C protease cleavage site, a Twin-Strep-tag, and an 8×His-tag was cloned into vector pcDNA3.1 (Thermo Fisher Scientific, MA, USA) according to previous publications [24 (link)]. The protein was purified from Expi293 cells (Thermo Fisher Scientific, MA, USA) using affinity chromatography followed by Strep-Tactin resin (IBA, Göttingen, Germany, Cat 2-1201-500), detailed as described previously [25 (link)].
Sars cov 2 spike protein
Manufactured by GenScript
Sourced in China, United States
The SARS-CoV-2 spike protein is a structural protein found on the surface of the SARS-CoV-2 virus. It plays a key role in the virus's ability to bind to and infect human cells. This protein is a critical component for research and development related to COVID-19 diagnostics, therapeutics, and vaccine development.
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Lab products found in correlation
Nunc maxisorp, by Thermo Fisher Scientific (3 mentions)
Multiskan ascent, by Thermo Fisher Scientific (3 mentions)
Tetramethylbenzidine, by R&D Systems (3 mentions)
Prism 8, by GraphPad (2 mentions)
Human neuropilin 1 fc, by Thermo Fisher Scientific (2 mentions)
Anti his probe hrp, by Thermo Fisher Scientific (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Peroxidase affinipure goat anti rabbit igg 111 035 003, by Jackson ImmunoResearch (1 mentions)
Human alpha thrombin ht 1002a, by Enzyme Research (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hemagglutinin ha protein of influenza a h1n1 a california 04 2009, by Sino Biological (1 mentions)
Streptavidin hrp, by Jackson ImmunoResearch (1 mentions)
Eg00229, by Bio-Techne (1 mentions)
Biotinylated human vegf a165, by R&D Systems (1 mentions)
Neuropilin 1 fc, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary ab, by Merck Group (1 mentions)
Nisin, by MoBiTec (1 mentions)
Freestyle 293 f, by Thermo Fisher Scientific (1 mentions)
10 protocols using sars cov 2 spike protein
1
SARS-CoV-2 Variant Antigen Production
2
SARS-CoV-2 Spike Protein Binding Assay
3
SARS-CoV-2 Spike Protein Binding Assay
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Reagents were purchased from Sigma-Aldrich or indicated suppliers. Anti-β-actin mouse (sc-47778) and anti-LL-37 mouse monoclonal antibodies (sc-166770) were purchased from Santa Cruz Biotechnology. Anti-cathelicidin rabbit monoclonal antibodies (ab207758) were purchased from Abcam. Peroxidase-AffiniPure goat anti-rabbit IgG (111-035-003), peroxidase-AffiniPure goat anti-mouse IgG (115-035-003), and fluorescein (FITC)-AffiniPure donkey anti-mouse IgG (715-095-151) were purchased from Jackson ImmunoResearch Laboratories. The SARS-CoV-2 spike protein (Z03481) was purchased from GenScript. Human alpha thrombin (HT 1002a) and human factor Xa (HFXa 1011) were purchased from Enzyme Research Laboratories.
4
SARS-CoV-2 Spike Protein Induced PBMC Response
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PBMCs collected from healthy subjects were incubated in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (Gibco), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO2 (v/v). Indicated concentration of extra cellular domain truncation of SARS-CoV-2 Spike protein (GenScript) or Hemagglutinin/HA protein of Influenza A H1N1 (A/California/04/2009) (Sino Biological) was then added to the cultured PBMCs for 12 h, followed by RNA extraction and qRT-PCR assay.
5
SARS-CoV-2 Spike Protein Binding Assay
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Plates (96 well, Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated with human Neuropilin-1-Fc (10 ng per well, Cat# 50-101-8343, Fisher, Hampton, NH) and incubated at room temperature overnight. The following day, the plates were washed and blocked with 3% BSA in PBS to minimize non specific adsorptive binding to the plates. SARS-CoV2 Spike protein (100 nM, S1 domain aa16–685, Cat# Z03485, Genscript, Piscataway, NJ), EG00229 (Cat#6986, Tocris) or the indicated compounds were added at 12.5μM and incubated for 30 min at room temperature prior to adding biotinylated human VEGF-A165 (Cat#BT293, R&D systems) at 10 nM. As a negative control, some wells received PBS containing 3% BSA. The plates were incubated at room temperature with shaking for 1 h. Next, the plates were washed with PBS to eliminate unbound protein. Bound biotinylated VEGF was detected by streptavidin-HRP (Cat#016-030-084, Jackson immunoResearch). Tetramethylbenzidine (Cat#DY999, R&D Systems, St. Louis, MO) was used as the colorimetric substrate. The optical density of each well was determined immediately, using a microplate reader (Multiskan Ascent; Thermo Fisher Scientific) set to 450 nm with a correction wavelength of 570 nm. Data were normalized to the background and to the signal detected for VEGF-A165 alone.
6
SARS-CoV-2 Spike Protein Binding Assay
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Plates (96-well, Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated with human Neuropilin-1-Fc (10 ng per well, Cat# 50-101-8343, Fisher, Hampton, NH) and incubated at room temperature overnight. The following day, the plates were washed and blocked with 3% BSA in PBS to minimize non-specific adsorptive binding to the plates. SARS-CoV-2 Spike protein (S1 domain aa16-685, Cat# Z03485, Genscript, Piscataway, NJ) was added at concentrations ranging from 0.07 to 500 to nM. As a negative control, some wells received PBS containing 3% BSA. The plates were incubated at room temperature with shaking for 3 h. Next, the plates were washed with PBS to eliminate unbound protein. Bound SARS-CoV-2 Spike was detected by anti-His probe HRP (Cat#15165; Thermo Fisher Scientific). Some wells received no antibody (no antibody control). Tetramethylbenzidine (Cat#DY999, R&D Systems, St. Louis, MO) was used as the colorimetric substrate. The optical density of each well was determined immediately, using a microplate reader (Multiskan Ascent; Thermo Fisher Scientific) set to 450 nm with a correction wavelength of 570 nm. Data were analyzed by non-linear regression analysis using GraphPad Prism 8 (GraphPad, San Diego, CA, USA).
7
Optimizing SARS-CoV-2 Spike Protein Expression
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HCR spike gene expression was induced by supplementation of various nisin concentrations (MoBiTec GmbH, Goettingen Germany) to determine the optimal condition for protein expression. Various nisin concentrations (0–80 ng/mL) were added to the culture medium at the OD600 of around 0.8 followed by incubation at for 18 h at 30 °C. Bacterial cells and culture supernatants were separated by centrifugation, and the cells were sonicated five times. The expression of the intracellular and extracellular proteins was analyzed using SDS-PAGE. Furthermore, Western blotting was performed using a primary antibody (Ab) against the SARS-CoV-2 spike protein (0.5 ug/mL, GenScript, New Jersey, USA), followed by incubation with a horseradish peroxidase-conjugated secondary Ab (1:100.000; Sigma, St. Louis, MO, USA) for detection of the intracellular spike protein.
8
SARS-CoV-2 Spike Protein Expression and Antibody Binding
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SARS-CoV-2 spike-expressing FreeStyle™ 293-F (RRID:CVCL_D603; Invitrogen cat. no. R79007) cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein (GenScript) matching the amino acid sequence having mutations E156G, D614G, P681R, T19R, T478K, L452R, D950N, and 157–158 del for Delta. Stable transfectants were single-cell sorted and selected to obtain a high-level Spike surface expressing clone (293F-Spike-Delta). 293F-Spike-Delta cells were incubated with 100 μl of 4-fold serial dilutions of plasma starting at 100-fold for 30 min at 4 °C. Cells were washed twice and stained with anti-human IgG PE, anti-human IgM Alexa Fluor 647, and anti-human IgA FITC (Southern Biotech, Birmingham, AL, USA, cat. nos. 2040-09, 2020-31, and 2050-02). Cells were then fixed with 4% formaldehyde solution (Tousimis, Rockville, MD, USA, cat. no. 1008B) and fluorescence was evaluated on a LSRII (BD Bioscience).
9
SARS-CoV-2 Spike Protein Assay
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SARS-CoV-2 spike-expressing CEM-NKR cells (RRID:CVCL_X622; NIH AIDS Reagent Program cat. no. ARP-458) were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein (GenScript) matching the amino acid sequence having mutations E156G, D614G, P681R, T19R, T478K, L452R, D950N, and 157–158 del for Delta. Stable transfectants were single-cell sorted and selected to obtain a high-level spike surface expressing clone (CEM-Spike-Delta). CEM-Spike-Delta cells were plated at 100,000 per well in round bottom 96-well plates and incubated with 100 μl of diluted plasma (100-fold) for 30 min at 4 °C. Cells were washed and 200,000 Jurkat-Lucia NFAT-CD16 cells (RRID:CVCL_A7ZT; Invivogen, San Diego, CA, USA, cat. no. jktl-nfat-cd16) were added to each well in 100 μl of IMDM (Gibco, Burlington, ON, Canada, cat. no. 12440-053) 10% FBS. The cells were then centrifuge for 1 min at low speed and co-cultured for 24 h at 37 °C. 50 μl of Quanti-Luc (Invivogen, cat. no. rep-qlc2) was added to 20 μl of co-culture and luminescence was measured immediately on a luminometer (2104 Multilabel reader, PerkinElmer).
10
SARS-CoV-2 Spike Protein Antibody Detection
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The SARS-CoV-2 spike protein (GenScript, Z03481-100) was coated onto a 96-well microplate at 50 ng per well (Corning, Cat. 9018) overnight at 4°C. The uncoated spike protein was removed, and then the wells were blocked with 200 µl of blocking buffer (1X PBS with 1% BSA) for 2 h at room temperature. The plates were washed with 200 µl of PBS-T again, three times. Then, 10000-fold diluted HRP-conjugated donkey anti-mouse IgG, HRP-conjugated rabbit anti-mouse IgG1, and IgG2a antibodies (Invitrogen, Cat. 31430) were added to the wells and incubated
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