PBMCs were isolated as described above and cultivated in complete RPMI medium in a 12-well culture plate at 1×106 cells/mL for monocyte stimulation or in a 24-well culture plate at 2.5×106 cells/mL for T-cell stimulation. SARS-CoV-2 Nucleocapsid Protein (N protein, Cat# 40588-V08B), SARS-CoV-2 Spike Protein [S protein receptor binding domain (RBD), Cat# 40592-V08B], SARS-CoV-2 Spike Protein (S1 Subunit, Cat# 40591-V08B1), SARS-CoV-2 Spike Protein (S1+S2 extracellular domain, Cat# 40589-V08B1), and SARS-CoV-2 (2019-nCoV) Spike S1 (S1 protein mutant D614G, Cat# 40591-V08H3) (all from Sino Biological, Wayne, PA, USA) were added to their respective cell culture well at final concentrations of 20nM. A non-protein control, a positive control for T-cell activation [phytohemagglutnin (PHA; 5 μg/mL; Cat# L8902, Sigma-Aldrich)], and a positive control for monocytes [lipopolysaccharide of Escherichia coli (LPS; 100 ng/mL Cat#tlrl-peklps, Invivogen, San Diego, CA, USA)] were also included. Plates were incubated at 37°C and 5% CO2 for 24 h (for monocytes) or 48 h (for T cells). Protein transport inhibitor cocktail was added to the wells for the last 4 h of incubation. Cells were then collected, transferred into 5 mL Falcon tubes, and centrifuged at 300 x g for 5 min at 4°C. Cell supernatants were stored at −80°C for cytokine determinations and cell pellets were used for immunophenotyping.
Sars cov 2 spike protein
Manufactured by Sino Biological
Sourced in China, United States
The SARS-CoV-2 spike protein is a recombinant protein produced in HEK293 cells. It represents the S1 subunit of the SARS-CoV-2 viral spike protein, which is the key structure that facilitates the virus's entry into human cells.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Bio-Rad (4 mentions)
Rabbit anti mouse igg hrp, by Jackson ImmunoResearch (3 mentions)
Flow cytometry, by Beckman Coulter (2 mentions)
Brilliant violet 421 streptavidin conjugated abs, by BioLegend (2 mentions)
Nunc immuno maxisorp 96 well plate, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by LGC (2 mentions)
Stop solution, by LGC (2 mentions)
Goat anti hamster igg h l hrp, by Southern Biotech (2 mentions)
3 3 5 5 tetramethylbenzidine substrate solution, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated goat anti monkey igg γ chain specific, by Alpha Diagnostic (2 mentions)
Paldi lenti system, by Aldevron (2 mentions)
Vsv g, by Aldevron (2 mentions)
Tlrl peklps, by InvivoGen (1 mentions)
Spike rbd antibody, by Sino Biological (1 mentions)
Polyvinylpyrrolidone pvp, by Maclin Biochemical Technology (1 mentions)
1 3 dimethylaminopropyl 3 ethylcarbodiimide hydrochloride edc, by Energy Chemical (1 mentions)
Octet red384 system, by Sartorius (1 mentions)
Mers cov, by Sino Biological (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Sunrise microplate reader, by Tecan (1 mentions)
30 protocols using sars cov 2 spike protein
1
Evaluating SARS-CoV-2 Protein Immunogenicity
2
SARS-CoV-2 Spike RBD Binding Assay
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The receptor binding domain (RBD) of the SARS-CoV-2 spike protein (Sino Biologicals, Cat No 40592-V05H) [37 ] was expressed as a recombinant protein with the Fc region of mouse (mFc) at the C terminus end and its corresponding spike RBD antibody (Sino Biologicals, cat. no. 40592-T62) [37 ] was used for the binding affinity experiments. The RBD protein was prepared in sterile water at a stock concentration of 0.25 mg/mL, as per the manufacturer’s instruction. The spike RBD rabbit polyclonal antibody was prepared at a stock concentration of 1 mg/mL and diluted further for analysis.
3
Rapid Immunoassay for SARS-CoV-2 Detection
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AgNO3, NaBH4, Na2SO3, NaOH, KH2PO4, Na2HPO4, NaCl, H2O2, FeCl3, ethylene glycol, sodium acetate, and KCl were of AR grade and used without additional purification. Trisodium citrate (TSC), L-ascorbic acid (L-AA), and polyvinylpyrrolidone (PVP, MW = 10,000) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl4), sucrose, MBA, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Energy Chemical Co., Ltd (Shanghai, China). HS-(PEG)n-COOH (MW = 2000) was purchased from ToYongBio Tech. Inc. (Shanghai, China). SARS-CoV-2 spike protein, mouse anti-N protein antibody, and rabbit anti-human IgG were purchased from Sino Biological Inc. (Beijing, China). Mouse anti-Staphylococcus aureus antibody and mouse anti-Salmonella typhimurium antibody and were purchased from Sigma-Aldrich (Saint Louis, MO, USA). SARS-CoV-2 N protein antibody was purchased from Abcam. Bovine serum albumin (BSA) was purchased from Labgic Technology Co., Ltd. (Beijing, China). Tween-20 was purchased from EKEAR Bio@Tech Co., Ltd. (Shanghai, China). Glass fiber conjugate pad, nitrocellulose membrane (NC membrane), PVC substrate, and absorbent pad were purchased from Joey-biotech Co., Ltd. (Shanghai, China). Serum samples were taken from the affiliated hospital of Qingdao University and stored at − 20 ℃ for use.
4
SARS-CoV-2 Spike Protein RBD-Specific Memory B Cells
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Fresh peripheral blood samples were collected and centrifuged through a Ficoll density gradient (Histopaque; Sigma-Aldrich Corporation, St Louis, MO, USA). Brilliant Violet 421™ Streptavidin-conjugated Abs (BioLegend, San Diego, CA, USA) and biotinylated Abs against the RBD of the SARS-CoV-2 spike protein (Sino Biological, Beijing, China) at a molar ratio of 1:4 were used for identification of the MBCs. For flow cytometry (Beckman Coulter, Inc., Brea, CA, USA), the cells were washed with phosphate-buffered saline, suspended staining buffer containing 2% fetal bovine serum, and probed with Abs against IgG, IgM, cluster of differentiation 3 (CD3), CD19, CD21, and CD27 (all, Biolegend). The data were examined using FlowJo software (version 10.0.7; FlowJo, LLC, Ashland, OR, USA). The cell percentages were calculated for just MBCs, include RBD-specific B cell (CD3− CD19+ RBD+), RBD-specific MBCs (CD3− CD19+ RBD+ CD27+), RBD+ atypical MBCs (CD3− CD19+ RBD+ CD21− CD27−), RBD+-activated MBCs (CD3− CD19+ RBD+ CD21− CD27+), RBD+-resting MBC (CD3− CD19+ RBD+ CD21+ CD27+), and RBD+ intermediate MBC (CD3− CD19+ RBD+ CD21 + CD27−).
5
SARS-CoV-2 Spike Protein ELISA
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Nunc Immuno MaxiSorp 96-well plates (Thermo Scientific) were coated with SARS-CoV-2 Spike protein (Sino Biological) at 1μg/mL in PBS and incubated overnight at 4C. After incubation, plates were washed once with wash buffer (0.05% TWEEN-20 in 1X PBS), then blocked with casein for 2–3 hours at 25C. Mouse or hamster sera were diluted in casein block buffer starting at 1:25 followed by 3X serial dilutions and incubated in plates for 1 hr at 25C. Plates were then washed three times, and incubated with either rabbit anti-mouse IgG-HRP (Jackson Immuno) or goat anti-hamster IgG(H+L)-HRP (SouthernBiotech) diluted in casein for mouse or hamster samples, respectively. After 1 hour incubation at 25C, plates were wsaahed three times, then developed using TMB substrate (SeraCare). Development was halted using stop solution (SeraCare). For each sample, ELISA endpoint titer was calculated in Graphpad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value (450nm) of 0.2.
6
SARS-CoV-2 Spike Variants and Antibody Analysis
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The codon-optimized gene encoding reference strain (GenBank: QHD43416) SARS-CoV-2 Spike protein with C-terminal 19-amino acid deletion was synthesized by Sino Biological Inc (Beijing, China), and cloned into pCMV3 vector. D614G mutation was introduced using site-directed mutagenesis (denoted as pCMV3-S-D614G). SARS-CoV-2 B.1.617 and B.1.1.7 variant Spikes were codon-optimized and synthesized by GenScript Inc (Nanjing, China) and cloned into pCMV3 vector. The HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) constructed by Landau13 (link) was provided by Prof. Cheguo Cai from Wuhan University (Wuhan, China). The expression plasmid for human ACE2 was obtained from GeneCopoeia (Guangzhou, China). Anti-RBD monoclonal antibodies (mAbs) against the SARS-CoV-2 Spike protein were obtained from the blood samples of COVID-19 convalescent patients as described previously.14
7
SARS-CoV-2 Spike Protein Antibody Assay
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SARS-CoV-2 Spike protein was purchased from Sino Biologicals (40589-V08B1). High binding plates (96-well) were coated with 100 ng of S protein diluted at a concentration of 2 mg/mL in PBS. The plates were washed once and blocked with 3% non-fat milk for 1 h at room temperature. Sera samples serially diluted in 1% non-fat milk containing PBS were added to the plates and incubated at 37 °C for 1 h. The plates were washed 3X with PBS-T, and horseradish peroxidase conjugated goat anti-mouse IgG, IgG1, or IgG2c (SouthernBiotech, 1:6000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 1 h at room temperature. Wells were washed three times with PBS-T before addition of 3,3’,5,5’-tetramethylbenzidine substrate solution (Thermo Pierce). The reaction was stopped after 3 min by addition of 0.16 M sulfuric acid. The optical density at 450 nm was measured with a Bio-Rad microplate reader.
8
SARS-CoV-2 Spike Protein ELISA Protocol
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Nunc Immuno MaxiSorp 96-well plates (Thermo Scientific) were coated with SARS-CoV-2 spike protein (Sino Biological) at 1 µg/mL in PBS and incubated overnight at 4 °C. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1× PBS), then blocked with casein for 2 to 3 h at 25 °C. Mouse or hamster sera were diluted in casein block buffer starting at 1:25 followed by 3× serial dilutions and incubated in plates for 1 h at 25 °C. Plates were then washed three times and incubated with either rabbit anti-mouse IgG-HRP (Jackson Immuno) or goat anti-hamster IgG(H+L)-HRP (SouthernBiotech) diluted in casein for mouse or hamster samples, respectively. After 1-h incubation at 25 °C, plates were washed three times, then developed using TMB substrate (SeraCare). Development was halted using stop solution (SeraCare). For each sample, ELISA endpoint titer was calculated in GraphPad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value (450 nm) of 0.2.
9
Aptamer Selection for SARS-CoV-2 Spike Protein
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Aptamers were generated by in vitro selection, from a diverse starting library of 1014 different sequences. Aptamer selection was performed by Aptamer Group (York, UK) according to proprietary automated selection methods. These aptamers are commercially known as Optimer binders. Briefly, DNA aptamers were selected against the S1 domain of the SARS‐Cov‐2 Spike protein (Sino Biological, Eschborn, Germany) through 8 successive rounds of selection and preferential amplification. Following identification of the best performing individual aptamer sequences, the minimal functional fragment of the aptamers (the Optimers), were identified and assessed for binding to the SARS‐CoV‐2 S1 domain and the SARS‐Cov‐2 Spike protein trimer (Peak Protein, Macclesfield, UK) by Bio‐Layer Interferometry (BLI) using an Octet Red 384 system (Sartorius, Goettingen, Germany). Cross‐reactivity to the homologous SARS‐CoV, and MERS‐CoV (Sino Biological), was also assessed.
10
SARS-CoV-2 Spike Protein Antibody Assay
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SARS-CoV-2 Spike protein was purchased from Sino Biologicals. 96-well high binding plates were coated with 100 ng of S protein diluted at a concentration of 2 μg/ml in PBS. Next morning, the plates were washed once, blocked with 3% non-fat milk in PBS containing 0.1% Tween 20 (PBST) for 1 h at room temperature. Sera samples serially diluted in 1% non-fat milk containing PBST was added to the plates and incubated at 37°C for 1 h. The plates were washed 3X with PBST, horseradish peroxidase conjugated goat anti-monkey IgG (γ-chain specific, Alpha Diagnostics, 1:4,000 dilution), in PBS-T containing 1% non-fat milk was added and incubated for 1 hour at RT. Wells were washed 3x with PBST before addition of 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution. The reaction was stopped after 12 minutes by addition of 0.16 M sulfuric or 1 M hydrochloric acid. The optical density (OD) at 450 nanometers was measured with a Biorad microplate reader.
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