Sera from all mice were collected prior to immunization (week 0) and at weeks indicated after immunization and evaluated for SARS-CoV-2-S1-specific IgG, IgG1, and IgG2a antibodies using ELISA [34 (link)]. Briefly, ELISA plates were coated with 200 ng of recombinant SARS-CoV-2-S1 protein (Sino Biological) per well overnight at 4°C in carbonate coating buffer (pH 9.5) and then blocked with PBS-T and 2% BSA for 1 hour. Mouse sera were serially diluted in PBS-T with 1% BSA and incubated overnight. After the plates were washed, anti-mouse IgG-horseradish peroxidase (HRP) (1:10 000, Santa Cruz) was added to each well and incubated for 1 hour. The plates were washed three times, developed with 3,3′5,5′-tetramethylbenzidine, and the reaction was stopped. Next, absorbance was determined at 450 nm using a plate reader. For IgG1 and IgG2a ELISAs, mouse sera were diluted in PBS-T with 1% BSA and incubated overnight. After the plates were washed, biotin-conjugated IgG1 and IgG2a (1:1000, eBioscience) and biotin horseradish peroxidase (Av-HRP) (1:50 000, Vector Laboratories) were added to each well and incubated for 1 hour. The plates were washed three times and developed with 3,3′5,5′-tetramethylbenzidine, the reaction was stopped, and absorbance at 450 nm was determined using a plate reader.
Sars cov 2 s1 protein
Manufactured by Sino Biological
Sourced in China
The SARS-CoV-2 S1 protein is a recombinant protein produced using a mammalian expression system. It represents the S1 subunit of the SARS-CoV-2 spike protein, which is responsible for receptor binding during viral entry into host cells.
Automatically generated - may contain errors
Lab products found in correlation
Sars cov 2 s2 protein, by Sino Biological (2 mentions)
Anti mouse igg horseradish peroxidase hrp, by Santa Cruz Biotechnology (1 mentions)
Tetramethylbenzidine substrate, by Beyotime (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Epoch reader, by Agilent Technologies (1 mentions)
Tmb buffer, by Beyotime (1 mentions)
Synergy 2 multi detection microplate reader, by Agilent Technologies (1 mentions)
Streptavidin peroxidase polymer ultrasensitive, by Merck Group (1 mentions)
Ez link sulfo nhs lc lc biotin kit, by Thermo Fisher Scientific (1 mentions)
Infinite m200 pro multimode microplate reader, by Tecan (1 mentions)
Tmb single component substrate solution, by Solarbio (1 mentions)
Amicon ultra10 filters, by Merck Group (1 mentions)
Tmb substrate set, by BioLegend (1 mentions)
Hrp conjugated goat anti mouse igg, by BioLegend (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Trifluoroacetic acid tfa, by Merck Group (1 mentions)
Chymotrypsin, by Merck Group (1 mentions)
13 protocols using sars cov 2 s1 protein
1
SARS-CoV-2 S1-specific Antibody Quantification
2
SARS-CoV-2 S1 Protein Antibody Detection
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SARS-CoV2 S1 protein (Sino Biological Inc., BJ) was coated on a high-binding 96-well plate (Thermo Scientific) at 4°C overnight. Plates were blocked with 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with 1:100 diluted plasmas in dilution buffer (PBS, 2% non-fat milk, and 0.05% Tween-20). A 1:4,000 dilution of horseradish peroxidase (HRP)-conjugated mouse anti-human IgG, IgM, and IgA antibodies (BaiaoTong Experiment Center, LY) was added and incubated for 1 h at room temperature. Wells were washed six times between each step with 0.05% Tween-20 in PBS (PBST). Finally, wells were developed using tetramethylbenzidine substrate (Beyotime Inc., WH) and were stopped by the addition of stop solution, followed by reading at 450 and 570 nm. The sample OD was calculated as OD450 subtracted by OD570.
3
SARS-CoV-2 S1 Protein Serum IgG Assay
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For Study 1, recombinant SARS-CoV-2 S1 protein (1 µg/mL, Sino Biological, Eschborn, Germany) was coated overnight on MaxiSorp plates (Thermo Fisher Scientific, Drieieich, Germany) using sodium carbonate buffer. After a blocking step the next day, serum samples were incubated for 1 h at 37 °C to allow the binding to antigens. Bound rat IgG was detected using a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and tetramethylbenzidine (TMB) substrate (Biotrend, Köln, Germany). Data collection was performed using a BioTek Epoch reader and Gen5 software version 3.0.9. Each serum sample dilution was tested in duplicates. In parallel, a serial dilution of a polyclonal IgG rat isotype control (Southern Biotech, Birmingham, AL, USA) was implemented, allowing for concentration calculation as the sample signals were correlated to a standard curve of the isotype control.
4
SARS-CoV-2 S1 and S2 Protein ELISA
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ELISA protocol generally followed that of precious study.18 (link) Briefly, costar 96-well clear plates (Costar, 42592) were coated with 500 ng/mL SARS-CoV-2 S1 protein (Sino Biological, 40591-V08H) or SARS-CoV-2 S2 protein (Sino Biological, 40590-V08B) overnight at 4 °C. The next day, plates were blocked with 100 μL blocking buffer (5% FBS and 0.1% Tween 20 in PBS) at room temperature for 2 h. After washing with PBST buffer (0.1% Tween 20 in PBS), 1:100 diluted serum was then added to the plates and incubated for 1 h at room temperature. Serum was diluted in blocking buffer. Serum was heat inactivated at 56 °C for 30 min before added to the plate. Then, these plates were washed 5 times with 0.05% PBS-Tween 20. Then these ELISA plates were incubated with anti-human IgG HRP antibody (Bioss Biotech, 0297D) at room temperature for 1 h. Anti-human IgG antibody was used at a 1:3000 dilution. Then, these plates were washed 5 times with 0.05% PBS-Tween 20 and 100 μL TMB buffer (Beyotime, P0209) was added and reacted for 15 min at room temperature. These reactions were stopped with 1 M H2SO4 stopping buffer. Plates were read on a Beckman Coulter Plate Reader at 450 nm, and ODs were background subtracted.
5
SARS-CoV-2 S1 Antibody ELISA Assay
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End-point titers of anti-S1 total IgG or its isotypes (IgG1, IgG2a and IgG2b) from immunized mice were determined by ELISA, as previously described [29 (link)]. Briefly, 96-well plates were coated with 1 μg/mL SARS-CoV-2 S1 protein (Sino Biological, Beijing, China) at 4 °C overnight. Plates were washed three times with PBS containing 0.1% Tween-20 (PBS-T) before blocking with 5% skim milk in PBS-T for 1 h at room temperature. After washing, 2-fold serial dilutions of mouse sera, starting from 1:100, were added to wells and incubated for 1 h at 37 °C. Then, peroxidase-conjugated rabbit anti-mouse IgG secondary antibodies (Sigma, Germany) were added at the recommended concentrations and incubated for 1 h at 37 °C. After extensive washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD, USA) was added for 30 min to develop a colorimetric reaction. Finally, the reaction was stopped with 0.16 M sulfuric acid, and absorbance was read spectrophotometrically at 450 nm on a Synergy 2 Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA). End-point titers were determined as the reciprocals of the highest dilution with an OD above the cut-off value which was defined as the OD mean from the control group plus three standard deviations (SDs).
6
Luminex-based SARS-CoV-2 Variant Antigen Assay
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Antigens used for Luminex based assays included SARS-CoV-2 D614G spike protein (kindly provided by Erica Saphire, La Jolla Institute for Immunology), SARS-CoV-2 S1 protein (Sino Biological), SARS-CoV-2 S2 protein (Sino Biological), and SARS-CoV-2 RBD (kindly provided by Aaron Schmidt, Ragon Institute), as well as antigens from SARS-CoV-2 VOCs, such as Alpha (B.1.1.7) spike protein (LakePharma), Beta (B.1.351) spike protein (LakePharma), Gamma P1 spike protein (LakePharma), Kappa B.1.617.1 spike protein (Sino Biological) and Delta B.1.617.2 spike protein (kindly provided by Erica Saphire, La Jolla Institute for Immunology). Alpha (B.1.1.7), Beta (B.1.351), Gamma P1, Kappa (B.1.617.1) and Delta (B.1.617.2) RBDs were kindly provided by Florian Krammer, Icahn School of Medicine at Mount Sinai.
7
Epitope Correlation of SARS-CoV-2 Antibodies
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Competition enzyme-linked immunosorbent assay (ELISA) was performed to explore the epitope correlation of two antibodies. Briefly, the first antibody at the concentration of 2 μg/mL was coated on plates (BEAVER) and incubated at 4 °C overnight. Then, excess antibodies were washed away by PBS and blocked by 3% skim milk. SARS-CoV-2 S1 protein (Sino Biological) was biotinylated using an EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (ThermoFisher), followed by mixing with 50 μg/mL of the second competition antibodies or PBS blank control. After incubation at 37 °C for 1 h, plates were washed three times with PBS and the diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma) was added (1:2000) subsequently. Then the plates was incubated at 37 °C for 1 h again. TMB Single-Component Substrate Solution (Solarbio) was used to detect the S1 binding with the coated first antibodies. The absorbance at 450 nm was measured in an Infinite M200 PRO Multimode Microplate Reader (TECAN). Competitive percentage of two antibodies was calculated with reference to the PBS blank control.
8
Production and Purification of SARS-CoV-2 Spike Protein
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Genes encoding residues 319–532 of SARS-CoV-2 (GenBank accession number: QHD43416.1) spike protein fused with the genes of Fc or His in its N-terminal were inserted into the plasmid of PcDNA3.1. The recombinant expression plasmids were transfected into Expi293F cells, and then the cells were cultured for three days. After that, cell culture supernatants were collected and purified using affinity chromatography. The purified recombinant proteins were concentrated by ultrafiltration using Amicon Ultra-10 filters (Millipore, USA). Finally, the purified recombinant proteins were analyzed using SDS-PAGE. Briefly, 10% Tris-glycine SDS-PAGE was used to separate the proteins, and then the proteins in the gel were stained using Coomassie Brilliant Blue to visualize the protein lines. The SARS-CoV-2 S1 protein was purchased from Sino Biological (Beijing, China)
9
SARS-CoV-2 Antibody Quantification ELISA
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Ninety-six-well plates were coated with 1 μg/ml SARS-CoV-2 S1 protein (Sino Biological Inc.) or S1 RBD protein (Sino Biological Inc.) in PBS and incubated at 4°C overnight. After coating, plates were treated with 2% bovine serum albumin (BSA) (Sigma)/PBS for 2-5 h at 4°C. After blocking, serially diluted heat-inactivated sera or nasal washes (1%BSA/PBS) from the vaccinated or control mice were added to wells, and the plates were incubated for 1.5 h at RT, followed by washing 6 times with PBST and subsequent 1h incubation with an HRP-conjugated goat anti-mouse IgG (1:3,000 dilution; BioLegend) or goat anti-mouse IgA antibody (1:5,000 dilution; SouthernBiotech) in 1% BSA/PBS at RT. The wells were washed 6 times with PBST, and 100 μL of the TMB substrate set (BioLegend) was added to the immune complexes in each well. Plate development was then halted by the addition of 50 μL of 2N H2SO4 per well. The absorbance at 450 nm was recorded using a microplate reader. The ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2. (Log10 endpoint titers were given).
10
SARS-CoV-2 Spike Protein Western Blot Analysis
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Samples were run on a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% milk for 45 min at room temperature. The anti-SARS-CoV-2-S1 rabbit monoclonal antibody (Sino Biological) and the goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) (Sino Biological) were used as the primary antibody (1:2000 dilution) and secondary antibody (1:10,000 dilution), respectively, for the identification of proteins expressed in the PP samples. The sera of immunized mice and goat anti-mouse IgG-HRP (Sino Biological) were used as the primary antibody (1:1000 dilution) and secondary antibody (1:10,000 dilution), respectively, for the identification of SARS-CoV-2 spike recombinant RBD protein (Sino Biological) and SARS-CoV-2 S1 protein (Sino Biological). Blots were visualized by using Clarity™ Western ECL Substrate (Bio–Rad).
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