Antibody binding competition assay was performed using Bio-Layer Interferometry (BLI) on Octet QK instrument (ForteBio). The first mAb (30 μg/mL) was immobilized onto the anti-human IgG Fc capture (AHC) (Fortebio, China). After washing with PBS, the biosensor tips were immersed into a well containing the wildtype SARS-CoV-2 RBD Protein (SinoBiological, Beijing, China) at a concentration of 51.5 μg/mL and loaded into a well containing the secondary mAb at a concentration of 30 μg/mL.
Sars cov 2 rbd protein
Manufactured by Sino Biological
Sourced in China
The SARS-CoV-2 RBD protein is a recombinant protein derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The RBD is a critical region of the spike protein that mediates the virus's attachment to the human ACE2 receptor, which is the primary entry point for the virus into human cells.
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Lab products found in correlation
Anti hamster igg hrp, by Southern Biotech (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Beyotime (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Solarbio (2 mentions)
Incomplete freund s adjuvant, by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Octet qk instrument, by Molecular Devices (1 mentions)
Anti human igg fc capture ahc, by Molecular Devices (1 mentions)
O phenylenediamine tablets, by Merck Group (1 mentions)
Anti sars cov 2 spike elisa, by EUROIMMUN (1 mentions)
96 well plate, by Corning (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
S1 protein, by Sino Biological (1 mentions)
Tsr3000 membrane strip reader, by BioDot (1 mentions)
Sars cov 2 s protein, by Sino Biological (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Rbd pseudotyped lentivirus, by VectorBuilder (1 mentions)
Ace2 sars cov 2 spike inhibitor screening assay kit, by BPS Biosciences (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Sars cov 2 spike protein, by Sino Biological (1 mentions)
18 protocols using sars cov 2 rbd protein
1
Antibody Binding Affinity by BLI
2
SARS-CoV-2 Antibody Detection by ELISA
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A semi-quantitative in vitro determination of human IgG and IgA antibodies against the SARS-CoV-2 was performed on serum samples by using the Anti-SARS-CoV-2 Spike ELISA (EUROIMMUN), according to the manufacturer’s instructions. Values were then normalized for comparison with a calibrator. Results were evaluated by calculating the ratio between the extinction of samples and the extinction of the calibrator. Results are reported as the ratio between OD sample and OD calibrator. The ratio interpretation was as follows: < 0.8 = negative, ≥ 0.8 to < 1.1 = borderline, ≥ 1.1 = positive. To detect IgM anti-RBD we developed an in-house ELISA [17 (link)]. 96-well plates (Corning) were coated for 1 h at 37 °C with 1 μg/mL of purified SARS-CoV-2 RBD protein (Sino Biological). After washing with PBS 1 × /0.05% Tween and blocking with PBS 1 × /1% BSA, plates were incubated for 1 h at 37 °C with diluted sera (1:100). After washing again, plates were incubated for 1 h at 37 °C with peroxidase-conjugated goat anti-human IgM antibody (Jacksons ImmunoResearch Laboratories). The assay was developed with o-phenylenediamine tablets (Sigma-Aldrich) as a chromogen substrate. Absorbance at 450 nm was measured, and IgM concentrations were calculated by interpolation from the standard curve based on serial dilutions of monoclonal human IgM antibody against SARS-CoV-2 Spike-RBD (Invivogen).
3
SARS-CoV-2 RBD Antibody Detection by ELISA
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Antibody was tested by indirect ELISA with the SARS-CoV-2 RBD protein (Sino Biological Inc., China) and peroxidase conjugated goat anti-cat IgG (Sigma-Aldrich, USA). Briefly, ELISA plates were coated overnight at 4°C with RBD protein (1 μg/ml, 100 μl per well). After blocked with PBS containing 5% skim milk for 2 h at 37°C, the plates were added with sera at a dilution of 1: 40. After incubation for 30 min at 37°C, the plates were washed five times with washing buffer (PBS containing 0.05% Tween-20). A 1:20,000 diluted anti-cat IgG was added and incubated for an additional 30 min. After another 5 washes, TMB Substrate (Sigma-Aldrich, USA) was added and incubated for 10 min. Then the reaction was stopped, and optical density (OD) was measured at 450 nm. As the judgment method described previously[16 (link), 17 (link)], those sera were considered positive if the OD values were twice higher than the mean OD of the 39 sera collected between Mar. and May, 2019.
4
SARS-CoV-2 Protein Sensitivity Assay
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Strip sensitivity was determined using a serial diluted of SARS-CoV-2 RBD protein produced in this study (1 mg/mL) and S1 protein (1 mg/mL, Sino Biological Inc.). The protein of SARS-CoV-2 RBD protein produced in this study was diluted 2 times from 4,000 to 15.63 ng/mL with 0.01 M PBS and S1 protein (1 mg/mL, Sino Biological Inc.) was diluted 2 times from 4,000 to 62.5 ng/mL with 0.01 M PBS. Hundred microliter of each sample was added to the sample pad of the test strip and incubated for 10 min at room temperature. Test lines were scanned with TSR3000 Membrane Strip Reader (BioDot, USA) to obtain the relative optical density (ROD). The result was displayed as G/D × area (graphdensity × area)—ROD value, and it was considered positive if it was >10, otherwise it was negative.
5
Quantification of RBD-Specific Antibodies
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RBD-specific binding antibodies were assessed by ELISA essentially as described10 (link),11 (link). Briefly, 96-well plates were coated with 1 µg ml−1 of SARS-CoV-2 RBD protein (Aaron Schmidt, Massachusetts Consortium on Pathogen Readiness) or 1 µg ml−1 of SARS-CoV-2 S protein (Sino Biological) in 1× Dulbeccoʼs phosphate-buffered saline (DPBS) and incubated at 4 °C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween-20 in 1× DPBS) and blocked with 350 µl of casein block per well for 2–3 h at room temperature. After incubation, the block solution was discarded and plates were blotted dry. Three-fold serial dilutions of heat-inactivated serum in casein block were added to wells, and plates were incubated for 1 h at room temperature. Plates were washed three times and then subsequently incubated for 1 h with 0.1 µg ml−1 of anti-hamster IgG HRP (SouthernBiotech) in casein block at room temperature in the dark. Plates were washed three times, and then 100 µl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 µl of SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance two-fold above background.
6
SARS-CoV-2 RBD Protein Expression
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HeLa, HEK293T, Huh7 or Vero cells were seeded in 24-well plates at 200,000 cells/well. Eighteen hours later, the cells were transfected with RBD or control mRNA (2 μg/ml) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). Six hours later, the medium was replaced with Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific). The supernatant was collected at 48 hours after transfection, clarified by centrifugation at 1000 x g, and then mixed with 5 × SDS loading buffer (non-reducing). The samples were loaded for SDS-PAGE without heating. The secreted RBD protein was then detected by western blotting with a monoclonal antibody (mAb) against the SARS-CoV-2 RBD protein (Sino Biological).
7
Screening Assay for ACE2-SARS-CoV-2 Spike Inhibitors
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Corilagin (purity > 99%) was purchased from Chenguang Biological Technology Co., Ltd (Baoji, China) and was also verified by NMR and UPLC-UV (Fig. S1-3). Its chemical structure was shown in the Fig. 1 . DMSO was supported by Sigma-Aldrich (St. Louis, MO, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumromide) powder was obtained from Thermo Fisher (Waltham, MA, USA). FluorSave reagent was purchased from Calbiochem (San Diego, CA, USA). The ACE2: SARS-CoV-2 Spike Inhibitor Screening Assay Kit (Cat: #79936) was purchased from BPS Biosciences (San Diego, CA, USA). Purified ACE2-His tag protein and SARS-CoV-2 RBD protein were supported by Sino Biological (Beijing, China). ACE2-EGFP plasmid, ACE2-mCherry construct and RBD-pseudotyped lentivirus were purchased from Vectorbuilder (Chicago, IL, USA).Fig 1 ![]()
Chemical structure of corilagin.
8
SARS-CoV-2 RBD Antibody Detection ELISA
9
SARS-CoV-2 RBD Protein Immunization
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The animal studies were approved by the Institutional Animal Care and Use Committee at Chongqing Academy of Animal Sciences. CAMouseHG mice were subjected to multiple subcutaneous points primed with the SARS-CoV-2 RBD protein (Sino Biological, Beijing, China) (100 μg/mouse) in complete Freund’ adjuvant (Sigma-Aldrich) plus 25 μg of CpG (Sangon Biotech, China) and 1% (v/v) Alhydrogel (vac-alu-50, In vivogen). The mice were then successively boosted with the SARS-CoV-2 RBD protein in incomplete Freund’s adjuvant (Sigma-Aldrich) plus CpG and Alhydrogel at 1 week interval. Four days after the last injection, spleen cells were isolated and mixed with SP2/0 cells at a ratio of 1:5 to obtain hybridoma cells by electrofusion. Electrofusion was performed using BTX ECM2001 (BTX, San Diego, CA) by application a single 40-μs pulses at 900 V. The fused cells were added into ClonaCell™-HY Medium D (STEMCELL Technologies) to a final concentration of 0.9 × 107 cells/mL. and cultured at 37°C in a 5% CO2 incubator. After 8 days of culture, cell clones were picked and seeded into a 96-well plate. Cell supernatant was collected after 3-4 days culture for the further analysis.
10
SARS-CoV-2 RBD Protein ELISA Assay
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Enzyme-linked immunosorbent assay (ELISA) plates (Thermo Fisher, 446469) were coated with 50 ng SARS-CoV-2 RBD protein (Sino Biological, 40592-V08H) in 100 µL PBS per well overnight. Then, the ELISA plates were incubated with blocking buffer (5% fetal bovine serum + 0.1% Tween 20 in PBS) for 1 hour. Nasal mucosal samples (50 µL) were next added to each well and incubated for 1 hour. The ELISA plates were then washed with PBST (PBS + 0.1% Tween 20), incubated with horseradish peroxidase–conjugated goat antihuman immunoglobulin G (IgG) antibody (Bioss Biotech), and washed with PBST, and then TMB (Beyotime) was added. The ELISA plates were allowed to react with TMB for approximately 5 minutes and then stopped by 1 M sulfuric acid stop buffer. The optical density value was determined at 450 nm. In each ELISA assay, a range of serially diluted original/unmodified 35B5 mAb (0.00064, 0.0032, 0.016, 0.08, 0.4, 2, 10, and 50 µg/mL; 5-fold dilution) was used as positive control.
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