Recombinant SARS-CoV-2 Spike protein (50 ng, Sino Biological) diluted in carbonate buffer (0.1 M, pH 9.6) were coated into 96-well EIA/RIA plates (Coning) overnight at 4 °C. The plates were then washed with PBS-T (0.05% Tween-20) and were blocked with 10% goat serum in PBS for 2 h at 37 °C. Then, serum samples serially diluted in PBST containing 2% goat serum were added and incubated for 2 h at 37 °C. After washing, total IgG was evaluated using HRP-conjugated goat anti-mouse IgG Ab (1:10,000) or HRP-conjugated goat anti-NHP IgG Ab (1:50,000) for 1 h. In mice experiments, IgG subclasses were evaluated using biotinylated anti-mouse IgG2a mAb (clone: MG2a, Mabtech), biotinylated anti-mouse IgG1 mAb (clone: MG1, Mabtech), biotinylated anti-mouse IgG2c mAb (clone: MTG2c, Mabtech) for 1 h and further analyzed with Streptavidin-HRP (1:1000, Mabtech). TMB substrate (Solarbio) was used for development and the absorbance was read at 450 nm using SPECTROsta Nano (BMG) microplate reader. Endpoint titers were calculated as the dilution that exceed 2.1-folded value of the background.
Recombinant sars cov 2 spike protein
Manufactured by Sino Biological
Sourced in China
The Recombinant SARS-CoV-2 Spike protein is a laboratory reagent produced by Sino Biological. The spike protein is a structural protein found on the surface of the SARS-CoV-2 virus, which is responsible for the virus's ability to bind to and infect human cells.
Automatically generated - may contain errors
Lab products found in correlation
Tmb substrate, by Solarbio (1 mentions)
Streptavidin hrp, by Mabtech (1 mentions)
Multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
S1 protein, by Sino Biological (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
Pe labeled goat anti mouse igg fc antibody, by Jackson ImmunoResearch (1 mentions)
Versene solution, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Hrp conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Igg2a, by Jackson ImmunoResearch (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
6 protocols using recombinant sars cov 2 spike protein
1
SARS-CoV-2 Spike Protein IgG Assay
2
SARS-CoV-2 Spike and Envelope Protein Assays
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Bromelain API was manufactured by Mucpharm Pty Ltd (Kogarah, Australia) as a sterile powder. Acetylcysteine was purchased from Link Pharma (Cat# AUST R 170803; Warriewood, Australia). The recombinant SARS-COV-2 spike protein was obtained from SinoBiological (Cat# 40589-V08B1; Beijing, China). The recombinant envelope protein was obtained from MyBioSource (Cat# MBS8309649; San Diego, CA, USA). All other reagents were from Sigma Aldrich (St. Louis, MO, USA).
3
SARS-CoV-2 Spike Antibody Quantification by ELISA
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The titer of serum total anti-Spike antibodies was assessed on serum samples from the orbit vein of mice by ELISA. Blood samples, collected from the mice via orbit vein bleeding at seven days after three rounds immunization, were placed at room temperature for 1 h. After centrifugation at 6000 rpm for 5 min at 4 °C, supernatants were used for ELISA.
High binding ELISA plates were coated with 1 μg/mL recombinant SARS-CoV-2 Spike protein (Sino Biological, 40589-V08B1) for mouse Q1, Q2, Q3, Q6, and Q7 or S1 protein (Sino Biological, 40591-V02H) for mouse Q14 and Q15 at 4 °C overnight, and then blocked with 3% skim milk powder in PBST (PBS containing 0.05% Tween-20) at 37 °C for 1 h. Serially diluted serum (1:100, 1:500, 1:2500, and 1:12500, respectively) in PBST was added into the plates and incubated for 1 h at 37 °C. After washing two times, antibodies binding to coated proteins were detected by peroxidase Labeled Goat anti-human IgG (H + L) (KPL, 474-1006) for Q1, Q2, Q3, Q6, and Q7 or HRP-Goat anti-human Ig Fab (Southern Biotech) for Q14 and Q15. OD450 was read at Multimode Microplate Reader (Thermo Scientific varioskan flash).
High binding ELISA plates were coated with 1 μg/mL recombinant SARS-CoV-2 Spike protein (Sino Biological, 40589-V08B1) for mouse Q1, Q2, Q3, Q6, and Q7 or S1 protein (Sino Biological, 40591-V02H) for mouse Q14 and Q15 at 4 °C overnight, and then blocked with 3% skim milk powder in PBST (PBS containing 0.05% Tween-20) at 37 °C for 1 h. Serially diluted serum (1:100, 1:500, 1:2500, and 1:12500, respectively) in PBST was added into the plates and incubated for 1 h at 37 °C. After washing two times, antibodies binding to coated proteins were detected by peroxidase Labeled Goat anti-human IgG (H + L) (KPL, 474-1006) for Q1, Q2, Q3, Q6, and Q7 or HRP-Goat anti-human Ig Fab (Southern Biotech) for Q14 and Q15. OD450 was read at Multimode Microplate Reader (Thermo Scientific varioskan flash).
4
Quantifying hACE2 Expression in 3T3 Cells
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To examine hACE2 expression, 3T3 cells were transduced by AAV6/hACE2 or AAV9/hACE2 for 48 hours and dissociated by Versene solution (Thermo Fisher Scientific, MA, USA) at 37°C for 30 minutes. For each test, 2×105 cells were resuspended in 100 μl of Dulbecco’s phosphate buffered saline (DPBS) containing 1% FBS and then incubated with 0.2 μg of recombinant SARS-CoV-2 Spike protein (Sino Biological, Beijing, China) for 1 hour on ice. The recombinant protein consists of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein and a mouse Fc region at the C-terminus. Next, the cells were incubated with 100 μl PE-labeled goat anti-mouse IgG-Fc antibody (Jackson Immuno Research, PA, USA) for 30 minutes. After the staining procedure, cells were resuspended again in 300 μl of 1% FBS-DPBS containing 0.25 μg of 7-amino-actinomycin D (Thermo Fisher Scientific, CA, USA) to exclude non-viable cells, and then analyzed by LSRII flow cytometer (BD Biosciences, MA, USA).
5
SARS-CoV-2 Spike Protein IgG ELISA
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The specific antibody response against SARS-CoV-2 was determined by using ELISA. Briefly, 50 μL of 4 μg/mL recombinant SARS-CoV-2 spike protein (Sino Biological, cat# 40589-V08B1) in 0.1 M carbonate buffer (pH 9.5) was coated onto 96-well microplates by overnight incubation at 4 °C. The coated plates were washed twice with 0.05% Tween 20 in PBS and then blocked with 3% BSA in PBS at room temperature for 1 h. Diluted sera from immunized animals were added to the wells and incubated for 2 h at room temperature. HRP-conjugated goat anti-mouse IgG (1:10,000; Thermo Scientific, cat# 31430), HRP-conjugated rabbit anti-hamster IgG (1:7000; Arigo Biolaboratories, cat# ARG23730), HRP-conjugated goat anti-rat IgG (1:50,000; Bethy Laboratories, cat# A110-136P), HRP-conjugated mouse anti-rat IgG2a (1:6000; GeneTex, cat# GTX02893-01) or HRP-conjugated mouse anti-rat IgG2b (1:6000; GeneTex, cat# GTX02894-01) was used as the secondary antibody. The assay was developed by using SureBlue TMB 1-Component Peroxidase Substrate (KPL, cat# 52-00-00). The absorbance was measured using an ELISA reader at 450 nm.
6
SARS-CoV-2 Spike Protein IgG ELISA
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Nunc ELISAs plates (ThermoFisher Scientific) were coated with 100 ng of recombinant SARS-CoV-2 spike protein (Cat.# V0589-V08B, Sino Biological) in 50mM carbonate-bicarbonate buffer, pH 9.6. overnight at 4 °C. Plates were washed and blocked with 2% bovine serum albumin (BSA) in PBS for 2h at room temperature (RT). Plates were washed again and incubated with serial dilutions of mouse sera and incubated for 1h at 37°C. Plates were washed three times with PBS with 0.05% Tween 20 and then incubated for 1h at RT with horse radish peroxidase (HRP)- conjugated anti-mouse IgG (1:5,000, Cat.#62-6520, ThermoFisher Scientific) , IgG1 (1:5,000, Cat.# 115-035-205, Jackson ImmunoResearch) or IgG2a (1:5,000, Cat.# 115-035-206, Jackson ImmunoResearch) secondary antibody. After final wash, plates were developed using 50μl of 1-Step Ultra TMB (3,3’,5,5’-tetramethylbenzidine; ThermoFisher Scientific) and the reaction stopped with an equal volume of 2M sulfuric acid before the optical density (OD) was read at 405 nm using an Infinite M200Pro microplate reader (Tecan). The endpoint titers of serum IgG responses were determined as the dilution that emitted an optimal density exceeding average of OD values plus three standard deviations of negative serum samples.
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