Single cell suspensions of splenocytes from vaccinated mice were polyclonally stimulated for expansion of memory B cells in 96-well U bottom plates with 1ug/ml CLO97 and 10ng/ml rmIL-2 (Mabtech, Inc., Cat: 3825–2A) for 72 hours at 37°C, 5% CO2. Following 72 hours of polyclonal stimulation, splenocytes from vaccinated mice were washed and then incubated in 5ug/mL biotinylated RBD protein (Sino Biologicals, 40592-V08H-B) for 1 hr at room temperature (RT). After incubation, cells are washed and stained with APC-streptavidin (Cat: 405207) and PE-streptavidin (Cat: 405204) for 30 min at RT. Cells are washed and incubated with fluorochrome-conjugated antibodies for at least 15–20 min at 4 °C or on ice, protected from light. The following fluorochrome-conjugated anti-mouse antibodies were used: CD3 (Clone: 17A2, Cat: 100216), CD19 (Clone: 6D5; Cat: 115530), GL-7 (Clone: GL7; Cat: 144610), CD38 (Clone: 2H7; Cat: 102732), CD95 (Clone: SA367H8; Cat: 152612), B220 (Clone: RA3–6B2; Cat: 103241), IgD (Clone: 11–26c.2a; Cat: 405742), CD138 (Clone: 281–2; Cat: 142514), were all from Biolegend. LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) (Cat. Number: L34957) was used to gate on live cells. Samples were acquired on a BD LSR II and data were analyzed using FlowJo software (Treestar).
Rbd protein
Manufactured by Sino Biological
Sourced in China
The RBD protein is a recombinant protein produced by Sino Biological. It is a fragment of the SARS-CoV-2 spike protein that binds to the human ACE2 receptor, which is the primary entry point for the virus into human cells.
Automatically generated - may contain errors
Lab products found in correlation
Maxisorp plate, by Thermo Fisher Scientific (3 mentions)
K blue advanced substrate, by Neogen (2 mentions)
Hrp conjugated goat anti mouse igg h l secondary antibody, by Abcam (2 mentions)
Versamax, by Molecular Devices (2 mentions)
Biacore evaluation software, by GE Healthcare (2 mentions)
Biacore 8k system, by GE Healthcare (2 mentions)
Biacore 8k, by Cytiva (2 mentions)
Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
Prism 9, by GraphPad (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Peroxidase affinipure goat anti human igg h l antibody, by Jackson ImmunoResearch (1 mentions)
Octet data acquisition 9, by Sartorius (1 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Facs lsrfortessa, by BD (1 mentions)
Fitc conjugated anti cd3, by BD (1 mentions)
Apc conjugated anti ifn γ, by BD (1 mentions)
V500 conjugated anti cd4, by BD (1 mentions)
22 protocols using rbd protein
1
Memory B Cell Profiling of Vaccinated Mice
2
SARS-CoV-2 Spike Protein ELISA
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Fifty nanograms of S2P spike trimer or RBD protein (Sino Biological Inc. (Cat: 40,592-V08H121 and 40,592-V08H)) was coated on each well of an ELISA plate at 4°C overnight. Then the plates were blocked with 300 mL of blocking buffer (1% BSA and 10% bovine calf serum in PBS) at 37°C for 2 h. Afterward, serially diluted convalescent serum or Moderna vaccinee serum was added and incubated at 37°C for 1 h. Next, 100 μL per well of 10,000-fold diluted Peroxidase AffiniPure goat anti-human IgG (H + L) antibody (Jackson ImmunoResearch) was added and incubated for 1 h at 37°C. Between each step, the plates were washed with PBST three times. Finally, the TMB substrate (Sigma, St. Louis, MO) was added and incubated for 5 min at room temperature before the reaction was stopped using 1 M sulfuric acid. Absorbance was measured at 450 nm.
The serum endpoint dilutions that achieved an OD450 value > 3-fold over background were calculated by fitting the data in five-parameter dose-response curves in GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA).
The serum endpoint dilutions that achieved an OD450 value > 3-fold over background were calculated by fitting the data in five-parameter dose-response curves in GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA).
3
Kinetic Analysis of Antibody-RBD Interactions
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Antibodies to be tested were diluted to the concentration of 4 μg/mL with PBST and then immobilized onto Octet Fab2G biosensors for real-time association and dissociation. After arriving at the Signal Change Threshold 1.1 nm and washed in PBST biosensor tips were immersed into the wells containing RBD protein (40592-V05H, Sino Biological) of serial dilutions and allowed to associate for 200 s followed by a dissociation step of 400 s. KD was calculated using a 1:1 binding model in Octet Data Acquisition 9.0.0.49 (Sartorius AG). Experiments were performed in triplicate.
4
SARS-CoV-2 RBD Antibody ELISA
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Serum binding antibodies against SARS-CoV-2 RBD were determined by ELISA (HIPRA). MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with 100 ng/well RBD protein (Sino Biologicals, Beijing, China) and blocked with 5% non-fat dry milk (Difco Skim Milk, BD, Franklin Lakes, NJ, USA) in PBS. Wells were incubated with serial dilutions of the serum samples and the bound total IgG specific antibodies were detected by peroxidase-conjugated Goat Anti-Mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Finally, wells were incubated with K-Blue Advanced Substrate (Neogen, Lansing, MI, USA) and the absorbance at 450 nm was measured using a microplate reader (Versamax microplate reader, Molecular Devices, San Jose, CA, USA). The mean value of the absorbance was calculated for each dilution of the serum sample run in duplicate. Isotypes IgG1 and IgG2a were detected using Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 1 specific, and Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific, (Jackson ImmunoResearch, Cambridge, UK), respectively. The endpoint titer of RBD-specific total IgG binding antibodies was established as the reciprocal of the last serum dilution yielding 3 times the mean optical density of the negative control of the technique (wells without serum added).
5
Measuring RBD-specific T Cell Responses
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Splenocytes from each immunized mouse were seeded into 96-well plates (1 × 106 cells/well) in complete RPMI-1640 medium and stimulated with RBD protein (Sino Biological, Beijing, China; 5 μg/ml) in a total volume of 200 μl for 20 h. Stimulation with PMA (50 ng/ml) and ionomycin (1 μg/ml) was used as a positive control. To prevent cytokine release, brefeldin A (10 μg/ml) was added, and the cells were cultured for an additional 4 h. Cell surface markers were stained with FITC-conjugated anti-CD3, V500-conjugated anti-CD4 and PE-conjugated anti-CD8 (BD, New Jersey, USA) antibodies. After being fixed with 1% paraformaldehyde (20 min, RT) and permeabilized with 0.1% Triton X-100 (30 min, RT), the cells were stained intracellularly with APC-conjugated anti-IFN-γ (BD, New Jersey, USA) antibody. The stained cells were analyzed using a FACS LSRFortessa instrument (BD, New Jersey, USA) and the FlowJo program.
6
SARS-CoV-2 RBD Antibody Response in Mice
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RBD-specific IgG, IgG1 and IgG2a antibody titers in immunized mice serum were detected by ELISA. 20 μL RBD protein (Sinobiological: #40592-V05H, 3 μg/mL) were added to the 384-well plate and then incubated overnight at 4°C. After washing, the plates were blocked with blocking buffer (5% BSA plus 0.05% Tween 20) at 37°C for 1 hour and incubated with 20μL testing mice serum with ten-fold serial dilutions at 37°C for half an hour. Reacted mice serum were detected using HRP-conjugated Goat Anti-Mouse IgG H&L secondary antibody (Abcam: #ab6789, 1: 10000), HRP-conjugated Goat Anti-Mouse IgG1 H&L (Bethyl: #A90-105P, 1: 10000) and HRP-conjugated Goat Anti-Mouse IgG2a H&L (Bethyl: #A90-107P, 1: 10000) respectively.
7
Quantifying Antibody Levels in Mouse Samples
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The IgG and IgA antibody levels in mouse serum and BALF were measured by ELISA, as previously described55 (link). Briefly, ELISA plates were coated overnight at 4 °C with RBD protein of WT or BA.5 strain or HA protein of pH1N1 (100 ng/well, Sino Biological, Beijing, China). For lgG detection, serum samples were threefold serially diluted with 1:400 starting dilutions, and BALF samples were twofold serially diluted with 1:5 starting dilutions. For IgA detection, BALF samples were twofold serially diluted using an undiluted stock solution as the starting dilutions. Following 2-h incubation at 37 °C, secondary antibodies were added: HRP-conjugated anti-mouse IgG antibody (1:100000 dilution, ab6789; Abcam, Cambridge, UK); HRP-conjugated anti-mouse IgA (1:5000 dilution, 1040-05; Southern Biotech, Birmingham, AL, USA). Plates were again incubated for 1 h at 37 °C, followed by the addition of TMB substrate (NCM Biotech, Suzhou, China). Sulfuric acid (2 M H2SO4) solution was used to stop the reaction. The optical density (OD)450–630 was recorded using a microplate reader (Tecan, Männedorf, Switzerland). The binding antibody endpoint titer was determined as the reciprocal of the highest serum dilution that yielded an absorbance greater or equal to 0.1 OD unit above the absorbance of the pre-immune samples.
8
SARS-CoV-2 Anti-RBD Antibody Quantification
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Anti-RBD IgG antibodies in plasma samples were determined as described in (10.21203/rs.3.rs-1551351/v1) [11 (link)]. In brief, plasma samples were heat-inactivated and assayed in duplicate against the RBD protein (Sino Biological, Beijing, China) or antigen-free wells. A serially-diluted positive plasma sample was used as standard, and a pool of ten SARS-CoV-2-negative plasma samples were included as negative controls. Samples were incubated for two hours at room temperature. A horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific) (Jackson Immunoresearch, West Grove, PA, USA) was used as secondary antibody and O-phenylenediamine dihydrochloride (OPD) was used as a substrate (Sigma Aldrich, St. Louis, MO, USA). The enzymatic reaction of HRP was stopped with 2 M of H2SO4 (Sigma Aldrich). Signal was analyzed as the optical density at 492 nm with noise correction at 620 nm. Antigen-specific signal was calculated by subtracting background signal obtained for each sample in antigen-free wells. Results are shown as arbitrary units (AU)/mL.
9
SARS-CoV-2 RBD Antibody Detection
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RBD-specific IgG, IgG1, and IgG2a antibodies in mouse serum were detected by ELISA. Here, 20 μl RBD protein (Sinobiological) were added to the 384-well plate in duplets to make a final concentration of 3 μg/ml. The plates were incubated overnight at 4°C. After washing, the plates were blocked with blocking buffer [5% bovine serum albumin (BSA) plus 0.05% Tween 20] at 37°C for 1 h and incubated with 20 µl testing mouse serum with 10-fold serial dilutions at 37°C for 30 min. Reacted mouse serum was detected using HRP-conjugated Goat Anti-Mouse IgG H&L secondary antibody (Abcam, ab6789, 1:10,000), HRP-conjugated Goat Anti-Mouse IgG1 H&L (Bethyl, A90-105P, 1:10,000), and HRP-conjugated Goat Anti-Mouse IgG2a H&L (Bethyl, A90-107P, 1:10,000).
10
Measuring Antibody Affinity to SARS-CoV-2 Proteins
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Antibody affinity was measured on Pall ForteBio Octet RED96 system. Recombinant antibodies (20 μg/ml) was loaded onto the Protein A biosensors for 300 s. Following 10 s of baseline in kinetics buffer, the loaded biosensors were dipped into serially diluted (0.14–300 nM) RBD protein (Sino Biological, Cat: 40592-V08B) or the previously generated S protein for 200 s to record association kinetics5 (link). The sensors were then dipped into a kinetic buffer for 400 s to record dissociation kinetics. Kinetic buffer without antigen was set to correct the background. The Octet Data Acquisition 9.0 was used to collect affinity data. For fitting of KD value, Octet Data Analysis software V11.1 was used to fit the curve by a 1:1 binding model and use the global fitting method. Similarly, binding affinities to ACE2 by WT sCoV2-RBD protein, mutant sCoV2-RBD proteins, and sCoV-RBD protein were measured.
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