Plates were precoated with 5 µg/mL of hACE2 receptor overnight and washed with phosphate-buffered saline with 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA). Histidine-tagged S-RBD-Rhizavidin was threefold serially diluted and added to coated wells for 2 h at RT. Following washing, 100 µL of anti His-HRP (Bio-Rad, Hercules, CA, USA) was added to the plate. Plates were incubated for 1 h at RT, while SureBlue TMB Microwell Peroxidase Substrate (VWR, Radnor, PA, USA) equilibrated to RT. After a final wash, 100 µL TMB substrate was added to wells and development was stopped with 100 µL of 1N hydrochloric acid after 10 min at RT. The ELISA plates were read at an absorbance of 450 nm on a SpectraMax i3x Plate Reader using Softmax Pro 7.0.
Tbs startblock blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
TBS Startblock blocking buffer is a ready-to-use solution designed to block non-specific binding in western blotting and other immunoassay applications. It is a Tris-buffered saline (TBS) based formulation that effectively minimizes background signal.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax plus plate reader, by Molecular Devices (3 mentions)
T0440 il, by Merck Group (2 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (2 mentions)
Anti his hrp, by Bio-Rad (1 mentions)
Prism 7, by GraphPad (1 mentions)
Tmb stop solution, by ScyTek Laboratories (1 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (1 mentions)
4 protocols using tbs startblock blocking buffer
1
SARS-CoV-2 S-RBD Binding Assay
2
SARS-CoV-2 Spike Protein Binding Assay
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Ninety-six-well plates were coated with 100 μL SARS-CoV-2 S protein (2 μg mL−1) overnight at 4 °C. Plates were washed with phosphate-buffered saline with 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific). Histidine-tagged hACE2 and hDPP4 receptors were threefold serially diluted (5–0.0001 μg mL−1) and added to coated wells for 2 h at room temperature. The plates were washed with PBS-T. Optimally diluted (1:4000) horseradish peroxidase (HRP) conjugated mouse anti-histidine was added and color developed by addition of and 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) peroxidase substrate (T0440-IL, Sigma, St. Louis, MO, USA). Plates were read at an OD of 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax software. EC50 values were calculated by 4-parameter fitting using GraphPad Prism 7.05 software (San Diego, CA, USA).
3
SARS-CoV-2 Spike Protein IgG Titer Quantification
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An ELISA was used to determine anti-SARS-CoV-2 S IgG titers. Briefly, 96-well microtiter plates (Thermo Fisher Scientific, Rochester, NY, USA) were coated with 1.0 µg mL−1 of SARS-CoV-2 S protein. Plates were washed with PBS-T and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific). Mouse, baboon, or human serum samples were serially diluted (10−2 to 10−8) and added to the blocked plates before incubation at room temperature for 2 h. Following incubation, plates were washed with PBS-T and HRP-conjugated goat anti-mouse IgG (1:5000) or goat anti-human IgG (1:2000) (Southern Biotech, Birmingham, AL, USA) added for 1 h. Plates were washed with PBS-T and TMB peroxidase substrate (T0440-IL, Sigma, St. Louis, MO, USA) was added. Reactions were stopped with TMB stop solution (ScyTek Laboratories, Inc. Logan, UT). Plates were read at OD 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax software. EC50 values were calculated by 4-parameter fitting using SoftMax Pro 6.5.1 GxP software. Individual animal anti-SARS-CoV-2 S IgG titers and group GMTs and 95% confidence intervals (±95% CI) were plotted using GraphPad Prism 7.05 software.
4
SARS-CoV-2 S IgG Antibody ELISA
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An ELISA was used to determine anti-SARS-CoV-2 S IgG titers. Briefly, 96-well microtiter plates (ThermoFischer Scientific, Rochester, NY, USA) were coated with 1.0 µg mL−1 of SARS-CoV-2 spike protein. Plates were washed with PBS-T and blocked with TBS Startblock blocking buffer (ThermoFisher, Scientific). Mouse and baboon serum samples were serially diluted (10−2 to 10−8) and added to the blocked plates before incubation at room temperature for 2 h. Following incubation, plates were washed with PBS-T and HRP-conjugated goat anti-mouse IgG or goat anti-human IgG (Southern Biotech, Birmingham, AL, USA) added for 1 h. Plates were washed with PBS-T and 3,3’,5,5’-tetramethylbenzidine peroxidase substrate (TMB, T0440-IL, Sigma, St Louis, MO, USA) was added. Reactions were stopped with TMB stop solution (ScyTek Laboratories, Inc. Logan, UT). Plates were read at OD 450 nm with a SpectraMax®Plus plate reader (Molecular Devices, Sunnyvale, CA, USA), and data were analyzed with SoftMax® (Molecular Devices, Corp.) software. Data shown in the graph are the average of triplicate wells. EC50 values were calculated by 4-parameter fitting using SoftMax Pro 6.5.1 GxP software. Individual animal anti-SARS-CoV-2 rS IgG titers and group geometric mean titers (GMT) and 95% confidence interval (± 95% CI) were plotted GraphPad® Prism 7.05 software (Graphpad Software LLC).
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