96-well ELISA plates (Nunc Maxisorp 44-2404-21) were coated overnight at 4°C with 50 μL of Wuhan-1 RBD (Acrobiosystems SPD-C52H3) (2 µg/mL in PBS). Then, the coating solution was removed, and plates were blocked with 3% non-fat milk in PBS supplemented with 0.1% Tween-20 (P1379-500ML, Sigma) (PBST) for 1 hour at RT. Serum samples were pre-diluted in at 1:50 in 1% non-fat milk in PBST and incubated for 2 hours at RT. After three washes with 250 μL of PBST in a plate washer (Biotek), an anti-human IgG-horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) (GenScript A01854) was added for 1 hour at RT. Three washes were performed, and after an incubation of 2 min with 100 μL of TMB substrate (Thermo Scientific 34021), the reaction was stopped with 50 μL of stop solution (Thermo Scientific N600). The optical density (OD) was measured at 450 nm in a VictorNivo multimode plate reader (PerkinElmer) and shown OD values were calculated by subtracting the negative control values from all samples.
Spd c52h3
Manufactured by ACROBiosystems
The SPD-C52H3 is a centrifuge rotor designed for high-speed centrifugation applications. It features a capacity of 52 tubes and can achieve a maximum rotational speed of 52,000 rpm. The rotor is constructed from a durable, corrosion-resistant material to ensure reliable performance and long-term use.
Automatically generated - may contain errors
Lab products found in correlation
Nun c5227, by ACROBiosystems (2 mentions)
Plate washer, by Agilent Technologies (1 mentions)
P1379 500ml, by Merck Group (1 mentions)
Victor nivo multimode plate reader, by PerkinElmer (1 mentions)
Ab97225, by Abcam (1 mentions)
Ab97205, by Abcam (1 mentions)
Pr1200, by Solarbio (1 mentions)
A8020, by Solarbio (1 mentions)
C1058, by Solarbio (1 mentions)
S2n c52h5, by ACROBiosystems (1 mentions)
Spn c52h9, by ACROBiosystems (1 mentions)
P9416, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Il 1β, by Proteintech (1 mentions)
Ifn γ, by Proteintech (1 mentions)
Tnf α, by Proteintech (1 mentions)
Proteon xpr36, by Bio-Rad (1 mentions)
Spd c522g, by ACROBiosystems (1 mentions)
A00166, by GenScript (1 mentions)
S1n c52h3, by ACROBiosystems (1 mentions)
7 protocols using spd c52h3
1
SARS-CoV-2 RBD Antibody ELISA
2
SARS-CoV-2 Antibody Detection ELISA
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The SARS-CoV-2 S trimer, RBD, S2 and nucleocapsid protein (N) (Acro Biosystems SPN-C52H9, SPD-C52H3, S2N-C52H5, and NUN-C5227, respectively) were coated on 96-well plates at 0.5 μg/ml in PBS overnight at 4 °C. After the plates were blocked with buffer (1× PBS with 3% BSA (Solarbio, A8020) and 0.05% Tween-20 (Sigma, P9416)) for 1 h at 37 °C, plasma samples were added and incubated for 1 h at 37 °C. The plasma samples were assayed at a 1:100 starting dilution and 7 additional threefold serial dilutions in blocking buffer (1× PBS with 1% BSA (Solarbio, A8020) and 0.05% Tween-20). The plates were washed 5 times with washing buffer and then incubated with anti-human IgG (Abcam, ab97225) or IgM (Abcam, ab97205) secondary antibody conjugated to horseradish peroxidase (HRP) in blocking buffer at a 1:50,000 dilution (and IgG) or 1:20,000 dilution (IgM) for 0.5 h at 37 °C. After the plates were washed 5 times, the HRP substrate TMB (Solarbio, PR1200) was added for 10 min, followed by the addition of 50 μl of 1 M H2SO4 (Solarbio, C1058) to stop the reaction. The absorbance was measured at 450 nm with an ELISA microplate reader (TECAN Infinite 200 PRO).
3
SARS-CoV-2 Spike RBD Binding Assay
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The His-Tagged receptor binding domain (RBD) of the S1 SARS-CoV-2 spike protein (GenBank accession no. QHD43416.1; Arg319-Lys537; SPD-C52H3) was obtained from Acro Biosystems. Where indicated, Calu-3 cells were treated with 50 nM RBD and stimulated with the following tri-cytokine mix: 5 ng/mL TNFα (Proteintech, HZ-1014), 5 ng/mL IFNγ (Proteintech, HZ-1301), and 5 ng/ml IL1β (Proteintech HZ-1164).
4
SARS-CoV-2 RBD Binding Kinetics
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Surface plasmon resonance affinity and kinetic measurements were performed using the ProteOn XPR36 (Bio-Rad). Lanes of a general layer compact (GLC) chip were individually coated with 2 µg/mL SARS-CoV-2 Receptor Binding Domain (RBD) (#SPD-C52H3, ACRO Biosystems) in 10 mM sodium phosphate pH 6.0 and attached to the chip following the standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) coupling chemistry available from the manufacturer resulting ~ 1000RU of protein deposited. Binding kinetics of NIH-CoVnb-112 were tested at 25 °C by flowing six concentrations varying from 300 to 0 nM at 100 μL/min for 90 s or more then dissociation was monitored for at least 600 s. Following each run, the chip was regenerated by flowing 0.85% phosphoric acid (~ pH 3.0) across the surface. Data analysis was performed with ProteOn Manager 2.1 software, corrected by subtraction of an uncoated column as well as interspot correction. The standard error of the fits was less than 10%. Binding constants were determined using the Langmuir model built into the analysis software.
5
SARS-CoV-2 RBD Staining of Yeast Libraries
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Yeast libraries were stained with the SARS-CoV-2 RBD of the ancestral (SPD-C52H3, AcroBiosystems), delta (SPD-C5226, AcroBiosystems), omicron BA.1 (SPD-C522j, AcroBiosystems), or omicron BA.2 (SPD-C522g, AcroBiosystems) strains as previously described for omicron-specific enrichment using the FACSMelody. To normalize for antigen differences in background and signal, the binding-population threshold was set to 5% on the negative control for each antigen before it was applied to each of the anti-CoV-2 libraries with said antigen. Flow cytometry data were analyzed using FlowJo (BD Biosciences).
6
SARS-CoV-2 Antibody ELISA Assay
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ELISA assays were performed as previously described.30 (link) Briefly, MaxiSorp plates (96 wells; 442404, Thermo Scientific) were coated with recombinant SARS-CoV-2 S1 (S1N-C52H3–100 μg, ACROBiosystems), receptor-binding domain (RBD) (SPD-C52H3– 100 μg, ACROBiosystems) and the nucleocapsid protein (NUN-C5227–100 μg, ACROBiosystems) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4 °C. The primary antibodies used for the standard curves were human anti-spike (SARS-CoV-2 human anti-spike [AM006415; 91351, Active Motif]) and human anti-nucleocapsid (SARS-CoV-2 anti-nucleocapsid [1A6; MA5–35941, Active Motif]) and HRP anti-human IgG antibody (1:5,000; A00166, GenScript) was the secondary antibody.
7
Antibody Affinity Evaluation for SARS-CoV-2
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To evaluate the affinity of antibodies, His-tag labeled RBD of SARS-CoV and SARS-CoV-2 virus were separately immobilized on microbeads (Thermo Fisher, 10103D). 96-well plates containing RBD-coated beads were incubated with either 13I1 antibodies or VHH-72 nanobodies over a range of concentrations (3 pM to 12.5 nM) in PBSB for 2 h at room temperature. After primary incubation, the beads were washed with ice-cold PBSB and incubated with goat anti-human Fc AF 488 (Jackson ImmunoResearch, 109-545-008) for 4 min on ice. Following secondary incubation, the beads were washed twice with ice-cold PBSB and analyzed by flow cytometry. Similarly, 13I1 coated beads were assessed for binding to WT SARS-CoV-2 S1 protein as well as the S1 protein of variants of concern (B.1.1.7 and B.1.351) using commercially-available antigens (Acro Biosystems; SPD-C52H3, SPD-C52Hn, SPD-C52Hp). Briefly, 13I1-coated microbeads were blocked in PBSB with 10% milk for 1 h, washed with PBSB, and incubated with SARS-CoV-2 S1 proteins in 96-well plate format for 3 h at 25 °C and 225 rpm. Plates were washed with PBSB, incubated with His-tag antibody (Invitrogen, PA-9531) for 30 min on ice, washed with PBSB, incubated with detection antibody (Jackson ImmunoResearch, 703-606-155) for 4 min on ice, washed with PBSB, and analyzed by flow cytometry.
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