The S-RBD binding antibody in mouse sera was quantified using a multiplex microsphere-immunoassay (MMIA). AviTag-enzymatic biotinylated SARS-CoV-2 ancestral, Alpha, Beta, Gamma, Delta RBDs were custom-made by Genscript. The remaining RBDs were produced in-house by transfection of pCAGGS-expression plasmids (modified from pCAG-GFP, addgene, 11150) into the Expi293F system (ThermoFisher, A14527). The RBDs were purified using Nickel Sepharose columns (Cytiva, 17526802), and biotinylated using a BirA kit (Avidity, LLC, BirA500). The RBDs were coated on the Magplex avidin-microsphere (Luminex, MA-A012-01) at 25 μg per 5 million microspheres. The RBD-coated beads were pre-incubated with 1:100 diluted mouse sera for 1 h at 37 °C with agitation at 800 rpm. Following two 1% BSA PBS washes, the mouse IgG was immunostained with 1:1000 diluted PE-labelled anti-mouse IgG antibody (R&D systems, IC002P) for 1 h at 37 °C with agitation at 800 rpm. After two washes, the MFI signals were acquired using the Luminex MAGPIX reader.
Bira kit
Manufactured by Avidity
The BirA kit is a laboratory tool used for the site-specific biotinylation of fusion proteins. It contains the BirA enzyme, which catalyzes the attachment of biotin to a specific lysine residue within a target protein.
Automatically generated - may contain errors
Lab products found in correlation
Expi293 expression medium, by Thermo Fisher Scientific (2 mentions)
Histrap hp affinity column, by Cytiva (2 mentions)
Pcag gfp, by Addgene (1 mentions)
Magpix reader, by DiaSorin (1 mentions)
Expi293f system, by Thermo Fisher Scientific (1 mentions)
Magplex avidin microsphere, by DiaSorin (1 mentions)
3 protocols using bira kit
1
SARS-CoV-2 RBD Antibody Quantification
2
Recombinant RBD Construct Production
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BtKY72 RBD construct (BtKY72 S residues 318–520) was synthesized by GenScript into a CMVR plasmid with an N-terminal mu-phosphatase signal peptide and a C-terminal hexa-histidine tag (-HHHHHHHH) joined by a short linker (-GGSS) to an Avi tag (-GLNDIFEAQKIEWHE). BtKY72 mutant constructs T498W (BtKY72 S residue 487) and K493Y/T498W (BtKY72 S residue 482/487) were subcloned by GenScript from the BtKY72 RBD construct. BtKY72 and BtKY72 mutant RBD constructs were produced in Expi293F cells in Gibco Expi293 Expression Medium at 37 °C in a humidified 8% CO2 incubator rotating at 130 rpm. The cultures were transfected using PEI-25K with cells grown to a density of 3 million cells per ml and cultivated for 3–5 days. Proteins were purified from clarified supernatants using a 1 ml HisTrap HP affinity column (Cytiva), concentrated and then biotinylated using a commercial BirA kit (Avidity). Proteins were then purified from the BirA enzyme by affinity purification using a 1 ml HisTrap HP affinity column (Cytiva), concentrated and flash-frozen in 1× PBS, pH 7.4. Cell lines were not authenticated or tested for mycoplasma contamination.
3
BtKY72 RBD Construct Expression and Purification
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BtKY72 RBD construct (BtKY72 S residues 318-520) was synthesized by GenScript into a CMVR plasmid with a N-terminal mu-phosphatase signal peptide and a C-terminal hexa-histidine tag (-HHHHHHHH) joined by a short linker (-GGSS) to a Avi tag (-GLNDIFEAQKIEWHE). BtKY72 mutant constructs T498W (BtKY72 S residue 487) and K493Y/T498W (BtKY72 S residue 482/487) were subcloned by GenScript from the BtKY72 RBD construct. BtKY72 and BtKY72 mutant RBD constructs were produced in Expi293F cells in Gibco Expi293 Expression Medium at 37°C in a humidified 8% CO2 incubator rotating at 130 rpm. The cultures were transfected using PEI-25K with cells grown to a density of 3 million cells per mL and cultivated for 3-5 days. Proteins were purified from clarified supernatants using a 1mL HisTrap HP affinity column (Cytiva), concentrated, and then biotinylated with a commercial BirA kit (Avidity). Proteins were then purified from the BirA enzyme by affinity purification using a 1 mL HisTrap HP affinity column (Cytiva), concentrated, and flash frozen in 1x PBS, pH 7.4.
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