Recombinant bir a

Manufactured by Avidity

Recombinant Bir-A is a laboratory tool used for the site-specific biotinylation of proteins. It functions as a biotin ligase enzyme, catalyzing the covalent attachment of biotin to target proteins containing a specific recognition sequence.

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2 protocols using recombinant bir a

SARS-CoV-2 Spike and RBD Staining of PBMCs

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PBMCs were stained with SARS-CoV-2 Spike and RBD probes, as previously described (Juno et al., 2020 (link)). Probes were generated by sequential addition of streptavidin-phycoerythrin (PE) (Thermo Fisher Scientific) to trimeric S protein biotinylated using recombinant Bir-A (Avidity), while SARS-CoV-2 RBD was labeled to APC using an APC Conjugation Lightning-Link Kit (Abcam). PBMCs were surface stained with Aqua viability dye (Thermo Fisher) and monoclonal antibodies against CD19-ECD (#IM2708U, Beckman Coulter), IgM BUV395 (#563903), CD21 BUV737 (#564437), IgD PE-Cy7 (#561314), IgG BV786 (#564230), streptavidin-BV510 (#563261) (BD Biosciences), CD20 APC-Cy7 (#302314), CD14 BV510 (#301841), CD3 BV510 (#317332), CD8a BV510 (#301048), CD16 BV510 (#302048), CD10 BV510 (#312220) and CD27 BV605 (#302829) (BioLegend). Cells were washed, fixed with 1% formaldehyde and acquired on a BD LSRII Fortessa.

Nguyen T.H., Rowntree L.C., Petersen J., Chua B.Y., Hensen L., Kedzierski L., van de Sandt C.E., Chaurasia P., Tan H.X., Habel J.R., Zhang W., Allen L.F., Earnest L., Mak K.Y., Juno J.A., Wragg K., Mordant F.L., Amanat F., Krammer F., Mifsud N.A., Doolan D.L., Flanagan K.L., Sonda S., Kaur J., Wakim L.M., Westall G.P., James F., Mouhtouris E., Gordon C.L., Holmes N.E., Smibert O.C., Trubiano J.A., Cheng A.C., Harcourt P., Clifton P., Crawford J.C., Thomas P.G., Wheatley A.K., Kent S.J., Rossjohn J., Torresi J, & Kedzierska K. (2021). CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity. Immunity, 54(5), 1066-1082.e5.

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Workflow for SARS-CoV-2 S-specific B Cell Profiling

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Probes for delineating SARS-CoV-2 S-specific B cells within cryopreserved human PBMC were generated by sequential addition of streptavidin-PE (Thermofisher) to trimeric S protein biotinylated using recombinant Bir-A (Avidity). SARS-CoV-2 RBD protein was directly labelled to APC using an APC Conjugation Lightning-link kit (Abcam). Cells were stained with Aqua viability dye (Thermofisher). Monoclonal antibodies for surface staining included: CD19-ECD (J3-119) (Beckman Coulter), CD20 Alexa700 (2H7), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (O323) (Biolegend), IgA-Vio450 (clone) (Miltenyi). Cells were washed, fixed with 1% formaldehyde (Polysciences) and acquired on a BD LSR Fortessa or BD Aria II. Gating is shown in Supplementary Fig. 4.

Wheatley A.K., Juno J.A., Wang J.J., Selva K.J., Reynaldi A., Tan H.X., Lee W.S., Wragg K.M., Kelly H.G., Esterbauer R., Davis S.K., Kent H.E., Mordant F.L., Schlub T.E., Gordon D.L., Khoury D.S., Subbarao K., Cromer D., Gordon T.P., Chung A.W., Davenport M.P, & Kent S.J. (2021). Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19. Nature Communications, 12, 1162.

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