SARS-CoV-2 Spike RBD Antibody Measurement

Using luciferase immunoprecipitation system (LIPS) (33 (link)), we measured IgG binding to recombinant nanoluciferase-tagged antigens corresponding to SARS-CoV-2 Wuhan-Hu-1 spike RBD domains, as previously described (34 (link)). Viral sequences used in this study corresponded to the deposited sequence Genebank NC_045512.2 for SARS-CoV-2 Wuhan. Briefly, we cloned recombinant nanoluciferase-tagged antigens and expressed them by transient transfection into Expi293F™ cells (Expi293™ Expression System, ThermoFisher Scientific Life Technologies, Carlsbad, CA, USA). For LIPS, we incubated in liquid phase each antigen with test serum (1 µl) for 2 h and then captured immune complexes with rProtein A-sepharose. After washing (5 times) the sepharose pellets, we quantified bound IgG by measuring the recovered luciferase activity in a Berthold Centro XS3 luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) using the MikroWin version 5.22 software. We then converted raw data into arbitrary units (AU), using a local positive index serum for SARS-CoV-2-specific antibodies.

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Fovet C.M., Pimienta C., Galhaut M., Relouzat F., Nunez N., Cavarelli M., Sconosciuti Q., Dhooge N., Marzinotto I., Lampasona V., Tolazzi M., Scarlatti G., Ho Tsong Fang R., Naninck T., Dereuddre-Bosquet N., Van Wassenhove J., Gallouët A.S., Maisonnasse P., Le Grand R., Menu E, & Seddiki N. (2022). A Case Study to Dissect Immunity to SARS-CoV-2 in a Neonate Nonhuman Primate Model. Frontiers in Immunology, 13, 855230.

Publication 2022

Centro XS3 luminometer

Antibodies Antigen Cells Immune complexes Immunoprecipitation Luciferase Pellets Sars cov 2 Sepharose Serum Transfection Transient

Corresponding Organization :

Other organizations : Infectious Disease Models and Innovative Therapies, AR2i, IRCCS Ospedale San Raffaele

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Variable analysis

independent variables

Recombinant nanoluciferase-tagged antigens corresponding to SARS-CoV-2 Wuhan-Hu-1 spike RBD domains

dependent variables

IgG binding to recombinant nanoluciferase-tagged antigens

control variables

Viral sequences used in the study corresponded to the deposited sequence Genebank NC_045512.2 for SARS-CoV-2 Wuhan
Recombinant nanoluciferase-tagged antigens were expressed by transient transfection into Expi293F™ cells

positive control

A local positive index serum for SARS-CoV-2-specific antibodies

negative control

Not explicitly mentioned

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