Expi293F cells (Thermo Fisher, Cat# A14527) were transiently transfected with SARS-CoV-1 S-protein expression vectors (pcDNA3.3_CoV1_D28) using Expifectamine Enhancer according to the manufacturer’s protocol (Thermo Fisher). Two days later, to exclude dead cells from analysis, Expi293F were harvested, dispensed into a 96-well plate (3 × 105 cell/well), and stained for 30 min at room temperature (RT) with Live/Dead Fixable Aqua reagent (Invitrogen; Thermo Scientific) diluted 1:500. Following Live/Dead staining, cells were washed with PBS and incubated with nAbs candidates for 40 min at RT. Next, to identify the SARS-CoV-1 S protein mAbs binders, cells were washed and stained with the Alexa Fluor 488-labeled secondary antibody Goat anti-Human IgG (H + L) secondary antibody (Invitrogen) diluted 1:500. After 40 min of incubation, labeled cells were washed, resuspended in 150 μl of PBS, and analyzed using the BD LSR II flow cytometer (Becton Dickinson). Cells incubated with the SARS-CoV-1 nAb binder (S309) or incubated only with the secondary antibody were used as positive and negative controls respectively. Data were collected with the BD FACSDiva Software v9.0 (BD Biosciences) and analyzed with FlowJo™ Software (version 10).
Expifectamine enhancer
Manufactured by Thermo Fisher Scientific
Expifectamine Enhancer is a laboratory reagent designed to enhance the efficiency of transfection processes. It is intended to be used in conjunction with transfection reagents to improve the delivery and expression of nucleic acids in a wide range of cell types.
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2 protocols using expifectamine enhancer
1
SARS-CoV-1 S Protein mAb Screening
2
Complement Deposition Antibody Assay
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To investigate the antibodies’ ability to induce complement deposition, Expi293F cells (Thermo Fisher, Cat#A14527) were transiently transfected with SARS-CoV-2 original S protein, BA.1, or BA.2 expression vectors (pcDNA3.1_ spike_del19) using the ExpiFectamine Enhancer according to the manufacturer’s protocol (Thermo Fisher). Two days later, heated-inactivated plasma diluted 1/50 in complete Expi medium (Thermo Fisher) or monoclonal antibodies were incubated with HEK-S protein cells for 30 min at 37 °C, with 5% CO2 and 120 rpm shaker speed. Then, 50 µL of Expi medium containing 6% of baby rabbit complement (Cedarlane) was added, and cells were incubated at 37 °C, with 5% CO2 and 120 rpm shaker speed for 30 min. After incubation, cells were washed with PBS and stained with goat anti-rabbit polyclonal antibody against C3 (MP Biomedicals) for 1 h on ice. Cells were fixed with fixation buffer (BioLegend) for 15 min on ice. Then, cells were washed, resuspended in 100 µL of PBS1X, and acquired using the BD LSR II flow cytometer (Becton Dickinson). Plasma isolated from a prepandemic healthy subject was used as negative control. Single technical replicates were performed for each experiment. Results were reported as median fluorescence intensity of C3 deposition on the cells.
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