Bright lumi firefly luciferase reporter gene assay kit

Pseudo virus (PSV)-based neutralization assay was described previously [19 (link)]. Briefly, PSV of SARS-CoV-2 wild-type or mutant spike Δ19 (19 amino acids truncated at the C-terminus) with mCherry and Luciferase were first produced in HEK293T cells according to the reference. Then, HEK293T cells expressing hACE2 were seeded into a 96-well, white-opaque plate at a density of 1 × 10⁴ per well. Testing antibodies serially diluted in DMEM with 10% FBS (dilution factor: 3.16, from 200 nm to 6.3 fM) were incubated with an equal volume of PSV at 37 °C for 30 min. No PSV or PSV in the absence of antibodies were set as controls for normalization. 100 µL of the mixtures were transferred to the HEK293T/hACE2 cells. Fresh media was changed 16 h after treatment for an additional culture at 48 h. PSV transduction was evaluated by luciferase activity using the Bright-Lumi Firefly Luciferase Reporter Gene Assay Kit (Beyotime, #RG015M) according to the manufacturer’s instructions. Data fitting was carried out in GraphPad Prism 8.3. The combination index (CI) in the PSV neutralization assay was calculated by the CompuSyn program to evaluate the synergism according to the program instruction. CI > 1 indicates antagonism, CI = 1 indicates additive effect, and CI < 1 indicates synergism.

To determine the neutralization activity of VHH_5-05, the pseudotyped virus neutralization assay was performed. Briefly, 293T cells were co-transfected with psPAX2, pLenti-GFP, and plasmids encoding either SARS-CoV-2 S by using polyetherimide (PEI), as previously described [24 (link)]. The supernatants containing SARS-CoV-2-pseudotyped virus were harvested.
Pseudovirions were pre-incubated with serially-diluted VHH_5-05 for 1 h in 37 °C, then virus-VHH mixture was added onto 293/hACE2 cells (10⁴ per well) in a 96-well plate. About 40 h post inoculation, cells were collected and lysed with luciferase substrate (Bright-Lumi™ Firefly Luciferase Reporter Gene Assay Kit, #RG051M, Beyotime, Shanghai, China) at room temperature for 5 min, then the luciferase luminescence (RLU) of each well was measured with a luminescence microplate reader (SpectraMax i3x Microplate Reader, Molecular Devices, CA, USA). Half maximal inhibitory concentration (IC₅₀) value was determined by a four-parameter non-linear regression model using PRISM. All experiments were carried out in triplicates and repeated at least twice.

To detect the neutralization ability of selected antibodies against infection of coronavirus PSV, HEK293T/hACE2 cells were first seeded into 96‐well, white‐bottom plates at a density of 1 × 10⁴ per well and cultivated overnight. The PSV was pre‐incubated with an equal volume of different concentrations of selected antibodies (dilution factor: 3.16, from 200 nm to 200 fM for S‐B8 and S‐D4, 200 nm to 6.3 fm for S‐E6 in WT PSV detection, and others are as indicated) in DMEM at 37 °C for 30 min. DMEM with or without PSV in the absence of antibodies were set as controls. After incubation, the PSV mixture was transferred to the culture plates containing HEK293T/hACE2 cells. The DMEM media containing PSV and antibodies were replaced with fresh media after 16 h treatment, cells were incubated for an additional 48 h. PSV infection efficacy was evaluated by luciferase activity using Bright‐Lumi Firefly Luciferase Reporter Gene Assay Kit (Beyotime, #RG015M). Fifty microliter of luciferase substrate was added to each well, and the relative luminescence unit values were measured on an Envision plate reader (PerkinElmer, Ensight). The antibody concentration was first transformed into Log(X), and the least squares fit (Y = Bottom+(Top‐Bottom)/(1+10^((LogIC50‐X)*HillSlope))) was then used for non‐linear regression analysis in GraphPad Prism 8.3.