Renilla luciferase assay system

Neutralizing antibody titers against AAV-Spark100 and LK03 were determined using a cell-based luciferase reporter assay as described previously (28 (link)) with modifications. Serum samples were serially diluted in FBS and incubated with AAV-Spark100 or AAV-LK03 vectors expressing gaussia luciferase (GLuc) for 30 min at 37°C. The AAV-serum mix was then applied to freshly seeded 2V6.11 cells in a 96-well tissue culture plate. GLuc in the supernatant was collected and measured 24 hours after transduction using the Renilla Luciferase Assay System (Promega, Cat #E2829) kit with luminescence detection by a GloMax Discover plate reader (Promega). The antibody titer was determined as the highest serum dilution which inhibited transduction by 50% (IC50).

Approximately 4 × 10⁵ Vero-E6 cells were seeded in 100 μl volume of each well of a black-walled clear bottom 96-well plate (Corning) and incubated at 37°C for approximately 5 hours to allow cells to adhere. VSV pseudovirus at 1:10 final concentration volume/volume for unconcentrated virus (VSVpp-SARS1-S or VSVpp-MERS-S) or 1:20 for concentrated virus (VSVpp-SARS2-S). Virus was incubated with cells for 24 hours, and cells were subsequently lysed with Renilla Luciferase Assay System (Promega) according to manufacturer’s instructions. Luciferase activity was measured using a microplate reader (BioTek Synergy). Pseudovirus entry was normalized to VSV-G within each condition, and percent entry was calculated relative to WT Vero-E6 cells after normalization.

HEp-2 cells were seeded into a 96-well plate at a 20,000 per well (100 μL) one day before compound dosing. ALS-8112 and AZ-27 were first serially diluted in DMSO to 200 folds of their respective final concentrations. Briefly, ALS-8112 was diluted to 9 concentrations horizontally and AZ-27 was diluted to 7 concentrations vertically, in separate 96-well plates. For each compound, a column or a row for single drug treatment was set up as the control. The first row in each 96-well plate was DMSO treatment control. 5 μL of each compound was then added into 90 μL MEM (cell plate) to achieve a combination matrix with 10-fold of final concentration. Finally, 10 μL was transferred from the cell plate to HEp-2 cells in 96-well plates for overnight incubation. The next day, A2-RL-line19F virus was added to the plate at a multiplicity of infection of 2 for two days before measuring the reporter activity using the Renilla Luciferase Assay System (Promega) on a Perkin Elmer multilabel counter Victor 3V. Five replicate experiments were performed and data analyzed.

The luciferase assay was performed using the Renilla Luciferase Assay System from Promega (Fitchburg, WI, USA), and activity was measured using a Monolight 2010 luminometer. Cells were washed with PBS before directly lysing using passive lysis buffer and a cell scraper. 100 μl of Renilla luciferase assay reagent was added to a polycarbonate tube and luminescence was measured with 10 seconds of integration and a 2-second delay directly after adding 20 μl of cell lysate and mixing. Luciferase activity was performed in biological and assay duplicates for each time point.

VRPs were produced by co-electroporation of in vitro transcribed YFV-Gluc replicon RNA and in vitro transcribed SIN-flavi-prME/C packaging construct RNA into BHK-J cells using the electroporation conditions as described [39 (link)]. After electroporation, cells were kept at 32 °C in MEM complete for 72 h. The VRPs were harvested by centrifugation of the cell supernatant at 1200 rpm for 10 min to remove cell debris. The cleared supernatants containing the VRPs were aliquoted and stored at −80 °C.
For the determination of VRP titers TCID₅₀ analysis was performed. For this, the VRPs were serially diluted in tenfold steps in MEM with four replicates per dilution. In 96-well plates, an equal amount of BHK-J cell suspension (2 × 10⁴ cells/well) was added to each dilution and incubated at 37 °C, 5% CO₂ for 48 h. Readout was performed via measurement of the Gluc activity using the Renilla Luciferase Assay System (Promega, Madison, WI, USA) and a plate luminometer (BioTek Synergy 2, Agilent, CA, USA). Wells with luciferase activity at least two-fold above the threshold measured for mock-infected cells were counted as positive. TCID₅₀ was calculated by the Spearman and Kärber algorithm as described [40 ] and converted to focus forming units (FFU)/mL with FFU approx. 0.69 × TCID₅₀.

Cell lysates were subjected to a Renilla Luciferase Assay System (Promega) according to the manufacturer's protocol. In brief, 1 day after adenoviral transduction NRCM were transfected with a Renilla luciferase plasmid using GeneTrans II Transfection reagent (MoBiTec). After 48 h, cells were either subjected to a luciferase reporter assay or RNA was isolated for subsequent quantitative real‐time PCR. Luciferase activity was detected by using a Modulus Luminometer (Turner BioSystems) and normalized to total protein concentration.