In contrast with the low concentration combinations, the high concentrations of reagents did not saturate, suggesting a higher upper limit of detection than 1500 ng/mL of NP

In contrast with the low concentration combinations, the high concentrations of reagents did not saturate, suggesting a higher upper limit of detection than 1500 ng/mL of NP. In the presence of cells, S/B was elevated (Figure ?Figure22C,D and Supporting Information Figure S2) compared to media alone. for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, ), Brazilian/Japanese P.1 (Gamma, ), and Californian (Epsilon, ) variants of concern (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC. model efficacy studies. To date, assays including SARS-CoV-2 cytopathic effect (CPE),22 RT-PCR, and antigen immunostaining, among others, have been used in compound screens and evaluation of drug efficacy.23 While the RT-PCR assay does not have enough screening throughput, the SARS-CoV-2 CPE assay may not identify all of the active compounds that inhibit SARS-CoV-2 replication in the screen because CPE is cell type-dependent and is an indirect readout of viral infection. SARS-CoV-2 UC-1728 nucleocapsid protein (NP) is a structural protein that binds the positive strand viral genome within the JAM2 nucleocapsid core.24,25 Previously, we developed an AlphaLISA assay for the measurement of SARS-CoV-2 NP as an indicator of virus replication in infected cells.26 We used the NP as a target viral protein to develop a high throughput screening (HTS) assay that could be used in a biosafety level 3 containment facility to identify promising UC-1728 antiviral drugs targeting SARS-CoV-2 multiplication.27 Targeting the more conserved and highly expressed NP instead of the S would prevent detection failures due to newly emerging SARS-CoV-2 VoC with S mutations and increase sensitivity, respectively. In this work, we have developed a homogeneous time-resolved fluorescence (HTRF) assay that relies on the time-resolved fluorescence resonance energy transfer (TR-FRET) between a donor and acceptor fluorophore when they are in close proximity and proper orientation relative to each other.28,29 To specifically detect the SARS-CoV-2 NP, we conjugated two NP-specific monoclonal antibodies (MAbs) to either a donor or acceptor fluorophore that are brought into proximity when NP is present. We developed a microplate assay using recombinant NP added to cells, NP-transfected cells, and both tissue culture supernatants (TCS) and cell lysates UC-1728 from infected cells. The homogeneous format means that the whole well fluorescence emission ratio between the acceptor and donor fluorophores is read without washing using a plate reader with a TR-FRET module. The assay we have developed involves a single step; it is easy to use and scalable to HTS to identify SARS-CoV-2 antivirals. The assay can identify drugs targeting any of the steps of SARS-CoV-2 life UC-1728 cycle, hence enabling screening efforts to identify drugs with activity against emerging VoC. Methods Reagents Vero E6 cells [CRL-1586, Resource Research Identifier (RRID): CVCL0574] were purchased from American Tissue Type Collection. The following items were purchased from Corning TM: EMEM (10-009-CV), HI fetal bovine serum (FBS) (35-016-VC, and 0.25% Trypsin (25053CI). Pen/Strep (15140-122) was purchased from Gibco. Phosphate-buffered saline (PBS) (SH30256FS) was purchased from HyClone. The following items were purchased from Greiner Bio-One: white 384 well plate (781073), white half area 96-well plate (675083), and white 96-well plate (655083). The custom labeling of MAbs was performed by Columbia Biosciences. Rabbit anti-NP SARS-CoV-2 antibodies R001 (40143-R001, RRID number: AB_2827974), R004 (40143-R004, RRID number: AB_2827975), R019 (40143-R019, RRID number: AB_2827973), and R040 (40143-R040, RRID number: AB_2827976) were purchased from Sino Biological, and mouse anti-NP MAb 1C7C7 was provided by Dr. Tomas Moran and purchased from Leinco (LT7000). The following items were purchased from ThermoFisher: Goat anti-mouse HRP (A16072), goat-anti rabbit HRP (A16104), West Femto substrate (34094), Lipofectamine 2000 (11668019), and Optimem I Reduced Serum Media (31985070). Triton X-100 (100) was purchased from Sigma Aldrich. The cOmplete ULTRA protease inhibitor (PI) (05892791001) was purchased from Roche. Antibody Matrixing Four rabbit MAbs and one mouse Mab specific for SARS-CoV-2 NP were labeled with europium (donor Ab) or DyLight650 (acceptor Ab). IgG antibodies R001, R004, R019, and R040 were raised against recombinant SARS-CoV nucleocapsid phosphoprotein [NP (Sino Biological, 0143-V08B)] and expressed in HEK293 cells. Mouse MAb 1C7 (1C7C7, IgG2a) was expressed in HEK293 using recombinant NP as the immunogen. The labeled MAbs were then tested in HTRF assay, in triplicate wells, using a cross-matrix assay format. The data are presented as a median TR-FRET ratio (acceptor fluorescence/donor.