Following the virus binding period, the inocula and drugs were removed and the cell monolayers were washed with ice-cold PBS before fixation with pre-chilled 4% paraformaldehyde (PFA) selleck chemicals in PBS for 1 h on ice. At

that point, the wells were blocked with 5% bovine serum ARS-1620 cost albumin (BSA) at 4°C overnight to prevent any non-specific binding. Bound viruses on the cellular surfaces were then detected by ELISA assay whereby wells were incubated with the following respective mouse monoclonal primary antibodies (diluted in PBS containing 5% BSA) at 37°C for 1 h before washing with PBST (0.1% Tween 20 in PBS) three times: anti-HCMV gB antibody (1:10,000; Thermo Pierce, Rockford, IL, USA), anti-HCV E2 antibody (1:20,000; AUSTRAL Biologicals, San Ramon, CA, USA), anti-flavivirus group antibody (1:5,000) for DENV-2, anti-measles hemagglutinin antibody (1:5,000; Millipore), and anti-RSV fusion protein antibody (1:15,000). Samples were then subjected to incubation at 37°C for 1 h with goat anti-mouse IgG conjugated with horseradish peroxidase C59 manufacturer (HRP; Invitrogen), diluted at 1:20,000 (HCMV, DENV-2, MV-EGFP), 1:36,000 (HCV), or 1:30,000 (RSV) in PBS containing 5% BSA. The wells were afterwards washed with PBST three times and developed with a TMB (3,3′,5,5′-tetramethylbenzidine) Two-component Microwell Peroxidase Substrate Kit (KPL, Gaithersburg, MD) at room temperature for 20 min before stopping the reaction

with 1 M phosphoric acid (H3PO4). The plates were measured with an ELx800 Microplate reader (Instrument, Inc.; Winooski, VT, USA)

at 450 nm. Figure 5 Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and Lepirudin values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details. Viral penetration assay The viral penetration assay was performed as previously reported [33] and the incubation periods and viral dose used are indicated in Figure 4A.