The newly developed assay described here is rapid, low-cost, and time-saving, providing a useful tool for both basic research and epidemiological investigation. Methods Cells, virus and antibodies Baby hamster kidney cells (BHK-21) and African green monkey kidney (Vero) cells were cultured in Dulbecco’s Modified Essential Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin–streptomycin at 37°C in a 5% CO2. Human erythroleukemic K562 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10%

FBS (GIBCO) at 37°C https://www.selleckchem.com/products/Everolimus(RAD001).html in a 5% CO2. The reporter Luc-DENV has been previously described [9] and was prepared and tittered in Vero cells. The following characterized monoclonal antibodies (mAbs) against DENV were used in this study: 4G2, 2B8 and 2A10G6. Clinical samples Serum samples were collected from Rhesus monkeys (#175, #052) immunized with a single dose of a live attenuated DENV (unpublished data), and serum STA-9090 supplier from the unimmunized

animal was set as negative control (#NS). Human convalescent sera from DF patients (#19-20, #37-20, #37-30) and control serum negative for DENV (#NC) were from Guangzhou No.8 People’s Hospital, Guangzhou, China. All samples were inactivated at 56°C for 30 min before assay. Plaque reduction neutralization test (PRNT) PRNT were performed as previously described [12]. Briefly, 2 × 105 cells/well of AZD1480 datasheet BHK-21 cells were seeded into 12-well plates and incubated overnight. 100 μl serially diluted antibody samples were mixed with an equal volume of Luc-DENV containing 30 PFU. After 1 h incubation, 200 μL of antibody-virus mixture was added to BHK-21 cell monolayer in 12-well plates for another 1 h. Next, the supernatant was removed, and cells were overlaid with 1 mL of 1.0% (w/v) agarose (Promega) in DMEM containing 4% FBS. After further incubation at 37°C for 4 days, the overlay was removed,

and cells were fixed with 4% formaldehyde for 30 min, and stained with 1% (w/v) crystal violet. DMEM served as negative control, and each sample was assayed in triplicate. Plaques were counted and PRNT50 is defined as the antibody dilution resulting in 50% plaque reduction referred to negative control. Luc-base neutralization assay Luc-based neutralization assay was performed in 12-well plates, and the procedure was similar to the conventional PRNT assay. Briefly, virus-antibody mixture was Vasopressin Receptor added to BHK-21 cells in 12-well plates and adsorbed for 1 h at 37°C. Supernatant was removed and 1 mL DMEM-2% FBS was replenished onto cells. After 48 h incubation at 37°C, the supernatant was removed, cells were lysed with 250 μl lysates (Promega) per well for 15 minutes. 50 μl lysed suspension was assayed for enzyme activities after adding 100 μl substrate reagent. Data was collected using a continuous-read luminometer (GLOMAX 96 Microplate Luminometer, Promega) integrated over 10 seconds with a 2 second delay. Medium served as negative control, each sample was assay in triplicate.