Luciferase activities were measured in lysed BHK-21 cells after 48 h incubating to assay neutralization activities. Error bars indicate the standard deviations from two independent experiments. Three convalescent sera from DF patients (#19-20, #37-20, #37-30) were validated with the newly developed assay in DMXAA mouse K562 cells. As shown in Figure 5, all three samples were able to enhance DENV infection at dilutions from 2 × 10-2 to10-4 (#19-20), 10-2 to10-5 (#37-20), and 10-1 to10-4 (#37-30), respectively. Negative control (#NC) from healthy adult in varying dilutions showed no impact on

RLU as expected. Meanwhile, serum #19-20 and #37-20 showed strong neutralizing activities at a dilution of 10-2 or even lower, and LRNT50 was calculated to 80 and 10-fold dilution separately, whereas no neutralizing activity can be observed in serum #37-30 at detected dilutions. Together, these results indicate that the Luc-based assay

is suitable for detecting both neutralization and ADE activity of immune sera from vaccinated or infected individuals. Figure 5 Enhancing activity assay for patient sera using the new assay system. Samples #19-20, #37-20 and #37-30 were obtained from Chinese subjects positive to DENV, with a sample from healthy people #NS as a negative control. Sera in various dilutions were mixed with Luc-DENV and incubated for 72 h, and luciferase activities were measured in lysed K562 cells to assay enhancing activities. Error bars indicate the standard Lonafarnib clinical trial deviations from two independent experiments. Discussion A reliable, rapid, and high-throughput assay for DENV neutralization antibodies Inositol monophosphatase 1 is critical for laboratory and clinical studies of DENV infection and vaccine. Considering the limitations of plaque based assay, some novel methods for neutralizing assays have been described [12–18]. Che and coworkers recently developed a novel ELISPOT based neutralization test, demonstrating a well correlation with the conventional PRNT assay [19]. Pseudo infectious DENV reporter virus particles (RVP) carrying green fluorescent protein (GFP) reporter were also

used to measure neutralization antibodies with Selleckchem Fludarabine rapidity, stability and reproducibility [15, 16, 20]. Infection with RVP could be monitored by the GFP signals using flow cytometry. However, GFP is not suitable for real-time quantification, and production of RVP requires special cell lines and replicon based plasmids. Live reporter virus carrying luciferase reporter replicates almost the same as wild type virus, representing a more advanced tool. Many reporter viruses, including SARS-related corona virus, human hepatitis C virus, parainfluenza virus, HIV, adenovirus, have been described and well applied for antiviral screening, live imaging, or function studies [21–25]. Live reporter DENV engineering a reporter gene at the capsid gene has been developed [26].