2 (secondary infection).[12] The Roscovitine in vivo study protocol was approved by the institutional review board of the National Institute of Infectious Diseases, Japan.
Detection of DENV RNA by RT-PCR was performed as reported previously (Tables 1 and 3).[15, 16] Viral RNA was extracted using High Pure Viral RNA extraction kit (Roche Diagnostics, Mannheim, Germany) and DENV serotypes were determined by serotype-specific RT-PCR.[15, 16] Dengue-virus specific IgM antibody in serum samples was determined using IgM capture ELISA (Dengue Fever Virus IgM Capture ELISA, Focus Diagnostics, CA, USA) according to the manufacturer’s instructions. Dengue indirect IgG ELISA (Panbio, Queensland, Australia) was used for the detection of anti-DENV IgG antibody according to the manufacturer’s instructions.[15] Detection of the NS1 antigen was performed using Platelia Dengue NS1 Antigen (Biorad Laboratories, Marnes-la-Coquette, France) and Pan-E Dengue Early ELISA (Panbio) according to manufacturers’ instructions. The former kit was mainly used in the study. For the Platelia Dengue NS1 Antigen ELISA kit, 50 μL of serum sample, 50 μL of sample diluent (Diluent R7, phosphate buffer, Tween 20, and fetal calf serum supplemented with 0.15% ProClinTM 300) and 100 μL of diluted conjugate (anti-NS1 monoclonal www.selleckchem.com/products/ldk378.html antibody-coated
to horseradish peroxidase supplemented with 0.15% ProClinTM 300) were added to each anti-NS1 monoclonal antibody coated well. The assay plate was incubated at 37°C for 90 minutes. Positive controls and negative controls with calibrator sera were included in each assay. After six washings, 160 μL of tetramethylbenidine (TMB) substrate was added to each of the wells and the plate was further incubated at room temperature for 30 minutes in the dark. Reaction to was terminated with 100 μL of stop solution (1 N H2SO4). Optical density (OD) readings were obtained with a spectrophotometer at wavelengths of 450 nm/620 nm.
The index of each sample was calculated with the following formula: OD of samples/OD of calibrators. As the Biorad NS1 ELISA kit showed high sensitivity using 50 μL of patient serum samples, the serum sample volume was reduced and the assay was tested for detection rates. Serum samples were first diluted to 1:10 or 1:100 using diluent (Diluent R7, Platelia Dengue NS1 Ag, Biorad). The assay was then performed according to manufacturer’s instructions (Platelia Dengue NS1 Ag, Biorad). Results were interpreted in accordance with manufacturer’s recommendations. Sample ratios were determined by dividing the sample OD with the cut-off OD. Sample ratios of <0.5, 0.5–1.0, and >1.0 were classified as negative, equivocal, and positive, respectively. Equivocals were regarded as negative for analysis.