El abts chromogenic reagent kit

During the process of mouse vaccination, sera from immunized mice (n = 3) seven days after the last vaccination (day -3 prechallenge) were collected, and antibodies against B. pseudomallei Hcp1 were determined by enzyme-linked immunosorbent assay (ELISA) as described (30 (link)). Briefly, 96-well Maxisorp plates (Nunc) were coated overnight at 4°C with rHcp1 (50 ng/mL) solubilized in carbonate buffer (pH 9.6). The plates were blocked at room temperature for 1.0 h with StartingBlock T20 blocking buffer (Pierce). After blocking, plates were washed three times with TBS-T (Tris-buffered saline supplemented 10% StartingBlock T20 and 0.05% Tween 20, pH 7.5). Then, twofold dilutions of serum samples were made with TBS-T, added in triplicate to wells, and incubated for 2.0 h at 37°C. After washed three times with TBS-T, the plates were incubated for 1.0 h at 37°C with goat anti-mouse IgG-horseradish peroxidase conjugate (1:5,000 dilution, Solarbio). After incubation, the plates were washed three times with TBS-T and developed by the EL-ABTS Chromogenic Reagent kit (Sangon Biotech) and read at 405 nm by using a FLUOstar Omega microplate reader (BMG Labtech). The reciprocals of the highest dilutions exhibiting optical densities (OD) that were 2.1 times relative to the normal mouse serum levels were used to determine the endpoint titers of antibodies for individual vaccinated mice.

To find the key domain donated to DENV adsorption, we developed a purified protein-based ELISA for testing the binding activity of each vimentin segment to DENV-2 EDIII and DENV-2. The four above-mentioned purified vimentin proteins (1 μg) were suspended in ELISA coating buffer (Beijing Dingguo Changsheng Biotechnology, China) and immobilized overnight in 96-well Immulon 2 HB plates (Thermo, USA) at 4 °C. BSA and protein-devoid systems were used as controls. The wells were washed after discarding the protein solutions. All washes were completed with 0.1 M Tris-buffered saline (TBS) at pH 7.2 with 0.05% Tween 20. Virus and antibodies were diluted by TBS; all incubations were conducted at 4 °C unless specifically stated. The wells were blocked for 1 h with 200 μL of 3% skim milk in TBS buffer and then incubated with DENV-2 (1 × 10⁴ PFU) or 1 μg of the recombinant DENV-2 EDIII protein overnight, followed by three washes and 1 h of incubation with mouse anti-DENV-2 EDIII PcAb (1:500). After washing, the wells were incubated for 1 h with peroxidase-conjugated AffiniPure goat anti-mouse IgG (1:5000) and detected by EL-ABTS Chromogenic Reagent Kit (Sangon Biotech, China). Signals were recorded at 405 nm on a VERSAmax tunable microplate reader (Molecular Devices, USA). All experiments were repeated in triplicate and reported as average and S.D.