Evaluating Anti-Hcp1 Antibody Titers in Vaccinated Mice

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.