Blood samples were collected just prior to euthanization from mice at 1, 2, 4, 6, 8, and 10 weeks post exposure. Sera were separated and anti-Brucella antibodies, including IgG, IgG 1 , IgG 2a , IgM, and IgA were evaluated using ELISA. ELISA plates (Nunc) were coated with B. melitensis 16 M cell lysate. Briefly, 100 μl of 1 μg/ml lysate in bicarbonate/carbonate buffer (2.93 g NaHCO 3 , 1.59 g Na 2 CO 3 , 0.203 g MgCl 2 in 1 L of distilled water, pH 9.6) was added to microtiter wells (96 wells/plate) and incubated overnight at 4 • C (Pei and Collisson, 2005) . Non-specific binding was blocked via incubation with 5% (w/v) non-fat milk in PBS for 1 h at room temperature. The antigen-coated wells were incubated 2 h at room temperature (25 • C) with mouse sera diluted in PBS (pH 7.4) containing 0.05% (v/v) Tween-20 (PBS-T) 200 times for IgG1, IgG 2a , IgM, IgA detection or 1000 times for IgG detection. The plates were then incubated with HRP-conjugated goat anti-mouse immunoglobulins (IgG 1 , IgG 2a , IgM, IgA, and IgG from Kirkegard-Perry Labs (KPL) for 1 h at room temperature. The wells were washed with PBS-T between steps to remove any unbound materials. Color development followed the addition of 100 μl of TMB substrate KPL. The reaction was terminated by the addition of 50 μl of stop buffer (1 M H 2 SO 4 ) and OD 450 was determined using an ELISA reader (Bio-Rad).