Fig 3.

Antibody responses of mice immunized with recombinant vaccines determined by indirect ELISA. Microtiter plates were coated with the following proteins: (A) 100 ng well⁻¹ of recombinant proteins administered individually or a pooled protein sample containing 33.3 ng well⁻¹ of each protein included in the cocktail vaccines, totaling 100 ng, and (B) 100 ng well⁻¹ of each protein from the cocktail vaccines. Recombinant proteins were incubated with sera from immunized mice (diluted 1:50). Sera from mice immunized with PBS (negative control) and bacterin (positive control) were incubated with each protein individually, and a mean value was generated. Peroxidase-conjugated anti-mouse IgG (1:4000) was added as the secondary antibody. The data represent the mean optical density at 492 mm (OD₄₉₂) (n = 8) of the mouse sera collected at 105 DPI minus the mean OD₄₉₂ of the mouse sera collected at 0 DPI. All reactions were performed in triplicate. Different letters indicate significant differences (P < 0.01) among treatments according to the Tukey test, which was performed using GraphPad Prism 4 software.