2.2. Reference sera
2.3. Polyacrylamide gel electrophoresis and Western blot
2.4. rPA thermal treatment and M-344 formulation
rPA NR-2629 (at 1 mg/mL) was incubated during 30 min, one, four or 24 h at 22 (RT), 50 or 75 °C. Some treated samples (Table 1) and the control rPA were formulated into a vaccine as described previously [40]. rPA vaccine freshly thawed and then formulated at RT was used as a negative control. In brief, aluminum-containing adjuvant (Alhydrogel® Brenntag Biosector, Denmark; 1.42 mg/mL Al) was combined with 30 μg/mL rPA (control or treated). After vigorous agitation using a Vortex mixer, the suspensions were incubated at RT for 1 h.
Table 1.
2.5. Immunization and sera collection
With the approval of CBER’s Animal Care and Use Committee, a total of three hundred female CD-1 mice, four to six week old, were distributed in groups of twenty and then immunized once intraperitoneally with 6 μg of control or treated rPA, one group per treatment. Twenty-eight days post-immunization, mice were bled from the tail veins and sera separated. Samples were stored at −20 °C until use. A serum pool from non-immunized mice was used as negative control.
2.3. Polyacrylamide gel electrophoresis and Western blot
2.4. rPA thermal treatment and M-344 formulation
rPA NR-2629 (at 1 mg/mL) was incubated during 30 min, one, four or 24 h at 22 (RT), 50 or 75 °C. Some treated samples (Table 1) and the control rPA were formulated into a vaccine as described previously [40]. rPA vaccine freshly thawed and then formulated at RT was used as a negative control. In brief, aluminum-containing adjuvant (Alhydrogel® Brenntag Biosector, Denmark; 1.42 mg/mL Al) was combined with 30 μg/mL rPA (control or treated). After vigorous agitation using a Vortex mixer, the suspensions were incubated at RT for 1 h.
Table 1.
2.5. Immunization and sera collection
With the approval of CBER’s Animal Care and Use Committee, a total of three hundred female CD-1 mice, four to six week old, were distributed in groups of twenty and then immunized once intraperitoneally with 6 μg of control or treated rPA, one group per treatment. Twenty-eight days post-immunization, mice were bled from the tail veins and sera separated. Samples were stored at −20 °C until use. A serum pool from non-immunized mice was used as negative control.