FIG. 1.
Immunization of A/J mice results in high-titer anti-EF antibodies that are neutralizing in vitro and protective in vivo. A/J mice were immunized with either rEF or adjuvant alone on days 10, 24, and 38 and bled on days 14, 28, 42, and 111. (A) IgG anti-EF antibody titers of pooled sera from A/J mice immunized with rEF or adjuvant alone were assessed at specified time points to quantitate the IgG anti-EF response. Antibody levels were measured via standard ELISA at OD410/490. (B) Day 111 sera collected were subjected to an in vitro neutralization assay using a 3:1 ratio of PA to EF (EdTx). Serial twofold dilutions (1:50 to 1:1,600) of anti-EF sera were incubated with a 3:1 ratio of PA to EF (EdTx) for 1 h and added to RAW 264.7 mouse macrophages for 4 h. The cells were lysed, and intracellular cAMP was monitored by ELISA. Percent neutralization was calculated relative to the level for the control wells containing toxin alone. Sera demonstrate near-maximal neutralization of toxin activity on day 111 (73 days past the final booster immunization). The neutralization titer of rEF antisera was 800. (C) A/J mice immunized with rEF are partially protected from an in vivo EdTx challenge using four times the experimentally derived A/J LD50 (360 μg PA plus 144 μg EF, experimentally determined) for EdTx (P = 0.009).