Delaying time of delivery of human anti-plague mAbs provided better protection against a plague challenge One possible reason we observed less protection with the human anti-F1 and anti-V mAbs in the mouse plague model was that the level of the human IgGs may not have been sustained in the mouse over time compared to the mouse IgG mAbs, We tested this hypothesis in two separate studies. We first examined the concentration of the human antibody in mice directly, by measuring the level of m252 (anti-F1) and m253 (anti-V) in serum after they were given the human mAbs by i.p. injection. Mouse sera were collected at different time points after the initial dosing and human IgG levels were monitored by direct ELISA. As seen in Figure 8A , the anti-F1 m252 mAb appeared to have a half-life of approximately 8 days, and the half-life of m253 mAb was approximately 10 days. After 21 days, the levels of these two human antibodies were undetectable. In contrast, the level of both the mouse anti-F1 (F1m) and anti-V (7.3 m) mAbs may have decreased initially like the human anti-plague mAbs, but after 21 days, the levels were still approximately 40-50% of the initial concentration ( Figure 8B ). The half-live of human IgG mAbs in mice reported here is similar to what was found in another study where human mAbs were used against another biothreat agent [18] . In contrast, a human IgG molecule would have an average serum half -life of 21 days in a human [19] . Because of these findings we then administered the anti-F1 m252 mAb at different time points relative to the time of challenge. While the original regimen provided consistently modest protection, administration of the human m252 mAb 24 and 48 hours postchallenge provided increasing protection with the 48 hours schedule provided complete protection ( Figure 9 ). Antibody administration at even later time points was not performed since mice began to die 3-4 days after challenge without any treatment. However, we did evaluate the effect of a second dose of antibody at a later time point. In this group, mice first received an initial dose of human anti-F1 m252 mAb 24 hour before challenge as was done with the earlier protocols. These mice then received a second dose of the human anti-F1 m252 mAb 5 days after challenge. There was an increase in both the number of survivors (5/6) and MTD (20 . Epitope mapping of the human anti-F1 and anti-V antibodies by peptide binding assay. A. Each of twenty-seven peptides that covered the full length of the F1-antigen were used to coat an ELISA plate (0.05 ml of a 25 mg/ml solution of each peptide), and binding by the human anti-F1 m252 mAb (0.05 ml of a 10 mg/ml solution) was analyzed. The sample labeled F1 was the full-length antigen used to coat the plate as the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled Media was the negative control with no primary antibody added. B and C. Each of fifty-two peptides that covered the full length of the V-antigen was used to coat an ELISA plate (0.05 ml of a 25 mg/ml solution), and the human anti-V m253 (B) and m254 (C) mAbs were used (0.05 ml of a 10 mg/ml solution), respectively, to analyze for binding. The sample labeled V was the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled Media was the negative control without the primary antibody. doi:10.1371/journal.pone.0013047.g003 days) approaching the efficacy displayed by a single dose administered 48 hour after challenge. These data suggest that the optimum serum concentration of the human IgG1s was critically dependent on the time of administration, and that the optimum concentration of the human anti-plague IgG1s in turn determined the outcome of the treatment protocol.