To do this, we initially set up a new luciferase based assay to measure the neutralization of large numbers of EV particles. B126 was confirmed to neutralize EV in the presence of C’ as had been previously reported. We also tested another anti-B5 mAb and found that it was unable to neutralize EV in the presence of C’. Benhenia et al, reported that B126 was an IgG2a Benzethonium Chloride isotype and this afforded it the ability to activate C’ as mAbs of IgG1 isotype did not. Benzoylaconine Interestingly, VMC-30 is an IgG2b isotype, which should also be capable of activating C’ and other effector functions similar to IgG2a. However, in this case, the isotype of the mAb did not predict functional activity in vitro and therefore highlights the importance of testing functional activity of mAb and not relying solely on the prediction of isotype analysis. When passively transferred into mice, VMC-30 also did not protect against challenge, again demonstrating the need for effector function for protection in vivo. We confirmed the isotype of VMC-30 and speculate that it may have been unable to neutralize VACV in the presence of C’ due to potential amino acid changes in the 
Fc region of the mAb which could abrogate functional activity. However the IgG2b isotype could interact with Fc receptors, so other factors like affinity may be playing a role in its inability to protect. The role of IgG2b in mediating protection from vaccinia virus infections in vivo is currently unknown. This may be interesting to examine further in the future. As others had previously found in BALB/c mice, we observed that protection in vivo by protein vaccination was correlated with the production of IgG2a antibodies. Therefore, we predicted that these antibodies would neutralize EV in the presence of C’ similar to B126, but we needed to fully examine this given the lack of C’-fixing activity with mAb VMC-30. We found that sera containing IgG2a from mice vaccinated with A33 or B5/CpG/alum could utilize C’ to neutralize large numbers of EV particles in vitro. Sera lacking IgG2a antibody from mice vaccinated with proteins and alum only was unable to neutralize virus in the presence of C’. This confirms the importance of isotype and strengthens the correlation between protection of mice and production of IgG2a isotype antibodies. Further examination of the mechanism of C’-mediated neutralization revealed that anti-A33 and anti-B5 antibody responses utilized C’ to neutralize EV in different ways. In agreement with previous reports, A33 antibody required C’ activation steps that could lead to virolysis, while B5 antibody and C’ could neutralize through opsonization. Benhnia et al. provided a model of anti-B5 antibody- C’ mediated neutralization whereby B5 protein was not in high enough density on the surface of EV to allow for the basic occupancy model of antibody neutralization to succeed. They reasoned that deposited C’ components enhanced the footprint of antibody bound to B5 protein on the virus surface to assist in neutralization. This model explained why virolysis was not needed. Lustig et al. provided a model of C’ assisted EV neutralization for anti-A33 antibody. In this model, C’ lyses the outer envelope of EV, which provides anti-MV antibody access to the MV virion within. An anti-MV antibody was required to be present during the assay for C’ assisted neutralization to occur. Because our luciferase based EV neutralization assay always contains an anti-MV antibody to eliminate contaminating MV in the EV preparations, we were unable to examine neutralization in the absence of an anti-MV antibody.