The two-step pretargeting method utilized biotinylated G1 phage and 111In-DTPA-SA as previously described by Newton et al. [23]. The DTPA-SA conjugates generated contained, on average, two DTPA chelates per SA, and the G1 phages were biotinylated with, on average, five biotins per phage particle. The added mass of five biotins did not significantly alter the final mass of the phage particle (23.6 MDa). The two-step pretargeting method required, in brief, tumor-bearing SCID mice to receive VX-809 tail vein injection of 1×1011 phage. The biotinylated G1 phages were allowed to circulate within the mice for 4 h, at which time the mice received a second tail vein injection containing 111In-DTPA-SA (Fig. 3). Each mouse was then sacrificed 24 h postinjection of the 111In-DTPA-SA for investigation of either the biodistribution of the radiolabel or for SPECT/CT imaging. In comparison, the three-step pretargeting protocol delivered the radiolabel via the small molecule 111In-DOTA-biotin (～840 Da), instead of the much larger molecular weight 111In-DTPA-SA (～60,000 Da) (Fig. 3). The tumor-bearing SCID mice being treated with the three-step pretargeting method first received an intravenous injection of biotinylated G1 phage. Four hours postinjection of the biotinylated phage, the tumor-bearing mice received a genotype second injection containing avidin. Twenty four hours later, the same mice then received the final injection of 111In-DOTA-biotin. Half an hour postinjection of the radiolabeled biotin, the mice were then subjected to biodistribution studies or SPECT/CT imaging.