反应详情

QFP-phage and control insertless phage were amplified to a high titer for labeling with 64Cu radionuclide via the macrocyclic bifunctional chelator 2-(4-isothiocyanato benzyl)-1,4,7,10 tetraazacyclo dodecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) (Macrocyclics, Dallas, Tex.). The isothiocyanate functionality of the p-SCN-Bn-DOTA reacts with primary amino groups on the phage coat protein pVIII to produce a covalent attachment of DOTA to the phage surface (Jakubowski et al. (2008) J Anal At Spectrom 23(5):1497-1507). To carry out this functionalization procedure, phage (1×1011 pfu) were re-suspended in 400 μl conjugation buffer (carbonate-bicarbonate buffer, pH=9) and 4 μl p-SCN-Bn-DOTA (100 mM stock) were added. The conjugation reaction was conducted overnight at 35° C. DOTA-phage intermediate was separated from the unreacted DOTA through a 50K microcone filter by centrifugation at 14000 g for 10 minutes. DOTA-phage conjugates were re-suspended in 400 μl 10.1M sodium acetate buffer (pH=5.5) and incubated with 64CuCl2 (2 mCi per reaction, decay corrected) for 50 minutes at 50° C. Unbound 64Cu2+ was removed by ultracentrifugation as described above. 64Cu labeled phage were reconstituted in sterile PBS. Activity was determined using a γ-counter (Packard) immediately before injecting into mice. Complexes of DOTA-phage with cold CuCl2 were prepared to test phage infectivity and target binding upon labeling.