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N(C1C=CC(C[CH:9]2[CH2:20][N:19]([CH2:21][C:22]([OH:24])=[O:23])[CH2:18][CH2:17][N:16]([CH2:25][C:26]([OH:28])=[O:27])[CH2:15][CH2:14][N:13]([CH2:29][C:30]([OH:32])=[O:31])[CH2:12][CH2:11][N:10]2[CH2:33][C:34]([OH:36])=[O:35])=CC=1)=C=S.[N-]=C=S>>[CH2:9]1[N:10]([CH2:33][C:34]([OH:36])=[O:35])[CH2:11][CH2:12][N:13]([CH2:29][C:30]([OH:32])=[O:31])[CH2:14][CH2:15][N:16]([CH2:25][C:26]([OH:28])=[O:27])[CH2:17][CH2:18][N:19]([CH2:21][C:22]([OH:24])=[O:23])[CH2:20]1참가 물질
실험 절차
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.
출처 및 근거
- ORD 기록
- ord-865c3c4533d24bbf9db973ef007ac0d8
- 출처 데이터셋
- uspto-grants-2015_12
- 특허 번호
- US09198975B2
연결된 노트
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