Liang F H, Virzi F, Hnatowich D J
University of Massachusetts Medical Center, Department of Nuclear Medicine, Worcester 01605.
Int J Rad Appl Instrum B. 1987;14(6):555-61. doi: 10.1016/0883-2897(87)90025-0.
Previously we investigated the use of DTPA-coupled proteins to simplify labeling with 99mTc but especially to improve the stability of the label. These investigations have now been extended to include several N2S2 ligands such as N,N'-bis(2-methyl-2-mercaptopropyl)ethylenediamine (DADT) and a novel ligand of similar structure with a propylene bridge between two amines, 2-hydroxy-N,N'-bis(2-methyl-2-mercaptopropyl)propylenediamine++ + (DADT-3C-2OH). The condition of labeling of free ligand (pH, buffer and tin concentration) was optimized to provide 100% chelation with 99mTc at reasonable ligand concentrations (100 micrograms/mL or less). Labeling was determined by paper chromatography, reverse-phase and size-exclusion HPLC. After incubation in fresh serum, 37 degrees C for 24 h, repeat analysis showed less than 5% dissociation of the chelate. By contrast, the DTPA chelate shows instability towards oxidation during this period. DADT derivatized on an ethylene carbon showed almost identical serum stability as DADT itself whereas when derivatized on a nitrogen greater instabilities were apparent. Using identical labeling conditions, free DADT was chelated in the presence of IgG at different ligand: protein molar ratios. Non-specific binding of 99mTc to IgG at a 10:1 DADT-HM:IgG molar ratio was as little as 5% and was essentially zero at a 2:1 DADT:IgG molar ratio when labeling was by transcomplexation from 99mTc-EDTA. The DADT-3C-2OH ligand showed superior performance both in regard to serum stability and the absence of non-specific binding. In conclusion, the N2S2 ligands form more stable chelates with 99mTc than does DTPA with reduced non-specific binding and may therefore represent an attractive alternative for labeling proteins with 99mTc by the bifunctional chelate approach.
此前我们研究了使用二乙三胺五乙酸（DTPA）偶联蛋白来简化用99m锝（99mTc）标记的过程，尤其是为了提高标记的稳定性。现在这些研究已扩展至包括几种N2S2配体，如N,N'-双(2-甲基-2-巯基丙基)乙二胺（DADT）以及一种结构类似、在两个胺之间有一个丙烯桥的新型配体，即2-羟基-N,N'-双(2-甲基-2-巯基丙基)丙二胺（DADT-3C-2OH）。对游离配体的标记条件（pH值、缓冲液和锡浓度）进行了优化，以便在合理的配体浓度（100微克/毫升或更低）下实现与99mTc的100%螯合。通过纸色谱法、反相高效液相色谱法和尺寸排阻高效液相色谱法测定标记情况。在新鲜血清中于37℃孵育24小时后，重复分析显示螯合物的解离率低于5%。相比之下，在此期间DTPA螯合物对氧化表现出不稳定性。在乙烯碳上衍生化的DADT显示出与DADT本身几乎相同的血清稳定性，而当在氮上衍生化时则明显更不稳定。在相同的标记条件下，游离DADT在不同的配体:蛋白质摩尔比下于免疫球蛋白（IgG）存在的情况下进行螯合。当通过从99mTc-乙二胺四乙酸（99mTc-EDTA）进行转络合标记时，在DADT-HM:IgG摩尔比为10:1时，99mTc与IgG的非特异性结合低至5%，而在DADT:IgG摩尔比为2:1时基本为零。DADT-3C-2OH配体在血清稳定性和无非特异性结合方面均表现出卓越性能。总之，N2S2配体与99mTc形成的螯合物比DTPA更稳定，非特异性结合减少，因此可能是通过双功能螯合方法用99mTc标记蛋白质的有吸引力的替代物。
