Thakur M L, DeFulvio J, Richard M D, Park C H
Department of Radiation Oncology and Nuclear Medicine, Thomas Jefferson University, Philadelphia, PA 19107.
Int J Rad Appl Instrum B. 1991;18(2):227-33. doi: 10.1016/0883-2897(91)90083-w.
We have evaluated five compounds, stannous chloride (SnCl2), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with 99mTc. The reduction of 99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 +/- 1% for an IgM and 82.6 +/- 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such 99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no 99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 +/- 2.9% per gram for 125I-TNT-1 (IgG) and 6 +/- 5.1% per gram for the same MoAb labeled with 99mTc using AA. The AA technique promises to label antibodies with 99mTc and perhaps with 186Re, by a simple "kit" procedure.
我们评估了五种化合物，即氯化亚锡（SnCl₂）、2-巯基乙醇（2-ME）、二硫苏糖醇（DTT）、二硫赤藓糖醇（DTE）和抗坏血酸（AA），用于还原单克隆抗体MoAb（二硫键），并比较它们用⁹⁹ᵐTc标记MoAb的效果。在pH 11条件下用连二亚硫酸盐还原⁹⁹ᵐTc几乎是定量的。对于所检测的三种IgG和三种IgM抗体，使用摩尔比为3500:1的AA，标记效率大于95%。因此无需纯化。通过特异性抗原测定确定的AA制剂的免疫特异性，对于一种IgM为84±1%，对于一种IgG为82.6±1.1%，在所有测试试剂中最高。通过用半胱氨酸、二乙三胺五乙酸（DTPA）和人血清白蛋白等对⁹⁹ᵐTc有亲和力的试剂处理产物来评估示踪剂的稳定性。通过高效液相色谱（HPLC）分析，当螯合剂与蛋白质的摩尔比高达500:1时，没有⁹⁹ᵐTc发生转螯合。在两组各五只小鼠中，注射后4小时，¹²⁵I-TNT-1（IgG）的肝脏摄取平均为每克6.8±2.9%，使用AA用⁹⁹ᵐTc标记的相同MoAb的肝脏摄取平均为每克6±5.1%。AA技术有望通过简单的“试剂盒”程序用⁹⁹ᵐTc标记抗体，也许还能用¹⁸⁶Re标记。
