Reisser-Rubrecht Laetitia, Torne-Celer Caroline, Rénier Wendy, Averseng Olivier, Plantevin Sophie, Quéméneur Eric, Bellanger Laurent, Vidaud Claude
CEA Valrhô, DSV/IBEB/Service de Biochimie et de Toxicologie Nucléaire, BP 17171, F-30207 Bagnols sur Cèze, France.
Chem Res Toxicol. 2008 Feb;21(2):349-57. doi: 10.1021/tx700215e. Epub 2007 Dec 22.
Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO2(2+), Fe (3+), Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO2(2+) equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups.
单克隆抗体（mAb）已被证明是研究蛋白质-金属相互作用的有价值模型，先前的报告描述了针对螯合金属离子（包括铀酰）的非常特异性的抗体。我们制备了针对UO2(2+)-DCP-BSA（DCP，1,10-菲咯啉-2,9-二羧酸）的特异性单克隆抗体，以生成可能与不同环境中各种络合形式的铀酰发生交叉反应的新抗体集，以便在毒理学领域进一步应用。通过用UO2(2+)-DCP-酪蛋白进行反向筛选，我们选择了两种针对铀酰-DCP的高度特异性单克隆抗体（KD 10-100 pM）：U04S和U08S。在不同金属离子（UO2(2+)、Fe(3+)、Zn2+、Cu2+和Ca2+）存在下的竞争试验表明，溶液中的铀酰可以作为良好的竞争者，这表明一些抗体能够与UO2(2+)赤道配位平面中除DCP之外的螯合基团发生交叉反应。有趣的是，其中一种抗体可用于揭示细胞样品中的铀酰阳离子。免疫标记后的荧光激活细胞分选分析揭示了铀酰与人肾细胞HK2的相互作用。使用荧光标记的单克隆抗体进行金属免疫染色可以直接观察到铀酰在细胞内的积累。我们的结果表明，U04S单克隆抗体的表位大多包括铀酰部分，其互补位可以容纳多种螯合基团。
