RADIOLABELING OF FABS BY PHOTOCHEMICAL CONJUGATION

描述：拟议的研究是制备和评价双功能 设计用于光标记抗体的螯合剂， 具有放射性核的抗体片段（Tc-99 m和Re-186/Re-188）。 这些 研究是基于一个重大发现， 具有全氟芳基叠氮化物光探针的蛋白质和抗体可以 以1：1的光电探针： 抗体，并几乎完全保留免疫反应性的 抗体的 这些性质是理想的辐射标记，并建议 光标记具有真实的潜力， 治疗和诊断放射性核素。 该研究提出了几个 目的：放射性标记光探针的合成; 抗体和抗体片段;以及体外和体内测试 稳定性、免疫反应性、生物定位和清除。 初始 努力将是将光不稳定部分连接到核特异性 螯合剂，其已知具有良好的稳定性。 附件将 使用光探针和螯合剂之间的连接体来实现， 确保电子和空间隔离。 还将设计连接器 以促进放射性同位素清除后，catenib B有机体。 这些合成的双功能螯合剂将用以下标记： 用于测试稳定性和光标记效率的所需放射性金属。 这些用于抗体标记的双功能放射性标记的活力将 用B72.3抗体和由有限的 蛋白水解或在细菌中重组表达。 标签效率将 通过使用UV和/或辐射监测器的尺寸排阻HPLC测定 使用较早开发的方法。 那些显示高度依恋的标签 将使用体外稳定性研究进一步评价效率， 免疫反应性。 最后，标记的材料，显示最大的 承诺，将进行体内生物分布测试， 间隙

DESCRIPTION: The proposed research is to prepare and evaluate bifunctional chelating agents designed for the purpose of photolabeling antibodies and antibody fragments with radionuclei (Tc-99m and Re-186/Re-188). These studies are based on the significant discovery that the photolabeling of proteins and antibodies with a perfluoroaryl azide photoprobe could be achieved with high efficiency (75-80%) in a 1:1 ratio of photoprobe: antibody, and with nearly complete retention of immunoreactivity of the antibody. These properties are ideal for radionuclear labeling and suggest that photolabeling has real potential for labeling of antibodies with therapeutic and diagnostic radionuclei. The proposed research has several objectives: synthesis of the radiolabeled photoprobe; attachment to antibodies and antibody fragments; and in vitro and in vivo testing of stability, immunoreactivity, biolocalization, and clearance. The initial effort will be the attachment of the photolabile moiety to nuclei-specific chelating agents, which are known to have good stability. Attachment will be achieved using linkers between the photoprobe an the chelating agents to insure electronic and spatial isolation. The linkers will also be designed to promote radioisotope clearance following catabolism b the organism. These synthesized bifunctional chelating agents will be labeled with the desired radiometal for testing of stability and photolabeling efficiency. The viability of these bifunctional radiolabels for antibody labeling will be tested with the B72.3 antibody and antibody fragments produce by limited proteolysis or recombinant expression in bacteria. Labeling efficiency will be determined by size exclusion HPLC using UV and/or radiation monitors using methods developed earlier. Those labels showing high attachment efficiency will be further evaluated using in vitro studies of stability and immunoreactivity. Finally, the labeled materials, which show the greatest promise, will be subjected to in vivo testing for biodistributon and clearance