Abstract
抗體具高度的親和力與專一性，以抗體做為放射核種之載體可準確地到達 病灶；本論文以2-亞胺環硫丁烷 (2- iminothiolane ; 2-IT) 修飾人體 免疫球蛋白G (immuno- globulin G ; IgG) ，植入額外的硫醇基，以利 鎝-99m之鍵結；藉各項特性分析，研究反應之動力參數並依最佳條件製備 適當的注射小瓶，以作為發炎病灶診斷之照影劑。本研究之特性分析包括 以瞬間薄層層析(ITLC)及高效率液相層析(HPLC)來鑑定標幟效率，以親和 層析測定免疫活性，以凝膠電泳(SDS-PAGE)分析其降解情形，藉紫外╱可 見吸光分析測定硫醇基之數目。根據反應參數之探討，適當的乾粉注射小 瓶配方為：取1 mg已修飾 IgG，加入 0.25 ml酒石酸亞錫飽和溶液為還原 劑，並添加1 mg甘露醇為穩定劑。經冷凍乾燥後加入過鎝酸根食鹽水溶液 標幟之，標幟效率高達99％，免疫活性為85％。修飾抗體以2-IT的莫耳數 為IgG 之1100倍，室溫下培育三小時，經 PD-10管柱純化製備之。其穩定 度測試分別與0.67 mM 、6.7 mM之cysteine或glutathione 進行競爭反應 ，於37℃下培育24小時，經cysteine考驗之解離率分別為13％及30％，而 glutathione 之解離率為10％及20％。分析不同時間之生物分佈，顯示此 標幟藥物係由腎、肝、脾等器官代謝。以2-IT修飾抗體分子，可植入有限 個硫醇基 (單位抗體約4∼8) ，不會改變抗體原來之特性；其標幟過程簡 單、可靠且容易製備注射小瓶，由其HPLC及SDS-PAGE分析，並無任何碎裂 或聚合反應發生，可保持抗體之完整性，故此2-IT修飾程序反應相當溫和 ，可應用於標幟抗體注射小瓶之製備，極具臨床應用潛力。 Based on high affinity and specificity of the antibodies, radiopharmaceuticals using antibodies as the carrier of radionuclides can be preferent- ially localized in the target organ. In this study,Tc-99m is labelled to human immunoglobulin G(IgG) where the antibody is modified by 2-iminothiolane (2-IT) to generate sulfhydryl groups for Tc-99m binding. The reaction parameters for the Tc-99m labelling are investigated and aimed to an optimum preparation of a kit which can be used as a clini- cal imaging agent for diagnosing infection and inflammation. The labelling efficiency and proper- ties of the Tc-99m-IgG previously produced are determined by the following analytical methods: HPLC, ITLC, affinity chromatography, SDS-PAGE, sul- fhydryl determination, cysteine and glutathione challenge reaction etc. According to observed study, the suitable formulation of a kit is that each vial is composed of 1mg of 2-IT modified IgG, 0.25 ml of the saturated solution of tin tartrate,and 1 mg of mannitol before lyophilization. The 2-IT modified IgG is prepared by mixing 2-IT and IgG with the molar ratio of 1100:1, incubated at room temperature for 3 hr, and purified by PD-10 column chromatography. The labelling efficiency of Tc-99m -IgG of the kit reconstituted with a saline solu- tion of TcO4- is higher than 99%, and its immuno- reactivity can reach ca. 85%. The stability of the Tc-99m-IgG is determined by a challenge test with 100 and 1000 molar excess of cysteine and glutath- ione. After incubating at 37 degree for 24hr, the corresponding dissociation of Tc-99m is 13% and 20% in the medium of cysteine, 10% and 20% in the medi- um of glutathione, respectively.