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題名 | 簡易製備細胞凋亡藥物碘-123-annexin V=A Feasible Method to Prepare 123I-Annexin V for Imaging Apoptosis |
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作者 | 廖美秀; 詹東榮; 魏孝萍; 江昭志; 戚謹文; 傳應凱; 沈立漢; | 書刊名 | 核子醫學雜誌 |
卷期 | 19:4 民95.12 |
頁次 | 頁231-238 |
分類號 | 418.346 |
關鍵詞 | 碘-123-annexin V; 超離心純化法; 放射化學純度; 123I-annexin V; Ultracentrifugation; Radiochemical purity; |
語文 | 中文(Chinese) |
中文摘要 | 背景:藉由annexin V與phosphatidylserine (PS)的特異性結合能力而發展的放射性標幟annexin V可用於觀測細胞凋亡(apoptosis)時的PS細胞膜外表現。許多放射性標幟annexin V使用雙官能基(土方攵虫)合劑修飾annexin V,需要毫克量價格昂貴的annexin V。本研究評估以碘-123標幟微量annexin V合併使用超離心(ultracentrifugation)技術純化標幟物製備碘-123-anncxin V之可行性。 方法:置10µg annexin V、40µ1 0.1M磷酸二氫鉀溶液及1~10 mCi碘-123-碘化銨於一塗佈50µg Iodo-Gen之試管內,室溫下反應10分鐘,再以Nanosep(上標 TM)分離管進行超離心純化。以ITLC-SG放射薄層色層分析法(分別以0.9%氣化鈉溶液及85%乙醇溶液展開)以及Ultrahydrogel 250放射FPLC(流動相為pH 7.4之0.01 M磷酸二氫鉀溶液,流速為每分鐘0.8 ml)進行放射化學純度量測及標幟產率分析。以sodium dodecyl sulphate-polyacrylamide凝膠電泳(SDS-PAGE)(gel electrophoresis)確認碘-123-annexin V分子大小。以試管試驗分析碘-123-annexin V是否仍保有對PS的特異結合能力。 結果:六次標幟實驗結果顯示以碘-123-碘化銨進行碘化反應的標幟效率為59~87%。以Nanosep(上標 TM)超離心純化後之碘-123-annexin V放射化學純度均大於90%,且具有至少48小時之安定性。SDS-PAGE分析碘-123-annexin V分子量與人類annexin V(35.8kDa)相當。碘-123-annexin V放射活性滯留在含PS之試管明顯高於不含PS之對照組(P<0.03),顯示碘-123-annexin V仍保有對PS的特異結合能力。 結論:使用微量annexin V及Iodo-Gen氧化性碘化反應,並合併使用Nanosep(上標 TM)超離心法純化標幟物,可以快速製備高穩定性及具PS結合能力的碘-123-annexin V。 |
英文摘要 | Background: The exposure of phosphatidylserine (PS) on the cell surface is one of the early stages in apoptotic cells. Annexin V is a protein with a high affinity for PS, therefore, annexin V-mediated detection of PS becomes more important for monitoring cells under apoptosis. Many annexin Vbased radiotracers have been developed by conjugation of annexin V with various bifunction chelators. However, the conjugation required several mg of very expensive recombinant human annexin V. This study evaluated a feasible method that prepared 123I-annexin V by using μg-level protein for oxidative radioiodination and followed by ultracentrifugation for purification. Methods: Radioiodination was carried out for 10 min at room temperature by mixing 10 μg of annexin V, 1~10mCi of 123I-ammonium iodide and 40μl of 0.1M KH2PO4 in a test tube precoated with 50mg of Iodo-Gen. The radioiodinated annexin V was purified by Nanosep(superscript TM) ultracentrifugation, and its radiochemical purity was determined by radio-instant thin layer chromatography (ITLC) and fast protein liquid chromatography (FPLC), respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular size of 123Iannexin V. The specific binding of the radiotracer for PS was verified by incubating and counting 123I-annexin V in the test tubes with and without PS. Results: The radiolabeling yield ranges 59~87% for six preparations. Following purification with Nanosep(superscript TM) ultracentrifugation, the radiochemical purity of 123I-annexin V was more than 90%. The in-vitro stability of 123I-annexin V was verified for up to 48h. The result of SDS-PAGE revealed that the molecular weight of the radiotracer was similar to that of human annexin V (35.8kDa). The significant retention of radioactivity in PS-containing test tubes than those in PS-free control (P<0.03) demonstrated the specific biding capability of 123I-annexin V for PS. Conclusions: By using μg-level annexin V for oxidative radioiodination and followed by employing ultracentrifugation for purification, 123I-annexin V can be rapidly prepared in high radiochemical purity, with high stability and capability to bind PS specifically. |
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