- 期刊
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
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.
並列摘要
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.
延伸閱讀
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