Grierson John R, Yagle Kevin J, Eary Janet F, Tait Jonathan F, Gibson Don F, Lewellen Barbara, Link Jeanne M, Krohn Kenneth A
Department of Radiology, University of Washington, Seattle, Washington 91895, USA.
Bioconjug Chem. 2004 Mar-Apr;15(2):373-9. doi: 10.1021/bc0300394.
Recombinant human-annexin-V was conjugated with 4-[F-18]fluorobenzoic acid (FBA) via its reaction with the N-hydroxysuccinimidyl ester (FBA-OSu) at pH 8.5. A series of reactions using varying amounts of annexin-V, unlabeled FBA-OSu, and time produced products with different conjugation levels. Products were characterized by mass spectrometry and a cell-binding assay to assess the effect of conjugation. In each case, the conjugated protein was a mixture of proteins with a range of conjugation. Annexin-V could be conjugated with an average of two FBA mole equivalents without decreasing its affinity for red blood cells (K(d) 6-10 nM) with exposed phosphatidylserine. An average conjugation of 7.7 (range 3-13) diminished the binding 3-fold. Large-scale production and purification of [F-18]FBA-OSu from [F-18]fluoride was accomplished within 90 min and in 77% radiochemical yield (decay-corrected to the end of cyclotron bombardment). The conjugation reaction of annexin with [F-18]FBA-OSu was studied with respect to activity level, protein mass, and concentration. Under the most favorable conditions, >25 mCi [F-18]fluoroannexin (FAN) was isolated in 64% yield (decay-corrected for a 22 min conjugation process) from labeling 1.1 mg of annexin-V. A pilot PET imaging study of [F-18]fluoroannexin in normal rats showed high uptake in the renal excretory system and demonstrated sufficient clearance from most other internal organs within 1 h. [F-18]Fluoroannexin should prove useful in imaging targeted apoptosis.
重组人膜联蛋白-V在pH 8.5条件下通过与N-羟基琥珀酰亚胺酯（FBA-OSu）反应与4-[F-18]氟苯甲酸（FBA）偶联。使用不同量的膜联蛋白-V、未标记的FBA-OSu以及不同时间进行了一系列反应，产生了具有不同偶联水平的产物。通过质谱和细胞结合试验对产物进行表征，以评估偶联的效果。在每种情况下，偶联的蛋白质是一系列具有不同偶联程度的蛋白质混合物。膜联蛋白-V平均可与两个FBA摩尔当量偶联，而不会降低其对暴露有磷脂酰丝氨酸的红细胞的亲和力（解离常数K(d)为6 - 10 nM）。平均偶联7.7（范围为3 - 13）会使结合力降低3倍。从[F-18]氟化物大规模生产和纯化[F-18]FBA-OSu在90分钟内完成，放射化学产率为77%（经衰变校正至回旋加速器轰击结束）。研究了膜联蛋白与[F-18]FBA-OSu的偶联反应在活性水平、蛋白质质量和浓度方面的情况。在最有利的条件下，从1.1 mg膜联蛋白-V标记中以64%的产率（经衰变校正用于22分钟的偶联过程）分离得到>25 mCi的[F-18]氟膜联蛋白（FAN）。对正常大鼠进行的[F-18]氟膜联蛋白的PET成像初步研究显示，在肾排泄系统中有高摄取，并且在1小时内从大多数其他内部器官有足够的清除。[F-18]氟膜联蛋白应被证明在靶向凋亡成像中有用。
