A fluorescence-based one-step assay for serum non transferrin-bound iron
A fluorescence-based one-step assay for serum non transferrin-bound iron
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DOI：
10.1006/abio.2001.5378
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发表时间：
2001-12-15
影响因子：
2.9
通讯作者：
Cabantchik, ZI
中科院分区：
文献类型：
--
作者：
Breuer, W;Cabantchik, ZI
我们介绍了一种监测非转铁蛋白结合铁（NTBI）的方法，NTBI是一种与铁代谢不平衡相关的不稳定和潜在毒性形式的血清铁。该测定采用荧光素标记的脱铁转铁蛋白（Fl-aTf），其在结合铁后经历荧光淬灭。它的优点是简单，高灵敏度，并检测那些形式的NTBI，坚持在血清中低转铁蛋白饱和度。由于NTBI不易用于检测，因此可通过10 mM草酸盐进行动员。内源性血清脱铁转铁蛋白，能够结合叶酸动员的NTBI，被0.1 mM镓（III）阻断。这种金属，像铁一样，与Fl-aTf结合，但它既不淬灭其荧光，也不干扰铁的淬灭。将血清和含有草酸盐、Ga（Cl）（3）和Fl-aTf的试剂在多孔板中混合，1小时后在酶标仪中测定荧光。为了补偿由血清颜色引起的人为荧光变化，用过量的未标记的脱铁转铁蛋白制备平行样品，其清除样品中的所有铁。从8个血色素沉着症患者的血清进行了测试NTBI通过本试验，并通过一个既定的替代方法，定性相似的结果。该测试的一个潜在应用是从有发展NTBI风险的患者中筛选大量样本。(C)2001年，爱思唯尔科学。
We introduce a method for monitoring non-transferrin-bound iron (NTBI), a labile and potentially toxic form of serum iron associated with imbalanced iron metabolism. The assay employs fluorescein-labeled apotransferrin (Fl-aTf), which undergoes fluorescence quenching upon binding iron. It has the advantages of simplicity, high sensitivity, and detection of those forms of NTBI that persist in sera with low transferrin saturations. Since NTBI is not readily available for detection, it is mobilized by 10 mM oxalate. Endogenous serum apotransferrin, capable of binding oxalate-mobilized NTBI, is blocked by 0.1 mM gallium(III). This metal, like iron, binds to Fl-aTf, but it neither quenches its fluorescence nor interferes with quenching by iron. Serum and reagent containing oxalate, Ga(Cl)(3), and Fl-aTf are mixed in multiwell plates and fluorescence is determined after 1 h in a microplate reader. To compensate for artifactual fluorescence changes caused by serum color, parallel samples are prepared with excess unlabeled apotransferrin, which scavenges all iron in the sample. Sera from eight hemochromatosis patients were tested for NTBI by the present assay and by an established alternative method, with qualitatively similar results. A potential application of the test is for screening large numbers of samples from patients at risk of developing NTBI. (C) 2001 Elsevier Science.