Capillary electrophoresis of insulin-like growth factors: enhanced ultraviolet detection using dynamically coated capillaries and on-line solid-phase extraction.
Capillary electrophoresis of insulin-like growth factors: enhanced ultraviolet detection using dynamically coated capillaries and on-line solid-phase extraction.
复制标题
胰岛素样生长因子的毛细管电泳：使用动态涂层毛细管和在线固相萃取增强紫外线检测。
DOI：
10.1006/abio.1997.2553
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发表时间：
1998
影响因子：
2.9
通讯作者：
Landers,JP
中科院分区：
文献类型：
--
作者：
Roche,ME;Anderson,MA;Oda,RP;Riggs,BL;Strausbauch,MA;Okazaki,R;Wettstein,PJ;Landers,JP
胰岛素样生长因子-I和胰岛素样生长因子-II(IGF-I和IGF-II)由于在结构和免疫上的同源性，很难分离和测定。许多与IGFS高亲和力相互作用的结合蛋白(Bps)使IGFS的准确和重复性测量能力变得复杂。目前测量IGF的方法是基于免疫的，包括从IGFS解离，通过样品酸化去除结合蛋白，通过固相吸附去除。然而，最终的结果是一种耗时的分析，充其量也是半定量的。为了提高IGF-I和IGF-II测定的重复性和准确性，对使用动态涂层和裸露二氧化硅毛细管的电泳系统进行了评估。在存在或不存在阳离子添加剂溴化十六烷基铵的情况下，在裸二氧化硅毛细管中的分离对IGF-I和IGF-II的分离无效。然而，当毛细管动态涂上聚丙稀膜时，IGF-I和IGF-II可以在低pH缓冲液中在BSA样品基质中被拆分。尽管IGFS可以在使用IGF-I类似物作为内标的情况下得到解决，但仅在运行12次后就需要聚溴重新涂覆，并且观察到涂层对涂层的重现性很差。使用聚二烯丙基二甲基氯化铵(PDMAC)作为动态阳离子涂层和含有0.5%PDMAC的低pH缓冲液被发现要有效得多，提供了IGF-I和IGF-II的可重复分离。发现PDMAC不需要包括在分离缓冲器中以获得关于IGF分离的可重现分析。随后，在需要重新涂覆之前，功能保持不变的连续分析多达35-40次。无需在缓冲液中加入PDMAC，即可实现在线固相萃取-毛细管电泳法检测低至195 ng/ml的IGF-I和IGF-II。
Insulin-like growth factor-I and -II (IGF-I and IGF-II) are difficult to separate and measure as a result of their homology, both structurally and immunologically. A number of binding proteins (BPs) which interact with the IGFs with high affinity complicate the ability to measure the IGFs accurately and reproducibly. Current methodology for measuring IGF is immuno-based and involves dissociation from the IGFs and removal of the binding proteins through sample acidification and removal by solid-phase adsorption. However, the net result is an assay that is time-consuming and, at best, semiquantitative. In an attempt to improve the reproducibility and accuracy of IGF-I and -II measurement, electrophoretic systems employing dynamically coated and bare silica capillaries were evaluated. Separations in bare silica capillaries in the presence or absence of the cationic additive, decamethonium bromide were ineffective for resolving IGF-I and IGF-II. However, when the capillary was coated dynamically with polybrene, IGF-I and -II could be resolved in a BSA sample matrix using a low pH buffer. Despite the fact that the IGFs could be resolved in the presence of an IGF-I analog used as an internal standard, polybrene recoating was required after as few as 12 runs and poor coating-to-coating reproducibility was observed. Use of polydiallyldimethylammonium chloride (PDMAC) as a dynamic cationic coating and a low pH buffer containing 0.5% PDMAC was found to be much more effective, providing reproducible separation of IGF-I and -II. It was found that PDMAC need not be included in the separation buffer to obtain reproducible analyses regarding IGF separation. Subsequently, functionality remained intact for as many as 35–40 consecutive analyses before recoating was required. Without the need for PDMAC in the buffer, on-line solid-phase extraction–capillary electrophoresis could be accomplished for detection of IGF-I and -II at concentrations as low 195 ng/ml.