Immunoaffinity capillary electrophoresis applications of clinical and pharmaceutical relevance
Immunoaffinity capillary electrophoresis applications of clinical and pharmaceutical relevance
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DOI：
10.1007/s00216-003-2326-y
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发表时间：
2004-01-01
影响因子：
4.3
通讯作者：
Guzman, NA
中科院分区：
文献类型：
--
作者：
Guzman, NA
在过去的十年中，几种CE方法已经取代了临床实验室中的一些传统程序，特别是对血清蛋白、许多与法医相关的物质以及以专门的分子诊断试验为特征的分析物的鉴定和定量[1，2，3，4，5，6，7，8]。每一种CE模式都被成功地用于产生大量的临床和制药应用。然而，最近一种全自动的二维毛细管电泳法已经开始引起人们的兴趣，这种方法提供了更好的分离和灵敏度。一种方法是用荧光试剂标记蛋白质，然后用两根毛细管分离蛋白质组分，一根用中性pH值的分离缓冲液，另一根用高pH值的分离缓冲液[12]。另一种概念，称为免疫亲和毛细管电泳法(IACE)，正在几个实验室得到普及。这种在线预富集CE的概念已被用作一种很有前途的技术，用于捕获、浓缩、净化和分析细胞、细胞器、组织和/或生物液中存在的各种物质[11，13，14，15]。将免疫亲和力偶联到CE上，用于免疫吸附和分离选择性免疫活性物质(S)[11，13，14，15]的二维分离方法可以比作使用强大的磁铁从干草堆中分离出针。抗体是生物“磁铁”，正被用作强大的工具，为发展药物、法医和临床应用提供免疫分析和CE之间的协同作用。已经报道了一种提供多肽和药物的多步骤分离和分析的仪器[16]。该仪器包含一种或一系列用于在线亲和毛细管电泳法的固相微萃取装置。每个设备设计成十字形或十字形配置，有四个端口，包括一个用于输送样品和洗涤缓冲液的大口径管，以及一个用于分离分析物的小口径熔融石英毛细管。在传送管和分离管的交叉处制造了一个小腔，称为分析物浓缩器-微反应器，它包含四个多孔壁或半透膜(管的每个入口和出口一个)，允许将珠子或合适的微结构限制在受限区域内。分析物浓缩器中的珠子表面携带吸附化学或亲和配基材料(例如抗体、凝集素、酶等)的分子识别。这种吸附固定固相(S)的作用是捕获存在于简单或复杂基质中的目标分析物，该基质对结合物质具有选择性的高亲和力。一旦所需分析物被结合的亲和配体保留，用适当的缓冲液将盐和其他不需要的或污染的物质从传输毛细管中洗掉，使目标分析物非共价结合到固定化的亲和配体上。最后，使用最佳分离缓冲液调节每个毛细管。然后，用一小块解吸缓冲液洗脱目标配体，通常是300 mM甘氨酸缓冲液，pH 3.4或含有有机溶剂的中性pH缓冲液。然后对分离毛细管内的洗脱化合物(S)进行毛细管电泳法，一次一根毛细管。分离出的化合物(S)在离开毛细管时，使用一个或一组检测器(包括质谱仪)进行监测。免疫亲和毛细管电泳法使用具有多个毛细管的仪器进行，每个毛细管连接到带有…的设备内的分析物浓缩器
In the last decade, several CE methods have replaced a number of traditional procedures in the clinical laboratory, in particular the identification and quantification of serum proteins, many substances of forensic relevance, and analytes characterized by specialized molecular diagnostic tests [1, 2, 3, 4, 5, 6, 7, 8]. Every mode of CE has been used successfully to generate a large number of clinical and pharmaceutical applications. However, recently a fully automated, two-dimensional CE approach that provides improved separation and sensitivity has begun to generate interest. One approach is to label proteins with a fluorogenic reagent, and separate the components using two capillaries, one with a separation buffer at neutral pH, the other with a separation buffer at high pH [12]. The other concept, termed immunoaffinity capillary electrophoresis (IACE) is gaining popularity in several laboratories. This concept of on-line preconcentration CE has been used as a promising technique to trap, enrich, clean, and analyze a variety of substances present in a cell, organelle, tissue, and/or biological fluid [11, 13, 14, 15]. A two-dimensional separation method, coupling immunoaffinity to CE for the immunoadsorption and separation of a selective immunoreactive substance (s)[11, 13, 14, 15] can be compared to the isolation of a needle from a haystack using a powerful magnet. Antibodies are biological “magnets” that are being used as powerful tools to provide synergy between immunoassay and CE for the development of pharmaceutical, forensic, and clinical applications. The description of an instrument that provides a multistep separation and assay of peptides and drugs has been reported [16]. The instrument contains one or a series of solid-phase, microextraction devices fabricated for use in on-line, affinity capillary electrophoresis. Each device, designed in a cross-shaped or cruciform configuration with four ports, includes a large-bore tube to transport samples and washing buffers, and a small-bore fused-silica capillary for separation of analytes. At the intersection of the transport and separation tubes, a small cavity was fabricated, termed the analyte concentrator-microreactor, which contains four porous walls or semipermeable membranes (one for each inlet and outlet of the tubes), permitting the confinement of beads or suitable microstructures within a restricted area. The surface of the beads in the analyte concentrators carried a molecular recognition adsorbing chemical or affinity ligand material (eg, antibody, lectin, enzyme, etc.). The role of this adsorbed stationary solid-phase (s) was to capture a target analyte present in a simple or complex matrix that has a selective high affinity for the bound material. Once the desired analyte is retained by the bound affinity ligand, salts and other unwanted or contaminating materials are washed away from the transport capillary with an appropriate buffer, leaving the target analyte noncovalently bound to the immobilized affinity ligand. Finally, each capillary is conditioned using an optimum separation buffer. Then, the target ligand is eluted with a small plug of a desorption buffer, usually 300 mM glycine buffer pH 3.4 or a neutral pH buffer containing organic solvent. Capillary electrophoresis is then performed for the eluted compound (s) within the separation capillary in a sequential mode, one capillary at the time. The separated compound (s) were monitored using one or an array of detectors, including a mass spectrometer, while exiting the capillary. Immunoaffinity capillary electrophoresis (IACE) performed with an instrument with multiple capillaries, each connected to an analyte concentrator within a device with a …