Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans
Simultaneous capillary electrophoresis competitive immunoassay for insulin, glucagon, and islet amyloid polypeptide secretion from mouse islets of Langerhans
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DOI：
10.1016/j.chroma.2011.05.006
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发表时间：
2011-07-01
影响因子：
4.1
通讯作者：
Roper, Michael G.
中科院分区：
文献类型：
--
作者：
Guillo, Christelle;Roper, Michael G.
建立了一种同时定量胰岛分泌胰岛素、胰高血糖素和胰岛淀粉样多肽（IAPP）的毛细管电泳竞争免疫分析法。优化了分离缓冲液和条件，以分离异硫氰酸荧光素（FITC）标记的胰高血糖素和IAPP免疫测定试剂，这些试剂用Ar（+）激光的488 nm线激发，用光电倍增管（PMT）在520 nm处检测。Cy 5标记的胰岛素免疫测定试剂由635 nm激光二极管模块激发，并在700 nm处用单独的PMT检测。使用20 mM碳酸盐分离缓冲液在pH 9.0下使用20 cm有效分离长度和500 V/cm的电场实现最佳分辨率。胰岛素、胰高血糖素和IAPP的检测限分别为2、3和3 nM。该方法用于监测在4、11和20 mM葡萄糖中孵育6 h后，这些肽从少至14个胰岛中同时分泌。对于胰岛素和IAPP，观察到分泌水平的统计学显著增加，而胰高血糖素水平在4和11 mM葡萄糖条件下显著降低。为了进一步证明该测定的实用性，证明了这些肽的Ca（2+）依赖性分泌，这与已发表的报道一致。检查多种肽分泌的能力可以允许确定胰岛内分泌过程的调节。(C)2011 Elsevier B. V.保留所有权利。
A capillary electrophoresis competitive immunoassay was developed for the simultaneous quantitation of insulin, glucagon, and islet amyloid polypeptide (IAPP) secretion from islets of Langerhans. Separation buffers and conditions were optimized for the resolution of fluorescein isothiocyanate (FITC)-labeled glucagon and IAPP immunoassay reagents, which were excited with the 488 nm line of an Ar(+) laser and detected at 520 nm with a photomultiplier tube (PMT). Cy5-labeled insulin immunoassay reagents were excited by a 635 nm laser diode module and detected at 700 nm with a separate PMT. Optimum resolution was achieved with a 20 mM carbonate separation buffer at pH 9.0 using a 20 cm effective separation length with an electric field of 500 V/cm. Limits of detection for insulin, glucagon, and IAPP were 2, 3, and 3 nM, respectively. This method was used to monitor the simultaneous secretion of these peptides from as few as 14 islets after incubation in 4, 11, and 20 mM glucose for 6 h. For insulin and IAPP, a statistically significant increase in secretion levels was observed, while glucagon levels were significantly reduced in the 4 and 11 mM glucose conditions. To further demonstrate the utility of the assay, the Ca(2+)-dependent secretion of these peptides was demonstrated which agreed with published reports. The ability to examine the secretion of multiple peptides may allow for the determination of regulation of secretory processes within islets of Langerhans. (C) 2011 Elsevier B.V. All rights reserved.