CAPILLARY ELECTROPHORESIS BASED IMMUNOASSAY TO DETERMINE INSULIN CONTENT AND INSULIN-SECRETION FROM SINGLE ISLETS OF LANGERHANS
CAPILLARY ELECTROPHORESIS BASED IMMUNOASSAY TO DETERMINE INSULIN CONTENT AND INSULIN-SECRETION FROM SINGLE ISLETS OF LANGERHANS
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DOI：
10.1021/ac00101a020
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发表时间：
1995-03-01
影响因子：
7.4
通讯作者：
KENNEDY, RT
中科院分区：
文献类型：
--
作者：
SCHULTZ, NM;HUANG, L;KENNEDY, RT
建立了一种基于毛细管区带电泳-激光诱导荧光检测（CZE-LIF）的胰岛素竞争免疫分析方法（FITC-胰岛素）作为示踪剂和抗胰岛素单克隆抗体的Fab片段作为免疫试剂，当CZE毛细管涂有中性亲水聚合物时，测定的重现性和可靠性得到改善。该测定法的平均相对标准偏差为3.4%，检测限为3 nM或约6 fg注射。使用该测定法，单个胰岛的胰岛素含量测定为35 +/- 4 ng，这与文献值非常一致。该测定还用于测定来自单个胰岛的胰岛素分泌。在5分钟静态孵育期间，3 mM葡萄糖引起0.8 +/-0.2ng的胰岛素释放，而16 mM葡萄糖引起3.2 +/-0.5ng的胰岛素释放。这些值也与先前报道的结果良好一致。这些应用证明了在复杂的生物微环境中快速测定低水平蛋白质的能力。
The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described, The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent, Reproducibility and reliability of the assay are improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected, Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets, During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release, These values are also in good agreement with previously reported results, These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.