Guillo Christelle, Roper Michael G
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA.
Electrophoresis. 2008 Jan;29(2):410-6. doi: 10.1002/elps.200700399.
A multianalyte CE competitive immunoassay using two-color detection was developed to measure insulin and glucagon in islets of Langerhans. Insulin was quantified with FITC-insulin (Ins*) and anti-insulin antibodies (Ins Ab) and glucagon was quantified with Cy5-glucagon (Glu*) and anti-glucagon antibodies (Glu Ab). A 3 mW Ar(+) laser at 488 nm and a 25 mW laser diode at 635 nm were used to excite FITC and Cy5, respectively. Fluorescence was split with a half-silvered mirror and passed through a 520 +/- 20 nm bandpass filter or a 663 nm longpass filter for the detection of insulin and glucagon, respectively. The two-color detection format enabled independent quantitation of both analytes even with concentrations of insulin immunoassay reagents 20-fold higher than glucagon reagents. Simultaneous calibration curves were generated and used to determine insulin and glucagon content in islets of Langerhans. Amounts of insulin and glucagon were 56.6 +/- 3.2 and 1.0 +/- 0.5 ng/islet, respectively. LODs were 7 nM insulin and 3 nM glucagon. The assay will be applicable to fast monitoring of multiple peptides secreted from islets of Langerhans and can be applied to other systems for the quantitation of multiple analytes with large differences in concentrations.
开发了一种采用双色检测的多分析物毛细管电泳竞争免疫分析法，用于测量胰岛中的胰岛素和胰高血糖素。胰岛素用异硫氰酸荧光素标记的胰岛素（Ins*）和抗胰岛素抗体（Ins Ab）进行定量，胰高血糖素用Cy5标记的胰高血糖素（Glu*）和抗胰高血糖素抗体（Glu Ab）进行定量。分别使用波长为488 nm的3 mW氩离子激光器和波长为635 nm的25 mW激光二极管激发异硫氰酸荧光素和Cy5。荧光通过半透半反镜分光，分别通过520±20 nm带通滤光片或663 nm长通滤光片用于检测胰岛素和胰高血糖素。即使胰岛素免疫分析试剂的浓度比胰高血糖素试剂高20倍，这种双色检测形式也能对两种分析物进行独立定量。生成了同步校准曲线，并用于测定胰岛中的胰岛素和胰高血糖素含量。胰岛素和胰高血糖素的含量分别为56.6±3.2和1.0±0.5 ng/胰岛。检测限分别为7 nM胰岛素和3 nM胰高血糖素。该分析方法适用于快速监测胰岛分泌的多种肽，也可应用于其他系统，用于定量浓度差异很大的多种分析物。
